Slide 1

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 2

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 3

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 4

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 5

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 6

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 7

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 8

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 9

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 10

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 11

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 12

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 13

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 14

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 15

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 16

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 17

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 18

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 19

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 20

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 21

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 22

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 23

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 24

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 25

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 26

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 27

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 28

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 29

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 30

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 31

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 32

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 33

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 34

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 35

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 36

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 37

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 38

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 39

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 40

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 41

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 42

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 43

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 44

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 45

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:6

School of Science and Technology, Online Counseling Resource…

Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

School of Science and Technology, Online Counseling Resource…

Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

School of Science and Technology, Online Counseling Resource…

A Flow Scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

School of Science and Technology, Online Counseling Resource…

Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

School of Science and Technology, Online Counseling Resource…

Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

School of Science and Technology, Online Counseling Resource…

A flow scheme for HPLC

School of Science and Technology, Online Counseling Resource…

High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

School of Science and Technology, Online Counseling Resource…

What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

School of Science and Technology, Online Counseling Resource…

Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

School of Science and Technology, Online Counseling Resource…

Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

School of Science and Technology, Online Counseling Resource…

Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

School of Science and Technology, Online Counseling Resource…

Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource…

Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource…

End of the Presentation

Thank You !


Slide 46

Online Counseling Resource
YCMOU ELearning Drive…

School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India

SEP –SBI084– CP01-04

Introduction
Programmes and Courses
 SEP –SBI084– CP04-U01

School of Science and Technology, Online Counseling Resource…

Credits
 Academic Inputs by

Sonali Alkari
 Faculty YCMOU Nagpur Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]

School of Science and Technology, Online Counseling Resource…

How to Use This Resource


Counselor at each study center should use this presentation to deliver

lecture of 40-60 minutes during Face-To-Face counseling.


Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.



Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.



Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.



Appear several times, for all the Self-Tests, available for this course.



Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.

4

School of Science and Technology, Online Counseling Resource…

Learning Objectives
After studying this module, you should be able to:

 Explain what is chromatography
 State principles of chromatography

 Explain basic technique of chromatography
 Explain gel Filtration Chromatography
 Explain ion exchange chromatography
 Explain

High

Performance

Liquid

Chromatography
© 2007, YCMOU. All Rights Reserved.

5

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:1
 It the individual component of a mixture have
widely dissimilar physical and chemical properties,
it is very easy to separate one from another.
 But as the individual components mixture get more
and more similar
in physical and chemical
properties, it become increasingly difficult to
separate them from one another.
 In such conditions chromatographic methods are
fruitful solution for separation.
 The term chromatography bunches together a
family of closely related extremely powerful
separation methods.

School of Science and Technology, Online Counseling Resource…

Introduction: Chromatography:2
 The first detailed description of chromatography is
credited to Michael Tsweet, a Russian biochemist,
who separated chlorophyll from a mixture of plant
pigments in 1906.
 The feature common to all chromatographic
method is that two mutually immiscible phases
are brought into contact with each other.
 One phase is stationary, while the other is mobile.
 The mobile phase either moves over the surface
or percolates through the interstices of the
stationary phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:1
 The sample mixture, introduced into the mobile phase
undergoes repeated interaction (partitions) between
the stationary and mobile phase while being carried
through the system by mobile phase.
 Different components of the sample mixture interact
with two phase differentially on the basis of small
differences in their phyico-chemical properties.
 Since these different rates of interactions govern the
migration of the sample components through the
system, each one on them migrates at a different rate.
 The compound which interacts more with mobile
phase and least with stationary phase migrates fast.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:2
 The component showing least interaction with mobile
phase while interacting strongly with the stationary
phase migrates slowly.
 This differential movement of the components is
responsible for their separation from each other.
 An analyte is in equilibrium between the two phases;
A mobile
A stationary
 Partition coefficient (also known as distribution
coefficient) is definitive term used to describe the way
in which a given compound distributes or partitions
between two immiscible phase, the stationary and
mobile phase.

School of Science and Technology, Online Counseling Resource…

Principles of Chromatography:3
The concentration of the compound in each of the
phase is described by the partition coefficient, K,
which is expresses as follows
K=

Cs

Cm

Where Cs is the concentration of the compounds in
stationary phase
Where Cm is the concentration of the compounds the
mobile phase.

School of Science and Technology, Online Counseling Resource…

Techniques of Chromatography
 There are two basic technique of chromatography:


Plane chromatography



Column chromatography

 Each of these techniques have their specific advantages,
application and modes of operation.

School of Science and Technology, Online Counseling Resource…

Planar Chromatography
 Planarchromatography is a
separation technique in which
the
stationary
phase
is
present as or on a plane.
 paperchromatography:The
plane can be a paper, serving
as such or impregnated by a
substance as the stationary
bed.
 Thin Layer Chromatograph: a
layer of solid particles spread
on a support such as a glass
plate

School of Science and Technology, Online Counseling Resource…

Column Chromatography-1
 Column chromatography is a separation technique
in which the stationary bed is within a tube.
 The particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column).
 Differences in rates of movement through the
medium are calculated to different retention times
of the sample .

School of Science and Technology, Online Counseling Resource…

Column Chromatography-2

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:1
 Gel filtration chromatography separates proteins,
peptides, and oligonucleotides on the basis of size.
 The support medium, a gel, consists of the porous
beds where pore size is strictly controlled.
 Molecules move through a bed of porous beads,
diffusing into the beads to greater or lesser
degrees.
 Smaller molecules diffuse further into the pores of
the beads and therefore move through the bed
more slowly, while larger molecules enter less or
not at all and thus move through the bed more
quickly.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:2
 Gel
Filtration
Chromatography may be
used
for
analysis
of
molecular
size,
for
separations of components
in a mixture, or for salt
removal or buffer exchange
from
a
preparation
of
macromolecules.
 Both molecular weight and
three dimensional shape
contribute to the degree of
retention.

School of Science and Technology, Online Counseling Resource…

Gel-filtration Chromatography:3

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 Gel-filtration chromatography is used to separate
three proteins whose molecular weights differ
significantly namely, alpha- and beta-amylase and
lysozyme.
 The enzymes separate cleanly into three peaks
even under the low-resolution conditions they use
in the lab.
 They can carry out the separation and assays all in
one period if they don't mess around.
 I set up in advance 1 x 17 cm columns of Sephacryl
S-300 in economy columns available from either
Bio-Rad or Kontes.
 The columns are equilibrated with 0.1 M NaCl.

School of Science and Technology, Online Counseling Resource…

Example of gel-filtration chromatography
 The sample mixture contains lysozyme (crystalline; 5
mg/mL sample), pancreatic amylase (alpha-amylase,
Sigma cat. # A3176; 5 mg/mL sample filtered
through glass fiber filter), and beta-amylase (Sigma
cat. # A7005; suspension in ammonium sulfate, 4
microliters/mL sample).
 0.4 mL of sample was put on top of the column;
 caution was taken for not disturbing the surface.
 the sample was washed onto the column with 4 mL
of 0.1 M NaCl, discarding the eluate.
 Then they elute the column with 0.1 M NaCl,
collecting ten fractions of twenty drops each.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:1
 This procedure was first developed by W.Cohn.
 Ion exchange chromatography is defined as the
reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
support medium.
 The ion exchanger consists of an inert support
medium couples covalently to positive (anion
exchanger) or negative (cation exchanger) functional
groups.
 To these covalently bound functional groups are
bound, through electrostatic attraction, oppositely
charged ions which will be exchanged with like
charges ions in the sample.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:2

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:3
 Ion
exchange
chromatography
separates
compounds according to the nature and degree of
their ionic charge.
 The column to be used is selected according to its
type and strength of charge.
 Anion exchange resins have a positive charge and
are used to retain and separate negatively charged
compounds.
 while cation exchange resins have a negative
charge and are used to separate positively charged
molecules

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:4

 Before the separation begins a buffer is pumped
through the column to equilibrate the opposing
charged ions.
 Upon injection of the sample, solute molecules will
exchange with the buffer ions as each competes for
the binding sites on the resin.

School of Science and Technology, Online Counseling Resource…

Ion Exchange Chromatography:5
 The length of retention for each solute
depends upon the strength of its charge.
 The most weakly charged compounds will elute
first, followed by those with successively
stronger charges.
 Because of the nature of the separating
mechanism,
pH,
buffer
type,
buffer
concentration, and temperature all play
important roles in controlling the separation.

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Ion Exchange Chromatography:6

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Affinity chromatography:1
 Affinity Chromatography is a separation
technique
based
upon
molecular
conformation, which frequently utilizes
application specific resins.
 These resins have ligands attached to their
surfaces which are specific for the
compounds to be separated.
 Most frequently, these ligands function in a
fashion similar to that of antibody-antigen
interactions.

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Affinity Chromatography:2
 This "lock and key" fit between the ligand and its
target compound makes it highly specific, frequently
generating a single peak, while all else in the sample
is unretained.
 A substrate analogue is couples to the gel matrix and
the cellular suspension is allowed to percolate
through.

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Affinity Chromatography:3
 The technique utilizes the specificity of an
enzyme for its substrate (also receptor for its
agonist, antibody for antigen) or substrate
analogue for the enzyme’s (other protein with
biological specification)separation.
 The enzyme which is specific for the
substrate analogue binds to the gel becoming
immobile while all other components move
down and out.
 The technique has very high resolution
power.

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Affinity Chromatography:4
 The

goal
of
affinity
chromatography
is
to
separate all the molecules of
a particular specificity from
the whole gamut of molecules
in a mixture such as a blood
serum.
 For example, the antibodies in
a serum sample specific for a
particular
antigenic
determinant can be isolated
by
the
use
of
affinity
chromatography

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High Performance Liquid Chromatography: 1
 High performance liquid
chromatography
is
basically
a
highly
improved form of column
chromatography.
 Instead of a solvent being
allowed to drip through a
column under gravity, it is
forced through under high
pressures of up to 400
atmospheres.
 That makes it much
faster.

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A Flow Scheme for HPLC

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High Performance Liquid Chromatography:2
 Injection of the sample is entirely automated, and
you wouldn't be expected to know how this is done
at this introductory level. Because of the pressures
involved, it is not the same as in gas
chromatography (if you have already studied that).
 The time taken for a particular compound to travel
through the column to the detector is known as its
retention time. This time is measured from the time
at which the sample is injected to the point at
which the display shows a maximum peak height
for that compound.

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High Performance Liquid Chromatography:3
 Different compounds have different retention times.
For a particular compound, the retention time will
vary depending on:
 the pressure used (because that affects the flow
rate of the solvent)
 the nature of the stationary phase (not only what
material it is made of, but also particle size)
 the exact composition of the solvent
 the temperature of the column
 That means that conditions have to be carefully
controlled if you are using retention times as a way
of identifying compounds.

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High Performance Liquid Chromatography:4
 There are several ways of detecting when a
substance has passed through the column.
 A common method which is easy to explain uses
ultra-violet absorption.
 Many organic compounds absorb UV light of various
wavelengths. If you have a beam of UV light
shining through the stream of liquid coming out of
the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how
much of the light is absorbed.
 The amount of light absorbed will depend on the
amount of a particular compound that is passing
through the beam at the time.

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Normal Phase HPLC
 The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for
example.
 A typical column has an internal diameter of
4.6 mm (and may be less than that), and a
length of 150 to 250 mm.
 Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds will.
 The non-polar ones will therefore pass more
quickly through the column

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Reversed Phase HPLC:1
 In this case, there will be a strong attraction between
the polar solvent and polar molecules in the mixture
being passed through the column.
 There won't be as much attraction between the
hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the
solution.
 Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
 Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of
van der Waals dispersion forces.

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Reversed Phase HPLC:2
 They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they
squeeze in between the water or methanol
molecules, for example.
 They therefore spend less time in solution in the
solvent and this will slow them down on their way
through the column.
 That means that now it is the polar molecules that
will travel through the column more quickly.
 Reversed phase HPLC is the most commonly used
form of HPLC.

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A flow scheme for HPLC

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High Performance Liquid Chromatography:6
 HPLC allows to use a very much smaller particle
size for the column packing material which gives a
much greater surface area for interactions between
the stationary phase and the molecules flowing past
it.
 This allows a much better separation of the
components of the mixture.
 The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and
extremely sensitive.

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What You Learn…
You have learnt :
 Chromatogrpahy is separation technique based on Partition
coefficient .
 Plane chromatography and Column chromatography are basic
technique of chromatography.
 Gel filtration chromatography separates proteins, peptides,
and oligonucleotides on the basis of size
 Ion
exchange
chromatography
separates
compounds
according to the nature and degree of their ionic charge.
 Affinity chromatgraphy utilizes the specificity of an enzyme for
its substrate.
 HPLC methods are highly automated and extremely sensitive

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Critical Thinking Questions
1. Describe the technique of chromatography in
details.
2. State
the
principles
of
gel—filtration
chromatography.
3. Write a short note on ion-exchange
chromatography.
4. Describe a chromatographic technique which
used specificity of an enzyme for its
substrate.
5. What is HPLC.
© 2007, YCMOU. All Rights Reserved.

41

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Hints For Critical Thinking Question
1. Principles of chromatography and techniques like
Column &Plane chromatography.
2.

separation on the basis of size.

3. Separation of compounds according to the nature and
degree of their ionic charge.
4. Affinity Chromatography
5. High performance liquid chromatography,
phase HPLC and Reversed phase HPLC
© 2007, YCMOU. All Rights Reserved.

Normal
42

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Study Tips:1
 Book1

 Title: Biophysical
techniques )

Chemistry

(principles

and

 Author: Upadhay. Upadhay.Nath

 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
 Author: Freifelder
 Publisher: W. H. Freeman and Company

to

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Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics


Author: Roy R.N.

 Publisher:New Central Book Agency

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Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia

http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia

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End of the Presentation

Thank You !