THIN LAYER CHROMATOGRAPHY

History

The origin of thin layer chromatography is a little obscure but was probably first
developed and utilized by Schraiber in 1939.when Izmailov and schraiber separated
plant extracts using 2mm thick and firm layer of alumina set on glass plate.

In 1944, Consden, Gorden and Martin used filter papers for separating amino acids.

In 1950, Kirchner identified terpenes on filter paper and later he used glass fiber
paper coated with alumina.

In 1958, Stahl developed standard equipment for analyzing by thin layer

chromatography.

Introduction

Thin-layer chromatography (TLC) is a very commonly used technique in synthetic

chemistry for identifying compounds, determining their purity and following the
progress of a reaction and used to separate mixtures. It is performed on a sheet of
glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent
material, usually silica gel, aluminium oxide, or cellulose. This layer of adsorbent is
known as the stationary phase. They all have a stationary phase (a solid or a
liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase
flows through the stationary phase and carries the components of the mixture with
it. Different analytes ascend the TLC plate at different rates, separation is achieved
Plate.

Principle

The principle of separation is “adsorption”. One or more compounds are spotted

on a thin layer of adsorbent coated on a chromatographic Plate. The mobile phase
solvent flows through because of capillary action (Against gravitational force). The
component move according to their affinities towards the adsorbent. The
component with more affinity towards the stationary phase travels slower. The
component with lesser affinity towards the stationary phase travels faster. Thus the
components are separated on a thin layer chromatographic plate based on the
affinity of the component towards the stationary phase.

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 Practical requirement

1. Stationary phases:

There are several adsorbent which can be used as stationary phases. Some
of the stationary phases, their composition and the ratio in which they have
to be mixed with water or other solvents to form slurry for preparing thin
layer chromatographic plates are given in the following table.

Name composition Adsorbent : water ratio

Silicagel H Silicagel without binder 1:1.5
Silicagel G Silicagel +CaSo4 1:2
Silicagel GF Silicagel +binder +fluorescent 1:2
indicator
Alumina Al2o3 without binder 1 :1.1
Neutral
Basic
Acidic
Al2o3 G Al2o3 + binder 1:2
Cellulose powder Cellulose without binder 1:5
Cellulose powder Cellulose with binder 1:6
Kieselguhr G Diatomaceous Earth + binder 1:2

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2. Glass plates:

Glass plates which are specific dimensions like 20 cm × 20 cm (full plate). 20

cm × 10 cm (Half plate). 20 cm × 5 cm (Quarter plate) can be used.
Microscopic slides can also be used for some applications like monitoring the
progress of chemical reaction.

3. Preparation and activation of TLC PLATES

The slurry, which is a mixture of stationary phase and water, is prepared by

using the ratio mentioned earlier. After preparing the slurry the TLC Plates
can be prepared by using any one of the following techniques: i.pouring
ii.dipping iii.spraying iv.Spreading.

i.pouring:

In this technique, the slurry is prepared and poured on to a glass plate which
is maintained on a leveled surface. The slurry is spread uniformly on the
surface of the glass plate. After setting, the plates are dried in an oven

DISADVATAGE

Uniformity in thickness cannot be ensured.

ii.dipping

In this technique, two plates are dipped on to the slurry and are separated
after removing from slurry and later dried.

DISADVATAGE

Large quantity of slurry is required even for preparing fewer plates.

iii.spraying

This technique resembles that of using a perfume spray on a cloth. The

suspension of adsorbent or slurry is sprayed on a glass plate using a sprayer.

DISADVATAGE

The layer thickness cannot be maintained uniformly all over the plate.

iv.Spreading

In this technique where TLC spreader is used. The glass plates of specific
dimension (20 cm × 20 cm (full plate). 20 cm × 10 cm (Half plate). 20 cm × 5
cm (Quarter plate) are stacked on a base plate .The slurry after preparation is
poured inside the reservoir of TLC plate. Thickness of the adsorbent layer is
adjusted by using a knob in the spreder.Normally; a thickness of 0.25mm is
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 used for analytical purpose and 2mm thickness for preparative purpose. the
spreader is rolled only once on the plates.

Plates are allowed for setting (air drying). This is done to avoid crack on the
surface of adsorbent. After setting, the plates are activated by keeping in an
oven at 100 0c to 120 0cfor 1 hr.

4. Application of Sample

Usually to get good spots, the concentration of the sample or standard

solution has to be minimum. 2-5µl of a 1% solution of either standard or test
sample is spotted using a capillary tube. The spots can be placed at random
or equidistant from each other by using a template, with markings.

The spots should be kept atleast 2 cm above the base of the plate and the
spotting area should not be immersed in the mobile phase in the
development tank.

5. Development Tank

For the purpose of development, a developing tank of different sizes to hold

TLC plates of standard dimension are used. These required more solvent for
developing the chromatogram. To solve this problem TLC develop in glass
beakers, specimen jars, to avoid more wastage of solvents.

The development chamber or tank should be lined inside with filter paper
moistened with mobile phase so as to saturate the atmosphere. If this type of
saturation of the atmosphere is not done, EDGE Effect occurs where the
solvent front in the middle of TLC plate moves faster than the edge.

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 (a) Edge effect
(b) Normal solvent front

a. (obtained when saturation of atmosphere with mobile phase is not done)

b. (obtained when saturation of atmosphere with mobile phase is not done)

6. Mobile phase

The mobile phase used depends upon various factors

i. Nature of the substances to be separated.

ii. Nature of the stationary phase.

iii. Mode of chromatography (Normal phase or reverse phase)

iv. Separation to achieved (Analytical or preparative)

E.g. of solvents used for mobile phase

Petroleum ether, Carbon tetrachloride, cyclohexane, carbondisulfide,

Ether, Acetone, Benzene, Toluene, Chloroform, Alcohols, Water.
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 Producing the chromatogram

A pencil line is drawn near the bottom of the plate and a small drop of a solution of
the dye mixture is placed on it. Any labeling on the plate to show the original
position of the drop must also be in pencil. If any of this was done in ink, dyes from
the ink would also move as the chromatogram developed.

When the spot of mixture is dry, the plate is stood in a shallow layer of solvent in a
covered beaker. It is important that the solvent level is below the line with the spot
on it.

The reason for covering the beaker is to make sure that the atmosphere in the
beaker is saturated with solvent vapour. To help this, the beaker is often lined with
some filter paper soaked in solvent. Saturating the atmosphere in the beaker with
vapour stops the solvent from evaporating as it rises up the plate.

As the solvent slowly travels up the plate, the different components of the dye
mixture travel at different rates and the mixture is separated into different colored
spots.

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 The diagram shows the plate after the solvent has moved about half way up it.

The solvent is allowed to rise until it almost reaches the top of the plate. That will
give the maximum separation of the dye components for this particular combination
of solvent and stationary phase.

Preparing the Plate

Do not touch the TLC plate on the side with the white surface. In order to obtain an
imaginary start line, make two notches on each side of the TLC plate. You can also
draw a thin line with pencil. Do not use pen. Why? The start line should be 0.5-1 cm
from the bottom of the plate.

Capillary spotters

Place a melting point capillary and in the dark blue part of the Bunsen burner flame.
Hold it there until it softens and starts to sag. Quickly remove the capillary from the
flame and pull on both ends to about 2-3 times its original length. If you pull the
capillary inside the flame, you will have a "piece of art", but not a good spotter.
Allow the capillary to cool down, and then break it in the middle. Make sure that you
break off the closed end on one of them. Do not use gloves when you pull
capillaries. You will have much better control without them.

What if the substances you are interested in are colourless?

There are two simple ways of getting around this problem:

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Using fluorescence

The stationary phase on a thin layer plate often has a substance added to it which
will fluoresce when exposed to UV light. That means that if you shine UV light on it,
it will glow.

That glow is masked at the position where the spots are on the final chromatogram
- even if those spots are invisible to the eye. That means that if you shine UV light
on the plate, it will all glow apart from where the spots are. The spots show up as
darker patches.

While the UV is still shining on the plate, you obviously have to mark the positions
of the spots by drawing a pencil circle around them. As soon as you switch off the
UV source, the spots will disappear again.

Showing the spots up chemically

In some cases, it may be possible to make the spots visible by reacting them with
something which produces a coloured product. A good example of this is in
chromatograms produced from amino acid mixtures.

The chromatogram is allowed to dry and is then sprayed with a solution of

ninhydrin. Ninhydrin reacts with amino acids to give coloured compounds, mainly
brown or purple.

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In another method, the chromatogram is again allowed to dry and then placed in an
enclosed container (such as another beaker covered with a watch glass) along with
a few iodine crystals.The iodine vapour in the container may either react with the
spots on the chromatogram, or simply stick more to the spots than to the rest of the
plate. Either way, the substances you are interested in may show up as brownish
spots.

Using thin layer chromatography to identify compounds

A small drop of the mixture is placed on the base line of the thin layer plate, and
similar small spots of the known amino acids are placed alongside it. The plate is
then stood in a suitable solvent and left to develop as before. In the diagram, the
mixture is M, and the known amino acids are labelled 1 to 5.

The left-hand diagram shows the plate after the solvent front has almost reached
the top. The spots are still invisible. The second diagram shows what it might look
like after spraying with ninhydrin.

How does thin layer chromatography work?

The stationary phase - silica gel

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Silica gel is a form of silicon dioxide (silica). The silicon atoms are joined via oxygen
atoms in a giant covalent structure. However, at the surface of the silica gel, the
silicon atoms are attached to -OH groups.

The diagram shows a small part of the silica surface.

The surface of the silica gel is very polar and, because of the -OH groups, can form
hydrogen bonds with suitable compounds around it as well as van der Waals
dispersion forces and dipole-dipole attractions. The other commonly used stationary
phase is alumina - aluminium oxide.

The aluminium atoms on the surface of this also have -OH groups attached.
Anything we say about silica gel therefore applies equally to alumina.

Application:

Thin layer chromatography finds many applications, including:

1. Determination of the components a plant contains

2. Monitoring organic reactions.

3. Analyzing ceramides and fatty acids

4. Detection of pesticides or insecticides in food and water

5. Analyzing the dye composition of fibers in forensics, or

6. Identifying compounds present in a given substance

7. Assaying the radiochemical purity of radiopharmaceuticals

Measuring Rf values

If all you wanted to know is how many different dyes made up the mixture, you
could just stop there. However, measurements are often taken from the plate in
order to help identify the compounds present. These measurements are the
distance travelled by the solvent, and the distance travelled by individual spots.

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When the solvent front gets close to the top of the plate, the plate is removed from
the beaker and the position of the solvent is marked with another line before it has
a chance to evaporate.

These measurements are then taken:

The Rf value for each dye is then worked out using the formula:

Where,

Rf (=retardation factor)

For example, if the red component travelled 1.7 cm from the base line while the
solvent had travelled 5.0 cm, then the Rf value for the red dye is:

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If you could repeat this experiment under exactly the same conditions, then the Rf
values for each dye would always be the same. For example, the Rf value for the
red dye would always be 0.34. However, if anything changes (the temperature, the
exact composition of the solvent) it will be change.

Advantage:

1. Simple method and cost of the equipment is low.

2. Rapid technique and not time consuming like column chromatography.

3. Separation of µg of the substance can be achieved.

4. Any type of compound can be analyzed.

5. Efficiency of Separation: Very small particle size can be used which increase
the efficiency of separation .flow rate is not altered because of the particle size
since it is not a closed column. It is a planer type having thin layer of adsorbent.

6. Detection is easy and not tedious.

7. Corrosive spry reagents can be used without damaging the plates.

8. Needs less solvent, stationary phase and time for every separation when
compared to column chromatography.

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