THIN LAYER CHROMATOGRAPHY

INTRODUCTION

This technique is more or less similar to

paper chromatography as far as most of the operations

involved are concerned. Thus, most of the previous discussion

applied here.

To briefly describe the technique, a thin

layer of a finely divided Substance is deposited on to a flute

glass plate. The sample to be separated that is spotted at one

end. The plate is dipped into the solvent in a glass jar & the

development carried out by the ascending technique. After the

development, the layer can be dried & the components detected

by various method available.

Thin layer chromatography may be either

carried out by the adsorption Principle (if the thin layer is

prepared by the adsorbent such as Kieselguhr, or alumina) or

by the partition principle (if the layer is prepared by a substance

such as silica gel which holds water like the paper). More recently,

thin layer gel filtration chromatography is also performed as we

will see later. In this case, the thin layer is made up of

controlled pore beads a various gels.

PREPARATION OF THE LAYERS

The glass plate on which the thin layer is

prepared should be even and is thoroughly washed and dried

before layer application. Modern thin layer chromatography kits

provide plastic or foil plates in lieu of glass plate. The material

of which the thin layer is to be made (silica gel) is usually

mixed with water in such proportion that a thick suspension,

known as slurry results. This slurry is applied to a plate

surface as uniform thin layer by means of plate “spreader”

starting at one end of the plate and moving to the other in

and unbroken uniform motion. The nature of the desired

chromatographic separation detected the thickness of the slurry layer

used. Thus, for analytical separations the thickness of the layer is

usually 0.25 mm, while for preparative separation the thickness of

the layer might be about 5 mm. Although thin layer technique can
be used for many different type of chromatographic separations

such as adsorption, gel filtrations, Ion exchange etc. It is usually

adsorption type which is most used with this technique.

Compounds Adsorbents Solvent systems (V/V)

Mono and Kieselguhr G Ethyl acetate/propan-1-

disaccharides (sodium acetate) ol(65/35)Butanol/acetone/phosphate

Kieselguhr G buffer pH 5 (40/50/10)

(sodium

Phosphate, pH Petroleum ether /diethyl

Triglycerides 5) ether/acetone(90/10/1)

Silica gel G Chloroform/methanol/water(65/25/4

Phospholipids )

Cholestrol esters Silica gel G Carbon tetrachioride/Chloroform

Silica gel G (95/5)

Amino acids 96%ethanol/water(70/30)

Silica gel G Butanol /aceticacid/water(80/20/20)

Plant pigments Petroleum ether/Propanol(99/1)

Kieselguhr
While preparing stationary phase for adsorption chromatography a

binder such as Calcium Sulphate is mixed with the slurry. The binder

helps in better adhesion of the stationary phase to the glass or foi

l plate. The plates are dried after application of the slurry (thin

layer for gel filtration is however, no t allowed to dry.). If

adsorption chromatography is to be performed, the thin layer is

activated by heating at 110 degree Celsius for several hours.

SAMPLE APPLICATION

This is absolutely similar to that described for

paper chromatography. Except that care should be taken not to

scrape the thin layer while applying the sample.

PLATE DEVELOPMENT
 The choice of solvent (as table A) and the

method of elution are much the same as for paper chromatography.

The procedure must of course be conducted in a close chamber to

prevent evaporation of the solvent and the technique used is a

sending out of necessity. Two dimensional chromatography may also

be carried out much in the same way as described for paper

chromatography. One of the greatest advantages of TLC is the

speed at which the separation is achieved. Generally 10-30 minutes

are sufficient. However, with certain compounds about 90 minutes

may be required.
DETECTION

Several detection methods are available. Many of

these have already been named in the section on paper

chromatography for example ultra violate absorption, fluorescence,

autoradiography, if the components are radio labeled, or production

of colors by chemical treatment. Those specific for TLC are:

1) Spraying the plate with 25-50 % Sulphuric acid in ethanol

and heating. These results in charring of most of compounds,

which show up as brown spots.

2) Iodine vapor is used extensively as a universal reagent for

organic compounds. The Iodine spot disappears rapidly but

can be made more permanent by spraying with 0.5% Benzedrine

solution in absolute ethanol.

Iodine vapor is seen concentrated in the form of a cloud over the

reason where the components have separated. This spot can then

be scraped out, eluted and analyzed quantitatively.

On plate quantification of the separated

components might be achieved employing a densitometer, which not

only measure the ultra violate or visible absorption of the separated

components but also gives a complete absorption spectrum of the

compound for identification purpose. Precision made densitometers

are now commercially available. In case the substance has been radio

labeled, radio- chromatogram scanning ight be employed to

quantitative the separated components.

ADVANTAGES AND APPLICATIONS

Compared to paper chromatography, thin layer is

more versatile, faster and more reproducible. Its often used as pilot

technique to quickly determine the complexity of a mixture. It may

otherwise be used as and aid in order to find out the best

conditions for large scales chromatography. Because of its speed and

simplicity, it is often used to follow the course of reaction. Thin layer

technique has often been used to identify drugs, contaminants and

adulterants. It has also been widely used to resolve plant extracts

and many other biochemical preparations.