INTRODUCTION

Chromatography is the separation of two or more compounds or ions by the

distribution between two phases, one which is moving and the other which is
stationary. These two phases can be solid-liquid, liquid-liquid or gas-liquid.
Although there are many different variations of chromatography, the principles
are essentially the same.

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 components
travel at different rates. We'll look at the reasons for this further down the page.

Thin layer chromatography is done exactly as it says - using a thin, uniform

layer of silica gel or alumina coated onto a piece of glass, metal or rigid plastic.

The silica gel (or the alumina) is the stationary phase. The stationary phase for
thin layer chromatography also often contains a substance which fluoresces in
UV light - for reasons you will see later. The mobile phase is a suitable liquid
solvent or mixture of solvents.

Thin-layer chromatography or TLC, is a solid-liquid form of chromatography

where the stationary phase is normally a polar absorbent and the mobile phase
can be a single solvent or combination of solvents. TLC is a quick, inexpensive
micro scale technique .

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 PRINCIPLE
The principle of separation is adsorption in Thin Layer Chromatography. 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 components 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 components
towards the stationary phase.

Measuring Rf values

Because the distance travelled by a substance, relative to the distance travelled

by the solvent front, depends upon the molecular structure of the substance,
TLC can be used to identify substances as well as to separate them.
The relationship between the distance travelled by the solvent front and the
substance is usually expressed as the Rf value:

The Rf values are strongly dependent upon the nature of the adsorbent and
solvent. Therefore, experimental Rf values and literature values do not often
agree very well. In order to determine whether an unknown substance is the
same as a substance of known structure, it is necessary to run the two
substances side by side in the same chromatogram, preferably at the same
concentration.

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A Retardation /Retention Factor value will always be in the range 0 to 1. If the
substance moves at all, it moves along the direction of the solvent (Mobile
Phase) does less, but cannot move further than the solvent does.

Retention factor values are only useful if they are between these two extremes.
An Retention factor value is characteristic of any given compound (Provided
hat the same stationary and mobile phases are used)

Reversed Phase: Reversed phase is the terminology used when the stationary
phase is silica bonded with an organic substrate such as a long chain aliphatic
acid like C-18 and the mobile phase is a mixture of water and organic solvent
which is more polar than the stationary phase.

Stationary Phase: This is the substance which is fixed in place for the
chromatography procedure. Examples include the silica layer in TLC.

Mobile phase: This is the phase which moves in a definite direction. It may be
a liquid phase or gas or supercritical fluid. The mobile phase consists of the
sample being separated / analysed and the solvent that moves the sample
through the column. Examples of Mobile phase are- petroleum ether,
cyclohexane, acetone, benzene, alcohol, water etc.

TLC Adsorbent: When choosing the most suitable grain size, the technical
preparation and migration times on the chromatogram must be considered and a
compromise arrived at. The grain size of most TLC adsorbent lies between 5
and 50 μm. The purity of the adsorbent is often significant for the adsorption
properties; occasionally inorganic or organic impurities interfere procedure if
were not removed. The adsorbent materials, usually silica gel, aluminium oxide
or cellulose. This layer of adsorbent is known as the stationary phase.

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 PREPARATION AND ACTIVATION OF TLC
PLATES
Although high quality pre coated plates of adsorbent on glass, aluminium or
plastic are readily available from laboratory equipment and chemical suppliers,
the preparation of plates may be easily carried out in the laboratory for a
fraction of the cost of commercial plates.
General Method:
The sizes of glass plates for use with commercially available spreaders are
usually 20x20, 20x10 or 20x5 cm.
Mix the adsorbent (30g) in a mortar to a smooth consistency with the requisite
amount of water or solvent specified in the manufacturer’s instruction and
transfers the slurry quickly to the spreader. Spread the mixture over 4 to 5 Plates
(20x20 cm) or a proportionately larger no. of smaller plates and allow the thin
layer to set (about CaSO4 is present) transfer the plates carefully to a suitable
holder and after a further 30 min., dry at 100 to 120 C for 1 hour to activate the
adsorbent. Cool and store the plates in a desiccator over silica gel. The thickness
of the moist thin layer should be about .25 mm.
Other Methods:
A) Pouring Technique: In this the slurry is prepared and poured on to a
plate which is maintained on a levelled surface. The slurry is spread
uniformly on the surface of the glass plate. After setting, the plates are
dried in an oven. The disadvantage is that uniformity in thickness cannot
be ensured.
B) Dipping Technique: In this technique two plates (either of standard
dimensions or microscopic slides) are dipped in to the slurry and are
separated after removing from slurry and later dried. The disadvantage is
that a larger quantity of slurry is required even for preparing fewer plates.
C) Spraying Technique: This technique resembles that of using a perfume
spray on a cloth. The suspension of adsorbent or slurry is sprayed on a
plate using sprayer. The disadvantage is that the layer thickness cannot be
maintained uniformly all over the plate.
D) Spreading Technique: This is the best technique here a TLC spreader is
used. The glass plates of specific dimensions as mentioned above are
stacked on a base plate. The slurry after preparation is poured inside the
reservoir o TLC spreader. The thickness of the adsorbent layer is adjusted
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 by using a knob in he spreader. Normally a thickness of 0.25mm is used
for analytical purpose and 2mm thickness for preparative purpose. Then
the spreader is rolled only once on the plates. The plates are allowed for
setting (air drying). This is done to avoid cracks on the surface of
adsorbent. After setting, the plates are activated by keeping in an oven at
100C to 120C for 1 hour.
This technique is used in the General Method mentioned above.

Activation:
Activation of TLC plates is nothing but removing water / moisture and other
adsorbed substances from the surface of any adsorbed by heating at high
temperature so that adsorbent activity is retained. The activated plates can be
stored in thermostatically controlled oven or in desiccator and can be used
whenever required.

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 APPLICATION OF SAMPLE AND
DEVELOPMENT OF PLATES

Application of sample:
The origin line to which the sample solution is applied, is usually located 2.0to
2.5cm from the bottom of the plate. The accuracy and precision with which the
sample ‘spots’ are applied is very important when quantitative analysis is
required. Usually to get good spots the concentration of the sample or standard
solution has to be minimum. 2to5microl of a 1% solution of either standard or
test sample is spotted using a capillary tube or micropipette. The spots can be
placed at random or equidistant from each other by using a template, with
markings. The spotting area should not be immersed in the mobile phase in the
development tank. Spot size is thus more uniform and results are found to be
more consistent.

Development of Plates:
The chromatogram is usually developed by the ascending technique or may be
one specially designed to allow a reproducible angle of inclination of the plate
in which the plate is immersed in the developing solvent to a depth of 0.5cm
(redistilled or chromatographic grade solvent should be used).
Allow the solvent to evaporate and transfer the plate to a previously prepared
developing tank; this preparation is done about 30 min before insertion of the
plate. It should be lined with filter paper dipping into the developing solvent so
as to maintain an atmosphere saturated with the solvent vapour in the tank. This
method helps to eliminate the ‘edge effect’ which occurs, particularly when
mixed solvents are used. In this effect a substance moves faster when near the
edge than when in the centre of the plate. Allow the solvent to rise a distance of
about 10 to 15 cm, remove the plate and allow to dry using heat or a current of
air as appropriate.

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 DETECTION OF COMPOUNDS

The two most important techniques used to locate colourless drug substances on
a thin layer chromatogram are:
A) Spraying with a reagent that reacts with the substance to produce a
coloured zone. A very large number of reagents have been proposed,
which have varying degrees of selectivity for certain functional groups in
the molecules. The use of highly corrosive non-selective spray reagents is
possible with silica gel, alumina and kieselguhr (but not cellulose) thin
layers, e.g. 70 to 80% sulphuric acid, which causes charring of organic
compounds on heating to 100C.
B) Examination under ultraviolet light of plates containing a fluorescent
indicator. Any substance that absorbs at the wavelength of excitation
(254nm or 365nm) quenches the fluorescence of the indicator and is
observed as a dark zone on a yellow- green back ground. The sensitivity
of detection is thus related to the absorptivity of the substance at the
wavelength or irradiation.

Detection at UV light

APPLICATIONS OF THIN LAYER

CHROMATOGRAPHY

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• TLC has several important uses. These are:

1) To establish that two compounds are identical

2) To determine the number of components in a mixture

3) To determine the appropriate solvent for a column-chromatographic

separation

4) To monitor the progress of a reaction

5) To detect the presence of foreign substances in drugs

6) To detect decomposition products in drugs

7) Separation of mixtures of drugs of chemical or biological origin,

plant etc.

REFERENCE

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E Stahl. Thin-layer Chromatography. A Laboratory Handbook: Springer –
Verlag Berlin
Heidelberg ,New York 1969:second edition 1990.

G Chatwal,S anand.Ihstrumental Methods of Chemical analysis: Himalaya

publishing
house,Delhi:Edition June 1995.

Dr.S. Ravi Sankar.Text book of pharmaceutical analysis: Rx

Publication,Tiruneveli:Edition
1997,1999,2001.

H.F.Waltor,J.Reyes:Modern Chemical analysis and instrumentation.

International Medical Book
distrubutor,Mumbai.

A.H. Backett, J.B.Stenlake, Practical Pharmaceutical Chemistry-Part two:

CBS Publication:Fourth Edition 20007.

D.Kealey, P.J.Haines, Analytical Chemistry:Viva Books Private Limited:

First Edition 2002

J Mendham,R.C. Denney, J.D. Barnes, M.J.K. Thomas, Vogel’s Textbook

of Quantitative Chemical Analysis, Vogel’s Textbook of Quantitative
Chemical Analysis: Pearson education: First Edition 2006.
Interrnet Source: website of wikipedia