Transcript HPLC - Professor Monzir Abdel

HPLC
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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
• High Performance Liquid Chromatography (HPLC) is one of the
most widely used techniques for identification, quantification and
purification of mixtures of organic compounds.
• In HPLC, as in all chromatographic methods, components of a
mixture are partitioned between an adsorbent (the stationary
phase) and a solvent (the mobile phase).
• The stationary phase is made up of very small particles contained
in a steel column. Due to the small particle size (3-5 um), pressure
is required to force the mobile phase through the stationary phase.
• There are a wide variety of stationary phases available for HPLC.
In this lab we will use a normal phase (Silica gel), although reverse
phase (silica gel in which a 18 carbon hydrocarbon is covalently
bound to the surface of the silica) columns are currently one of the
most commonly used HPLC stationary phases.
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Four main chromatographic techniques that use a
liquid mobile phase are covered under the
broad high performance liquid chromatographic
technique. These include:
1. Partition Chromatography
2. Liquid-Solid Chromatography
3. Ion-Exchange Chromatography
4. Size Exclusion (Gel Permeation)
Chromatography
The first of the abovementioned chromatographic
technique is most widely used. Generally,
HPLC uses very high pressures (up to 4000
psi) and very small particle size (down to 3
mm).
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Column efficiency in Liquid Chromatography
We have seen earlier that several factors affect
efficiency in chromatographic techniques
including:
1. Particle size
2. Flow rate
3. Thickness of stationary phase
4. Mobile phase viscosity
5. Diffusion of solute in mobile and stationary
phases
6. How well a column is packed
7. Sample size (mg sample/g packing)
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Extra-Column Band
Broadening
We have discussed three
reasons for intra
column band
broadening including:
1. Multiple paths effects
2. Longitudinal diffusion
3. mass transfer in
stationary and mobile
phases
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However, there are
other sources of
band broadening
unrelated to column
materials and occur
outside the column.
These include :
1. Fittings dead volume
2. Tubing length and
diameter
3. Detector volume
4. Injection volume
Instruments for Liquid Chromatography
Pumps
Three types of pumps are known:
1. Reciprocating pumps
2. Displacement Pumps
3. Pneumatic pumps
4. Reciprocating pumps are by far the most
widely used and practically 100% of the
pumps used in commercial HPLC
equipment are of the reciprocating type.
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Reciprocating pumps
In reciprocating pumps, a motor driven
reciprocating piston controls the flow of
mobile phase with the help of two ball
check valves that opens and closes with
the piston movement. The flow is thus not
continuous and damping of flow is
necessary. This is accomplished using
pulse dampers which are a long coiled
capillary tube.
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Displacement pumps
Displacement pumps, on the other hand, is
composed of a one directional motor
driven plunger that pushes the mobile
phase present in a syringe like chamber.
The volume of displacement pumps is
limited which lacks convenience. A
constant flow rate is usually obtained with
syringe like pumps.
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Sample Injection Valves
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Columns
Columns are almost always made from
stainless steel with most common
dimensions in the range from -25 cm long
and about 4.6 mm internal diameter.
Pellicular or porous packing materials are
usually used. Pellicular packings are
nonporous glass or polymer beads ranging
from 30 to 40 mm. Porous packings are
mostly silica based with particle diameters
from 3-10 mm.
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Chromatographic Column
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Detectors
Detector can be classified as bulk or property
detectors. Bulk detectors respond to a bulk
property of the mobile phase, like refractive
index, dielectric constant, conductivity, etc.
which is modified in presence of a solute. On
the other hand, solute property detectors
respond to a property of the solute like its UVVis absorption, fluorescence,
chemiluminescence, etc. that is not possessed
by the mobile phase.
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From Column
Po
P
To Waste
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HPLC-UV-Vis
Variable wavelength detector - monochromator
PMT
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HPLC-UV-Vis
Diode array detector - polychromator
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Fluorescence Detectors
From Column
lexc.
To Waste
lem
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Refractive Index Detectors, RI
These are very good detectors that responds to changes in
the refractive index of the mobile phase in presence of
an analyte.
Sample
Source
Mirror
Reference
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Detector
Bonded Phase Chromatography
In this case, the stationary phase is chemically bonded to
the solid support which implies the following:
1. No bleeding
2. Can use stationary phases of any chain length from a
C1 to C18 which reflects very well on the retention
characteristics of the column
3. The solid support surface is not completely covered with
stationary phase and residual silanol groups may
complicate retention mechanism and results in tailing
4. If damaged, the stationary phase can not be
regenerated in situ
5. Usually, bonded phase stationary phases are expensive
6. Most widely used (almost exclusively)
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Types of Bonded Phase
Chromatography
Two main techniques are usually used with
bonded phase chromatography:
Normal Phase Chromatography (NPC)
In this technique, the stationary phase is more
polar than the mobile phase where a hydroxyl,
amino, or cyano terminated stationary phase is
used while the mobile phase is a nonpolar
solvent like n-hexane. In this type of
chromatography, the least polar compound is
eluted first while the more polar compound will
be retained more.
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Reversed-Phase Liquid
Chromatography (RPLC)
In this technique, the stationary phase is less polar
than the mobile phase where a C3, C8, or a
C18 chain length stationary phase is used
while the mobile phase is a polar solvent like
methanol, acetonitrile, etc or mixtures with
water. In this type of chromatography, the more
polar compound is eluted first while the less
polar compound will be retained more. RPLC
is the most common technique in liquid
chromatography and several versions and
techniques had emerged from RPLC with
some modification of mobile phase.
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The separation mechanism in RPLC is relatively
simple where increasing percentage of organic
modifier in mobile phase decreases retention
time of less polar solutes, as the polarity of the
mobile phase decreases. In contrast, increasing
the polarity of the mobile phase increases the
retention time of less polar solutes and
decreases the retention time of the more polar
solutes. The separation mechanism in BPC is
simplified by assuming that the chemically
bonded stationary phase as if it were a
physically retained liquid.
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The chain length in RPLC can serve the following:
1. As the chain length of the stationary phase is
increased, the retention times of the less polar
solutes are increased.
2. Increasing the chain length of the stationary
phase controls the sample size where a C3
stationary phase can be used for separation of
a sample size about one half that of a C8
stationary phase, providing other conditions
are the same.
3. theoretically, shorter stationary phases result in
better efficiencies due to decreased HS.
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It should also be observed that the most
common solid support in BPC is silica. The
Si-O-Si (siloxane bonds) are not stable
outside the pH range from 3-8. Therefore,
the pH of mobile phases must not exceed
this limit, otherwise hydrolysis of silica
particles and release of stationary phase
will take place which results in
deterioration of the packing material and
hence the column.
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Mobile Phase Selection in
Partition Chromatography
Optimization of the mobile phase composition and
polarity is vital for obtaining good separations.
The optimization of the sedparation process
involves optimization of N, k', and a. Changing
the mobile phase composition can well control k'
and a as well as indirectly improving N. Initially,
k' is usually adjusted in the range from 2-5 and if
the required resolution is not obtained, one can
look at conditions that may change a.
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Effect of Solvent Strength on k'
Selection of a suitable mobile phase polarity is
very important for successful separations. The
polarity of the different solvents can be derived
from Snyder's polarity index where:
P'AB = fAP'A + fBP'B
Where, P'AB is the polarity index of the mobile
phase containing fA and fB are volume fractions
of solvents A and B while P'A and P’B are the
polarity indices of pure solvents A and B.
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The retention factor is also related to mobile
phase polarity by the relation:
log k'2/k'1 = (P'2 – P'1)/2 for RPLC, and:
log k'2/k'1 = (P'1 – P'2)/2 for NPC
Where, P’1 and P'2 are the initial and final
polarity indices of the mobile phase that
will result in bringing the value of the
retention factor from k'1 to k'2.
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A solute having a retention time of 31.3 min
is separated using a column with tM = 0.48
min and mobile phase composition of
30%methanol, 70% water. Find k', and the
mobile phase composition that can bring k'
to 5.
Solution:
K' = (31.3-0.48)/0.48 = 64
P'AB = fAP'A + fBP'B
Values of P' can be obtained from tables of
polarity indices
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P'AB = 0.30*5.1 + 0.70*10.2 = 8.7
log k'2/k'1 = (P'2 – P'1)/2
log 5/64 = (P'2 – 8.7)/2
P'2 = 6.5
P'AB = fAP'A + fBP'B
6.5 = x*5.1 + (1-x)*10.2
x = 0.73
% Methanol = 0.73*100 = 73%
% Water = 100-71 = 27%
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Therefore, the mobile phase composition
that will result in k' = 5 is 73% methanol
and 27% water. However, if k' was judged
suitable but still the two peaks overlap,
one should look at optimizing a while
keeping k' constant. This can be done by
changing the chemical nature of the
mobile phase, rather than its polarity (i.e.
by changing the nature of the organic
modifier say for example tetrahydrofuran
or dioxane instead of methanol).
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Size Exclusion Chromatography
(SEC)
This technique is also called gel permeation (GPC)
or gel filtration (GFC) chromatography. It is used
for the separation of high molecular weight
species (polymers, enzymes, proteins, etc.). No
interactions of solutes with the packing material
take place and retention is a function of
molecular size. The packing material is porous
and is characterized by certain range of pore
size. Large molecular weight species are
retained less since they do not enter the pores
while species which have dimensions smaller
than the pores will be retained more since they
travel through the pores.
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The separation takes place in a chromatographic
column filled with beads of a rigid porous
material (also called a gel). The technique can
also be used for polymer molecular-weight
determinations where highly crosslinked porous
polystyrene is the preferred packing materials.
The pores in these gels are of the same range
as the dimensions of analytes. A sample of a
dilute polymer solution is introduced into the
mobile phase. As the dissolved polymer
molecules flow past the porous beads, they can
diffuse into the internal pore structure of the gel
to an extent depending on their size and the
pore size distribution of the gel.
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Larger molecules can enter only a small fraction, if
at all, of the internal portion of the gel, or are
completely excluded; smaller polymer molecules
penetrate a larger fraction of the interior pores of
the gel. The different molecular species are
eluted from the column in order of their
molecular size.
A specific column or set of columns (with gels of
different pore sizes) is calibrated empirically to
give such a relationship relating retention to log
molecular weight. For convenience,
commercially available narrow-distribution
polystyrenes (anionic form) are often used as
standards.
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The total volume of a column packed with a porous
polymer is equal to:
Vt = Vg + Vi +Vo
Where,
Vg = Volume of the solid matrix
V0 = Void Volume (Volume in the system outside the
porous beads)
Vi = Internal Volume (Volume of solvent inside the pores)
The following cases can be identified:
a. For large molecules (larger than the pore size), full
exclusion takes place and the elution volume needed to
elute such high molecular weight compounds is:
Ve = Vo
Where, Ve is the elution volume
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b. For molecules of small size (smaller than the pore size)
the elution volume is:
Ve = Vi + Vo
c. For molecules of intermediate size (these will transfer to
some extent depending on their size and pore size
distribution, K, inside the solvent in the pores), the
elution volume is:
Ve = Vo + KVi
K = (Ve - Vo)/Vi = CS/CM
In other words:
K = 0 for molecules too large to enter the pores
K = 1 for molecules that can enter the pores unhindered
1> K >0 for solutes of intermediate size
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Exclusion
Limit
Log MW
Permeation
Limit
Vo
Vi
VR
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The permeation limit indicates molecular weight
below which all solutes have the same retention
time and thus will elute together as a single
peak. The exclusion limit indicates the molecular
weight of solutes above which all solutes having
a molecular weight greater than the exclusion
limit will elute at the same retention time as a
single peak. A specific column is practically
usable for separation of solutes with molecular
weights within the molecular weight window
between the exclusion and permeation limits
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A series of commercially available polymerized
polystyrenes (anionic form) is used as standards
for calibration. The elution volume corresponding
to a peak in the chromatogram is related to the
molecular weight of a particular polystyrene.
After assigning molecular weight of each
component to its elution volume, a plot of log
(MW) versus elution volume can be constructed.
A straight line should result and this is known as
the calibration curve.
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