Transcript THIN LAYER CHROMATOGRAPHY (TLC)

(In the name of GOD)
HIGH PERFORMANCE THIN
LAYER
CHROMATOGARPHY(HPTLC)
Dr. A.R.Bekhradnia
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HIGH
PERFORMANCE
THIN LAYER
CHROMATOGARPHY
(HPTLC)
Dr. A.R.Bekhradnia
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THIN LAYER CHROMATOGRAPHY
(TLC)
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Chromatography is a physical process of
separation in which the components to be
separated are distributed between 2 immiscible
phases a stationary phase which has a large
surface area and mobile phase which is in
constant motion through the stationary phase.
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THIN LAYER CHROMATOGRAPHY
(TLC)
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•
•
•
•
•
•
•
Mikhail Tsvet
Born
14 May 1872
Asti, Italy
Died
26 June 1919 (age 47)
Nationality Russia
Fields botany
Mikhail Semyonovich Tsvet (Михаи́л
Семёнович Цвет, also spelled Tsvett, Tswett,
Tswet, Zwet, and Cvet) (1872–1919) was a
Russian-Italian botanist who invented
adsorption chromatography.
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Invention of Chromatography by
M. Tswett
Ether
Chlorophyll
Chromatography
Colors
CaCO3
LAAQ-B-LC001B
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Comparing Chromatography to the
Flow of a River...
Light leaf
Heavy stone
Water flow
Base
LAAQ-B-LC001B
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Chromato-graphy / -graph / -gram /
-grapher

Chromatography:
 Chromatograph:
 Chromatogram:
 Chromatographer:
LAAQ-B-LC001B
Analytical technique
Instrument
Obtained “picture”
Person
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Three States of Matter and
Chromatography Types
Mobile phase
Gas
Liquid
Gas
chromatography
Liquid
chromatography
Solid
Gas
Stationary
phase
Liquid
Solid
LAAQ-B-LC001B
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Liquid Chromatography

Chromatography in which the mobile phase
is a liquid.
 The
liquid used as the mobile phase is
called the “eluent”.

The stationary phase is usually a solid or a
liquid.
 In general, it is possible to analyze any
substance that can be stably dissolved in
the mobile phase.
LAAQ-B-LC001B
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Interaction Between Solutes, Stationary
Phase, and Mobile Phase

Differences in the interactions between the solutes and
stationary and mobile phases enable separation.
Solute
Degree of adsorption,
solubility, ionicity, etc.
Stationary
phase
LAAQ-B-LC001B
Mobile phase
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Classification
According to the force of separation:
 Adsorption chromatography
 Partition chromatography
 Ion exchange chromatography
 Gel filtration chromatography
 Affinity chromatography

LAAQ-B-LC001B
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Column Chromatography and
Planar Chromatography
Separation column
Paper or a
substrate coated
with particles
Packing material
Column Chromatography
LAAQ-B-LC001B
Paper Chromatography
Thin Layer Chromatography (TLC)
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Output
concentration
Chromatogram
Time
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Intensity of detector signal
tR
Peak
t0
tR : Retention time
t0 : Non-retention time
h
A
A : Peak area
h : Peak height
Time
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Separation Process and Chromatogram
Output
concentration
for Column Chromatography
LAAQ-B-LC001B
Chromatogram
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Time
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THIN LAYER CHROMATOGRAPHY
Once the solvent is within ~1-2 cm of the top of
the TLC sheet, the TLC is removed from the
developing chamber and the farthest extent of
the solvent (the solvent front) is marked with a
pencil.
The solvent is allowed to evaporate from the
TLC sheet in the hood.
The spots are visualized using a UV lamp.
A fluorescent compound, usually Manganeseactivated Zinc Silicate, is added to the adsorbent
that allows the visualization of spots under a
blacklight (UV254). The adsorbent layer will
fluoresce light green by itself, but spots of analyte
quench this fluorescence and appear as a dark spot.
http://orgchem.colorado.edu/hndbksupport/TLC/TLCprocedure.html
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THIN LAYER CHROMATOGRAPHY - Visualization
As the chemicals being separated may be
colorless, several methods exist to visualize
the spots:
Chromatogram of 10 essential oils,
Stained with vanillin reagent.
•
Visualization of spots under a UV254 lamp. The
adsorbent layer will thus fluoresce light green by
itself, but spots of analyte quench this
fluorescence.
•
Iodine vapors are a general unspecific color.
•
Specific color reagents exist into which the TLC
plate is dipped or which are sprayed onto the
plate.
•
Once visible, the Rf value of each spot can be
determined
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THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
2.0 cm
5.0 cm
= 0.40
Rf (B) = 3.0 cm
= 0.60
Rf (A) =
Solvent Front
5.0 cm
Distance solvent
migrated = 5.0 cm
4.0 cm
Rf (C) = 0.8 cm = 0.16
Distance A
migrated = 3.0 cm
5.0 cm
Distance B
migrated = 2.0 cm
3.0 cm
Rf (D) = 4.0 cm = 0.80
5.0 cm
Distance C
migrated = 0.8 cm
Origen
x
A
x
B
0.8 cm
x x
U C
x
D
Rf (U1) = 3.0 cm = 0.60
5.0 cm
Rf (U2) =
0.8 cm
5.0 cm
= 0.16
The Rf is defined as the distance the center of the spot moved divided
by the distance the solvent front moved (both measured from the origin)
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THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
2.0 cm
5.0 cm
= 0.40
Rf (B) = 3.0 cm
= 0.60
Rf (A) =
Solvent Front
5.0 cm
Distance solvent
migrated = 5.0 cm
4.0 cm
Rf (C) = 0.8 cm = 0.16
Distance A
migrated = 3.0 cm
5.0 cm
Distance B
migrated = 2.0 cm
3.0 cm
Rf (D) = 4.0 cm = 0.80
5.0 cm
Distance C
migrated = 0.8 cm
Origen
x
A
x
B
0.8 cm
x x
U C
x
D
Rf (U1) = 3.0 cm = 0.60
5.0 cm
Rf (U2) =
0.8 cm
5.0 cm
= 0.16
The Rf is defined as the distance the center of the spot moved divided
by the distance the solvent front
(both measured from the origin)
Dr.moved
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THIN LAYER CHROMATOGRAPHY – Rf’s
Rf values can be used to aid in the identification of a
substance by comparison to standards.
The Rf value is not a physical constant, and
comparison should be made only between spots on
the same sheet, run at the same time.
Two substances that have the same Rf value may be
identical; those with different Rf values are not
identical.
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THIN LAYER CHROMATOGRAPHY – Rf’s
Absorption of Solutes
The adsorption strength of compounds increases with increasing polarity of
functional groups, as shown below:
-CH=CH2, -X, -OR, -CHO, -CO2R, -NR2, -NH2, -OH, -CONR2, -CO2H.
(weakly adsorbed)
(strongly adsorbed)
(nonpolar)
(more polar)
Elution Strength of Mobile Phase (e)
Elution strength is generally considered to be equivalent to polarity. A solvents
elution strength depends on Intermolecular Forces between the solvent and the
analytes and between the solvent and the stationary phase.
A more polar (or more strongly eluting solvent) will move all of the analytes to a
greater extent, than a less polar, weakly elution solvent.
For example,
the elution strength of hexane is very low;
the elution strength of ethyl acetate is higher;
the elution strength of ethanol is even higher;
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e = 0.01.
e = 0.45
e = 0.68
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Solvent Properties and Elution Strengths
Solvent
MF
MW
o
Bp ( C)
Density (g/mL)
Hazards*
Dipole
Hexane
CH3(CH2)4CH3
C6H14
86.17
68.7
0.659
Flammable
Toxic
0.08
Elution
Stength
(e)
0.01
Toluene
C6H5CH3
C7H8
92.13
110.6
0.867
Flammable
Toxic
0.31
0.22
Diethyl ether
CH3CH2OCH2CH3
C4H10O
74.12
34.6
0.713
Flammable
Toxic, CNS
Depressant
1.15
0.29
Dichloromethane
CH2Cl2
CH2Cl2
84.94
39.8
1.326
Toxic, Irritant
Cancer suspect
1.14
0.32
Ethyl Acetate
CH3CO2CH2CH3
C4H8O2
88.10
77.1
0.901
Flammable
Irritant
1.88
0.45
Acetone
CH3COCH3
C3H6O
58.08
56.3
0.790
Flammable
Irritant
2.69
0.43
Butanone
CH3CH2COCH3
C4H8O
72.10
80.1
0.805
Flammable
Irritant
2.76
0.39
1-Butanol
CH3CH2CH2CH2OH
C4H10O
74.12
117.7
0.810
Flammable
Irritant
1.75
0.47
Propanol
CH3CH2CH2OH
C3H8O
60.09
82.3
0.785
Flammable
Irritant
1.66
0.63
Ethanol
CH3CH2OH
C2H6O
46.07
78.5
0.789
Flammable
Irritant
1.70
0.68
Methanol
CH3OH
CH4O
32.04
64.7
0.791
Flammable
Toxic
1.7
0.73
Water
HOH
H2O
18.02
1.87
>1
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100.0
0.998
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Elution Strength of Mixed Solvents
The elution strength of the mixture is assumed to be the weighted average of the elution
strengths of the components:
where:
eonet = eoA (mole % A) + eoB (mole % B)
mole % A = (moles A) / (moles A + moles B)
Thus, to determine the eonet of a solvent mixture, the molar ratio of the solvents must first
be calculated. For example, the eonet of a solvent mixture prepared from 1.0 mL of ethyl
acetate plus 9.0 mL of hexanes is calculated as shown below:
eonet = eoEtOAc [(moles EtOAc)/(moles EtOAc+moles hexane)] +
eohexane [(moles hexane)/(moles EtOAc+moles hexane)]
where: moles EtOAc = [(volume EtOAc) (density EtOAc)] / [molecular weight of EtOAc]
thus:
eonet =
{0.45[(1.0mLEtOAc)(0.902g/mL)/(88.11g/mole)]+0.01[(9.0mLhexane)(0.659g/mL)/86.18g/mole)]}
{(1.0 mLEtOAc)(0.902g/mL)/88.11g/mole) + (9.0 mLhexane)(0.659g/mL)/86.18g/mole)}
and
eonet = 0.067
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Resolution
The separation between two analytes on a
chromatogram can be expressed as the
resolution, Rs and can be determined using
the following equation:
Rs = (distance between center of spots)
(average diameter of spots)
In TLC, if the Rs value is greater than 1.0, the
analytes are considered to be resolved.
x
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x
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Improving Resolution:
For two closely migrating components, optimum resolutions
are usually obtained when the Rf’s of both compounds
are between 0.2 and 0.5
* To Improve Rs, change the elution strength of the solvent
to optimize Rf’s
•
change eonet, all compounds will be effected similarly.
•
Alter the composition of the solvent system so that the
components affinity for the mobile phase vs. the solid
phase are differentially changed (= change in
selectivity).
•
Changing the chemical nature of the solvent system,
such as changing a hydrogen bonding solvent to a
solvent which cannot hydrogen bond to the analyte, is
often the most effective.
** Improve Rs by decreasing the diameter of the
analyte spots. This can be achieved by applying
smaller and less concentrated spots.
Dr. A.R.Bekhradnia
http://orgchem.colorado.edu/hndbksupport/
TLC/TLCprocedure.html
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HIGH
PERFORMANCE
THIN LAYER
CHROMATOGARPHY
(HPTLC)
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Introduction of HPTLC
•
HPTLC is the improved method of TLC which utilizes the
conventional technique of TLC in more optimized way.
•
HPTLC takes place in highspeed capillary flow range of
the mobile phase.
•
There are three main steps HPTLC procedure, they are
1] Sample preparation, volume precision and exact position are
achieved by use of suitable instrument.
2] Solvent (mobile phase) migrates the planned distance in layer
(stationary phase) by capillary action. In this process sample
separated into it’s components.
3] Separation tracks are scanned in densitometer with light
beams in visible or uv region
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Steps Involving in HPTLC
Sample Preparation
Selection of
chromatography layer
Pre-washing
Pre-conditioning
Application of sample
Chromatography development
Detection of spots
Scanning & documentation
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Sample preparation
• Normal phase chromatography: non polar solvent
• Reversed phase chromatography: polar solvent
Selection of chromatography
layer
Depends on nature of material to be
separated
Commonly used(silica gel, alumina)
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Pre-washing
• It is purification step
• Mainly methanol is used
• Essential for quantitative evaluation
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Linomat lV applicator
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Selection of HPTLC plates
• Previously hand made plates is used in TLC for both
qualitative and quantitative work. Certain drawbacks with
that is nonuniform layer, formation of thick layer, paved for
advent of precoated plates.
• Nowadays precoated plates are available in different format
and thickness by various manufactures. Precaoted plates can
be used for both qualitative and quantitative work in
HPTLC, they are
• GLASS PLATES
• POLY ESTER/POLYETHYLYNE
• ALUMINIUM PLATES
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Glass Plates:
 Offers superior flat and smooth surface.
- fragile
- high weight
- higher production cost
Polyester/polyethylene plates:
Thickness of plate is 0.2mm.
- It can be produced in roll forms.
- Unbreakable.
- Less packing material is required.
- Development of plate cann’t be above temperature
1200 c loses its shape.
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Aluminium plates:
- Thickness of plate is 0.1mm.
- It can be produced in roll forms.
- Unbreakable.
- Less packaging material is required.
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SORBENTS USED IN HPTLC PLATES:
sorbents which are used in convential TLC are
also used in HPTLC with or without modification.
- silica gel 65F
- highly purified silicagel 60
- aluminium oxide
- cellulose microcrystalline
- silica gel
- reversed stationary phase
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The layer thickness in HPTLC is around 100200cm,in conventional it is 250mm.
Layer prewashing:
- Ascending method
- Dipping method
- Continuous method
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
The plates are activated by placing in an oven at
1101200 C for 30 min, this step will removes water
that has been physically absorbed on surface at
solvent layer.

Freshly opened box of HPTLC plates usually
does not require activation.

Activation at higher temp and for longer time is
avoided which leads to very active layer and there
is risk of sample being decomposed.
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- Methanol (commonly used)
-
Chloroform:methanol:ammonia(90:10:1)
Chloroform:methanol(1:1)
Methylene chloride:methanol(1:1)
Ammonia(1%)solution
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
Usual concentration range is 0.1-1µg / µl,above
this causes poor separation.

Linomat IV (automatic applicator) - nitrogen gas
sprays sample and standard from syringe on TLC
plates as bands.

Band wise application - better separation - high
response to densitometer.
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Processes in the Developing Chamber
The «classical» way of developing a chromatogram is to place the
plate in a chamber, which contains a sufficient amount of
developing solvent.
The lower end of the plate should be immersed several millimeters.
Driven by capillary action the developing solvent moves up the layer
until the desired running distance is reached and chromatography is
stopped. The following considerations primarily concern silica gel as
stationary phase and developments, which can be described as
adsorption chromatography.
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Provided the chamber is closed, four partially competing processes occur:
Between the components of the developing solvent and their vapor, an
equilibrium will be established eventually (1). This equilibrium is called
chamber saturation. Depending on the vapor pressure of the individual
components the composition of the gas phase can differ significantly from
that of the developing solvent.
While still dry, the stationary phase adsorbs molecules from the gas phase.
This process, adsorptive saturation, is also approaching an equilibrium in
which the polar components will be withdrawn from the gas phase and
loaded onto the surface of the stationary phase (2).
Simultaneously the part of the layer which is already wetted with mobile
phase interacts with the gas phase. Thereby especially the less polar
components of the liquid are released into in the gas phase (3). Unlike (1) this
process is not as much governed by vapor pressure as by adsorption forces.
During migration, the components of the mobile phase can be separated by
the stationary phase under certain conditions, causing the formation of
secondary fronts.
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Also called Chamber Saturation
• Low polarity mob. Phase:- no need
• High polar mob. Phase:- desirable
• For reverse phase saturate chamber with polar
solvent
•
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LOW SOLVENT
CONSUMPTION
pre-equilibration
with
solvent vapor
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CAMAG Twin Trough Chambers offer several
ways to improve the results of TLC/HPTLC
developing techniques. It allows low solvent
consumption, reproducible pre-equilibration
with solvent vapor, equilibration performed
with any liquid and for any period of time,
and development is started only when
developing solvent is introduced into the
trough with the plate.
Twin Trough Chambers are available with
stainless steel lid or as a Light-Weight Twin
Trough Chamber made from highly
transparent sheet glass with a glass lid.
Start of
development
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
After development, remove the plate and
mobile phase is removed from the plate - to avoid
contamination of lab atmosphere.

Dry in vacuum desiccator - avoid hair
drier because essential oil components may
evaporate.
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


Detection under UV light is first choice non destructive.
Spots of fluorescent compounds can be
seen at 254 nm (short wave length) or at 366 nm
(long wave length).
Spots of non fluorescent compounds can
be seen - fluorescent stationary phase is used silica gel GF.
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

CATS STANDARD PROGRAM.
CATS PROGRAM OPTIONS
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
Non UV absorbing compounds like
ethambutol, dicylomine etc - dipping the plates in
0.1% iodine solution.

When individual component does not
respond to UV - derivatisation required for
detection .
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







100µm
HPTLC
High due to smaller particle
size generated
3 - 5 cm
Shorter migration distance
and the analysis time is
greatly reduced
Wide choice of stationary
phases like silica gel for
normal phase and C8 , C18
for reversed phase modes
New type that require less
amount of mobile phase
Auto sampler
Use of UV/ Visible/
Fluorescence scanner scans
the entire chromatogram
qualitatively and
quantitatively and the
scanner is an advanced type
TLC

250µm
Less
10 - 15 cm

Slower

Silica gel , Alumina

More amount

Manual spotting

Not possible


of densitometer
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
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







Pharmaceutical Researches
Biomedical Analysis
Clinical Analysis
Environmental Analysis
Food Industry
Therapeutic drug monitoring to determine concentration of drug
and it’s metabolite in blood, urine etc
Analysis of environmental pollutions levels
Quantitative determination of prostaglandin’s and thromboxanes
in plasma
Analysis of nitrosoamines in food and body fluids
Determination of sorbic acid in wine
Characterization of hazards in industrial waste
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