Thin layer

chromatography
NAME :
CLASS : B-PHARMACY IV YEAR- I SEMESTER
ROLL NO :
COLLEGE :
CHROMATOGRAPHY:
• The term chromatography (greek kromatos –colour and
graphos –written) meaning colour writing.

• Chromatography is a physical method of separation in which

the components to be separated are distributed between two
phases, one of which is stationary(stationary phase) while the
other(mobile phase) moves in a definite direction.
HISTORY:
• 1906-Michael tswett discovered the
chromatographic principle.
• 1931-Lederer and coworkers separated xanthenes
and lutein on a column of calcium cabonate powder.
• 1940 and 1946- Tiselius and Claeson classified
these into three groups based on the principle of
separation
• 1944-Martin,Consden,Gorden replaced silica gel
column by strips of filter paper and developed paper
chromatography.
• 1963-Giddings pointed out that efficiency of gas
chromatography have to be achieved in liquid
chromatography and then this led to the discovery of
HPTLC technique.
 classification:
i. Partition ii. Adsorption
chromatoraphy: chromatography:
Mobile phase: liquid or gas Mobile phase- liquid or gas.
Stationary phase: liquid or solid. Stationary phase- adsorbent
solid.
The operations include:
• Thin layer chromatography The operations include:
• Paper chromatography  Thin layer chromatography
• High performance liquid  Adsorption column
chromatography chromatography
• Gas-liquid chromatography  Gas-solid chromatography
• Partition column
chromatography
 iii. Ion exchange iv. PERMEation
chromatography : CHROMATOGRAPHY :

Mobile phase- liquid Mobile phase- liquid

Stationary phase- ion exchange Stationary phase- polymer matrix
resin
The operation includes:
The operations include: Gel permeation chromatography.
 Ion exchange chromatography.
 High performance ion
chromatography.
 Separation techniques:

I. ELUTION ANALYSIS:
• Common method used in column chromatography.
• In this method, a small volume of mixture to be separated is added on
top of column and mobile phase is allowed to flow through column
 II. FRONTAL IV. DISPLACEMENT
ANALYSIS: ANALYSIS:

Tiselius developed this technique In this, a small volume of

in 1940.In this method sample mixture is added to the column
mixture is added continuously on and elution is carried out by a
the column. No mobile phase is solvent containing a solute
used for development of column. which has high absorptives for
A complete separation can’t be column material.Here the
achieved. Only Partial separation adsorbed constituents of
is seen. mixture are displaced by the
solute from mobile phase.
Advantages:

• Capable of separating complex mixtures at low operating

temperatures.
• Large scale batch or continuous operation possible.
• Capable of separating materials according to size and/or
chemical properties.
• Can be used to separate delicate or heat labile
compounds.
• Separation can be achieved by a variety of methods.
• Very pure products can be recovered.
Disadvantages:
• Process scale-up is a problem (hence low throughput)
• Irreversible adsorption of materials creates problems
• To achieve efficient separations, high operating
pressures may be required.
• Over-loading of columns with feed material may cause
incomplete separation.
• Feed material is diluted by flowing mobile phase.
• Non-uniform column packing can lead to a significant
decrease in separation performance.
• Pre-filtration of feed material is usually required.
• Periodic column re-packing / regeneration required.
THIN LAYER
CHROMATOGRAPH
Y…
INTRODUCTION:

• TLC is one of the simplest, fastest, easiest and least expensive of

several chromatographic techniques used in qualitative and
quantitative analysis to separate organic compounds and to test the
purity of compounds.
TLC is a form of liquid chromatography consisting of:
 A mobile phase(developing solvent) and
 A stationary phase(a plate or strip coated with a form of silica gel)
 Analysis is performed on a flat surface under atmospheric pressure
and room temperature.
History:
Michael Tswett is credited as being the father of
liquid chromatography. Tswett developed his ideas in the
early 1900’s
1938- Izmailov and Shraiber described basic
principle and used it for separation of plant extracts.
1944- Consden, Gorden and Martin started using
filter papers for separation of amino acids.
1950- Kirchner who used impregnated glass plate
coated with alumina, identified terpenes.
1958- Ergon Stahl introduced a standard equipment
for preparing uniform thin layers of known thickness.
Advantages:

• Little equipment.
• Less (> I hour time) time.
• It is more sensitive, i.e. separation effects are usually
superior to those of others.
• Lower detection limit is > 1 decimal than paper
chromatography.
• Very small quantities of sample sufficient.
• Spraying with corrosive agents is permitted.
• Method used for adsorption, partition, ion exchange
chromatography as there is wide range of adsorbents
available.
• Components separated can be recovered easily.
Principle:
Thin layer chromatography included under both adsorption and partition
chromatographs.
• The component with more affinity towards the stationary phase travels
slower.
• The component with less affinity towards the stationary phase travels
faster.
TECHNIQUES :
• Draw a pencil line about a 1/4 inch from the bottom of the plate (along the
short side). Mark places along the line for each spot (pure reference spots
and your mixture).
• Dip the capillary into the solution and gently and quickly place a 1-2 millimeter
spot on the plate at the position you’ve marked. Keep the spots small!
• Pour approx. 3 mL of solvent into a screw-cap jar; place a piece of filter
paper in the jar and wet the paper with the solvent to saturate the
atmosphere. Make sure that the solvent is shallow enough that it will be below
the spot line on your plate.
• Place the plate carefully in the chamber (use tongs or tweezers), being
careful not to dunk the spots under the solvent. Put the cap on the jar and let
the plate develop.
• When the solvent front has almost reached the top of the TLC plate, remove
the plate and immediately mark the solvent front with a pencil line.
• Using tweezers, dip the TLC plate into the solution of PAA stain in a fume
hood. The plate should be dipped smoothly in one motion – your TA will
demonstrate.
• Wipe excess stain from the back of the plate (glass part) and place it on a
hot plate for ~20 sec. allow the solvent to evaporate and look at the spots
• Circle the spots in pencil, break out a ruler and calculate Rf values.
 STEP - 1

 Draw a pencil line about a 1/4 inch from the bottom of the plate (along
the short side). Mark places along the line for each spot (pure reference
spots and your mixture).
 STEP - 2
 Dip the capillary into the solution and gently and quickly place a 1-2
millimeter spot on the plate at the position you’ve marked. Keep the
spots small.
 STEP - 3
 Pour approx. 3 mL of solvent into a screw-cap jar; place a piece of filter
paper in the jar and wet the paper with the solvent to saturate the
atmosphere. Make sure that the solvent is shallow enough that it will be
below the spot line on your plate
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STEP - 4
Place the plate carefully in the chamber (use tongs or tweezers), being
careful not to dunk the spots under the solvent. Put the cap on the jar and
let the plate develop
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STEP – 5
When the solvent front has almost reached the top of the TLC plate,
remove the plate and immediately mark the solvent front with a pencil line
wipe excess stain from back of plate, place it on the hot plate for 20 sec.
Circle the spots and calculate the Rf value.
SELECTION OF ADSORBENTS:
 Solubility of compound e.g, hydrophilic or lipophilic.
 Adsorbent should not adhere to glass plate.
 Reactivity of compound with the solvent or adsorbent.
 Nature of substance to be separated i.e. whether it is acidic,
basic or amphoteric.
 Adsorbent particle size
 Chemical reactivity of compounds with binders.
Preparation of chromoplates:

GLASS PLATES :
Specific dimensions – 20cm*20cm,20cm*10cm,20cm*5cm
Microscopic slides can also be used.
Plates should be of good quality and withstand high temperatures.
The surface should be flat with irregularities.
The standard film thickness is 250um.
There are four methods of applying the thin layer of the adsorbent.
• Pouring
• Dipping
• Spraying
• Spreading.
METHODS FOR APPLICATION OF
ADSORBENT:

There are four methods of applying the thin layer of

the adsorbent.
• Pouring
• Dipping
• Spraying
• Spreading.
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POURING:

The adsorbent of finely divided and homogenous particle size is made into
slurry and is poured on a plate and allowed to flow over it so that it is
evenly covered.
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Dipping:
This technique is used for small plates by dipping the two plates at a time,
back to back in a slurry of adsorbent in chloroform or other volatile
solvents. Exact thickness of layer is not known and evenness of layer may
not be good.
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Spraying:
This technique is out of vogue as it is difficult to get uniform layer. Slurry is
further diluted for the operation of sprayer. This can be simply achieved by
spraying slurry on plate.
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SPREADING:
In this method, slurry is spread by using spatula or silica gel.
Thin and uniform layers can be achieved. Modern methods utilize the spreading
devices for preparation of uniform thin layers on glass plates.
A modified spreader gives layer thickness from 0.2-2.0mm.
Activation of plates:

• After spreading plate allowed to dry and activated by

heating about 100 degrees for 30 min.
• Plates made with volatile organic liquids may not require
this further drying.
• For activation plate is placed in hot oven at temperature
120-150 degrees for an hour.
• Long storage is not recommended.
Solvent system:

The solvent system performs the following:

To dissolve the mixture of substances.
To transport the substances to be separated across the
sorbent layer.
For the complete elution.
It should also fulfill the following requirements:
Adequate purity.
Adequate stability.
Low viscosity.
Vapour pressure neither very low or very high.
Low toxicity.
 The choice of the mobile phase depends on:

• Nature of substance to be separated.

• Nature of the stationary phase used.
• Mode of chromatography.
• Separation to be achieved.

Polarity of substances plays an important role.

Highly polar solvents are generally avoided to minimize adsorption.
Use of water as a solvent is avoided as it may loosen the adhesion of a
layer on a glass plate.
Application of sample:

• Sample solution in a non polar solvent is applied.

• Before the sample application, the starting point and finish line is
usually marked.
• The concentration of a sample or standard solution has to be
minimum of a 1% solution of either standard or test sample is spotted
using a capillary tube or micropipette.
• The area of application should be kept as small as possible for
sharper and greater resolution.
Development chambers:
• The TLC is placed vertically in a rectangular
chromatography tank or chamber
• According to the separation technique ,they are classified
as follows:
A. Tanks for ascending development.
B. Tanks for descending development.
C. Tanks for horizontal development.
D. Tanks for thin-layer electrophoresis.
 Development of
chromatograms:
• Ascending development: The plates are placed in chromatography
chamber containing solvent at the bottom. The flow of solvent-bottom to
top.
• Descending development: Flow of the solvent from reservior to the plate
is through a filter paper strip. The flow of solvent-top to bottom.
• Horizontal development: loose and non-sticky layer plates are used with
a use of shallow dish with a ground-glass cover. Solvent flows- bottom of
dish up to thin layer film.
• Step-wise and multiple development : This technique carried out by
developing the chromatogram in a given solvent. It is then removed from
chamber and solvent is allowed to evaporate. The plate is again developed
in the same solvent. This is repeated no. of times. If solvents used for first
and subsequent runs are same-multiple. If solvents used for first and
subsequent runs are different-step-wise.
 Two-dimensional development: If components of the mixtures are
not completely separated by development in a single direction, it is
possible to resolve by developing in second solvent in direction
perpendicular to the first development.
 Preparative TLC : In this method, sample is always applied in bands
or streaks and separation is affected by multiple development. After
the localization of spot , the band or streak is scrapped out and the
sample recovered by extraction with suitable solvent.
 Gradient elution: some times it is advantageous to change the
composition of the solvent continuously during the development and
specially designed tanks is used for gradient elution.
 Reverse phase TLC: chromoplates are prepared by immersing the
adsorbent layer very slowly in 5-10% of paraffin, silicone oil, and
petroleum ether or diethyl ether. After removing the plate and
evaporating solvent , the plate is ready for chromatography.
Location of spots:

Physical methods include ultraviolet, fluorescence or radioactive

counting.
Chemical methods include use of locating agents by spraying and not by
dipping.
During spraying care should be taken to prevent disturbing the layers.
Concentrated sulphuric acid can be used as locating agent as it
produces coloured spots which are visible in daylight as well as UV light.
Iodine vapour is used as locating agents for organic substances.
Evaluation of chromatograms:

• Qualitative-comparison of Rf values.
• Quantitative- These includes the following:
 Direct methods:
a) Visual comparison-Of spot size, intensity or combination of both
are done with standard spots.
b) Spot areas and weight relationship- From area, amount present
is calculated.
c) Spot densitometry-After development, plate is sprayed with
specific reagents and colour developed is measured directly in
densitometer.
d) Direct spectrometry-absorption or fluorescence of separated
zones directly on TLC plates at wavelength of max. absorption of
substance.
e) Spectral reflectance-Of dyes adsorbed on adsorbent.
 Indirect methods-After localization, areas containing substances are
scooped out with the help of vacuum cleaner. Adsorbent is eluted, the
eluate is then analyzed by colorimetry, spectrophotometry, flame
RETENTION FACTOR (Rf) values:
• The difference in rate of movement of components in
chromatography. It is measured by using the following formula:

It depends on several factors:

a) Nature of adsorbent
b) The mobile phase
c) Activity
d) Thickness of layer
e) The temperature
f) Equilibration
g) Loading
h) Dipping zone
i) Chromatographic technique.
Applications:
• Purity of sample-Direct comparison of sample and authentic sample
is done.
• Examination of reactions-Whether the reaction is complete or in
complete.
• Identification of compounds-It is increasingly employed in isolation,
purification, identification of various classes of organic compounds.
• Bio chemical analysis-In separation of biochemical metabolite or
constituent from its body fluids, blood plasma, serum, urine etc.
• In chemistry- For separation and identification of compounds which
are closely related to each other.
• In pharmaceutical industry- Detection of impurity(>130 drugs tested
by this method)
• Various drugs like hypnotics, sedatives, anticonvulsant, tranquillizers,
antihistaminic, analgesics, local anaesthetics are tested by TLC.
• In food and cosmetic industry, TLC used in separation and
identification of colours, preservatives, sweetening agents and various
cosmetic products.
 Chromatographic separation of
amino acids:
MATERIALS REQUIRED:

REAGENTS:
• 2% solution of individual amino acids.
• Solvent mixture of normal butanol, acetic acid and water in the ratio
12:3:5 by volume.
• Ninhydrin reagent.
REQUIREMENTS:
• TLC plate.
• TLC chamber.
• Capillary tubes.
• Reagent spray bottle.
• Conical flasks.
• Beakers.
PROCEDURE:

• Pour the solvent mixture in to the TLC chamber and close the chamber.
• The chamber should not be disturbed for about 30 minutes so that the
atmosphere in the jar becomes saturated with the solvent.
• Cut the plate to the correct size and using a pencil (never ever use a pen) gently
draw a straight line across the plate approximately 2 cm from the bottom.
• Using a capillary tube, a minute drop of amino acid is spotted on the line.
• Allow the spot to dry.
• Spot the second amino acid on the plate [enough space should be provided
between the spots].
• Repeat the above step for spotting the unknown acid.
• Place the plate in the TLC chamber as evenly as possible and lean it against the
side (immerse the plate such that the line is above the solvent). Allow capillary
action to draw the solvent up the plate until it is approximately 1 cm from the
end.
• Remove the plate and immediately draw a pencil line across the solvent top.
• Under a hood dry the plate with the aid of a blow dryer.
• Spray the dry plate with ninhydrin reagent.
• Dry the plates in hot air oven at 105°C for 5 min. [Ninhydrin will react with the
faded spots of amino acids and make them visible as purple coloured spots.]
• After some time, mark the center of the spots, then measure the distance of the
center of the spots from the origin and calculate the Rf values.
• The Rf values with butanol-acetic acid- water solvent are
as follows: alanine 0.24, glutamic acid 0.25, glycine 0.2,
leucine 0.58, valine 0.4, lysine 0.58, tyrosine 0.42.
Conclusion:
References: