Thin Layer Chromatography

CONTENTS
Introduction & History
Principal involved in TLC
Stationary phase & mobile phase
Advantages of TLC
Adsorbents
Mobile phase
Rf value
Factors affecting Rf value
References

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 1900s.
1958:- Ergon stahl introduced a standard equipment for preparing uniform thin layers
of known thickness.

Principle of TLC
It is based on the principle of adsorption chromatography or
partition chromatography or combination of both, depending on
adsorbent, its treatment and nature of solvents employed

The components with more affinity towards stationary phase travels

slower.
Components with less affinity towards stationary phase travels faster.

Separations in TLC involve distributing a mixture of two or more

substances between a stationary phase and a mobile phase
1.The stationary phase:
is a thin layer of adsorbent (usually silica gel or alumina) coated on a
plate.
2.The mobile phase:
is a developing liquid which travels up the stationary phase, carrying
the samples with it.
Components of the samples will separate on the stationary phase
according to:
how much they adsorb on the stationary phase versus
how much they dissolve in the mobile phase

TLC
The two most common classes of TLC are:
Normal phase
Reversed phase

Normal Phase
Normal phase is the terminology used when the
stationary phase is polar; for example silica gel,
and the mobile phase is an organic solvent or a
mixture of organic solvents which is less polar
than the stationary phase.

Reversed Phase
Reversed phase is the terminology used when the
stationary phase is a 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.

Advantages of TLC
Short analysis time
All spots can be visualized
Adoptable to most pharmaceuticals
Low cost
Uses small quantities of solvent
Requires minimal training
Reliable and quick
Minimum amount of equipment is needed

Steps in TLC Analysis

The following are the important components of a
typical TLC system:

Apparatus (developing chamber)

Stationary phase layer and mobile phase
Application of sample
Development of the plate
Detection of analyte

General Procedure (1)

Decide if you are going to do Normal or Reversed
phase chromatography
Prepare a plate or select a plate with the proper sorbent
material
Prepare the mobile phase
Mark the plate
Apply the sample
Develop the plate
Detect the analytes

PRACTICAL REQUIREMENTS
1.

STATIONARY PHASE

Adsorbents mixed with water or other solvents slurry

Silica gel H ( Silica gel with out binder )
Silica gel G ( Silica gel + CaSO4 )
Silica GF (Silica gel + binder + fluorescent indicator)
Alumina, Cellulose powder, Kieselguhr G( Diatomaceous earth
+ binder)

Coater, hand operated

2. GLASS PLATE
Specific dimensions20cm 20cm, 20cm 10cm, 20cm 5cm
Microscopic slides can also be used
Plates should be of good quality & withstand high temperatures

3. PREPARATION & ACTIVATION OF TLC

PLATES
Pouring ( simplest methods )
Dipping (used for small plates )
Spraying ( difficult to get uniform layers )
Spreading ( best technique ) TLC Spreader

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; pouring, dipping, spraying and spreading
Pouring 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 is used for spotting. The disadvantage is that

uniformity in thickness can not be ensured.

 Dipping technique: two plates (either of standard dimensions or

microscopic slides) are dipped in to 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.

Spraying 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. The disadvantage is that the layer thickness
cannot be maintained uniformly all over the plate.

Spreading: is the best technique where a TLC spreader is used. The glass plates of specific
dimensions (20cm X 20cm/10 cm/5cm) are stacked on a base plate. The slurry after
preparation is poured inside the reservoir of TLC spreader. The thickness of the adsorbent
layer is adjusted by using a knob in the spreader. Normally a thickness of 0.25cm is used for
analytical purpose and 2 mm thickness for preparative purpose. Then the spreader is rolled
only once on the plate. 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 100oC to 120oC for 1 hour

 Activation of TLC plates is nothing but removing

water/moisture and other adsorbed substances from the
surface of any adsorbent, 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.

4. APPLICATION OF SAMPLE
Using capillary tube or micropipette
Spotting area should not be immersed in the mobile phase

5. DEVELOPMENT TANK
Better to develop in glass beakers, jars to avoid more wastage
of solvents
When standard method is used, use twin trough tanks
Do chamber saturation to avoid edge effect

Selection of adsorbents
Solubility of compound e.g., hydrophilic or lipophilic
Nature of substance to be seperated i.e. whether it is acidic, basic
or amphoteric
Adsorbent particle size
Adsorbent should not adhere to glass plate
Reactivity of compound with the solvent or adsorbent
Chemical reactivity of compounds with binders.

Adsorbents for TLC

1.
2.
3.
4.

Inorganic:
Silica Gel
Kieselguhr
Aluminium Silicate
Bentonite

1.
2.
3.
4.

Organic:
Cellulose & its acetylates
Charcoal & activated C
Dextran Gel
Polyamides

Mobile phase
Nature of the substance to be seperated i.e polar or non polar
Nature of stationary phase used
Mode of chromatography
Nature of separation i.e. analytical or preparative
Solvent used should be of high purity.
Solvents used:- petroleum ether
benzene
carbon tetrachloride
chloroform

7) DEVELOPMENT TECHNIQUE
Different development techniques are used for efficient separations. They are
1. One dimensional development (vertical)
2. Two dimensional development

3. Horizontal development
4. Multiple development

1. One dimensional development (vertical)

Like conventional type, the solvent flows against gravity. The spots are kept at
the bottom portion of paper and kept in a chamber with mobile phase solvent at
the bottom

2. Two dimensional technique

This technique is similar to 2-Dimensional TLC. The paper is developed in one

direction and after development, the paper is developed in the second direction
allowing more compounds or complex mixtures to be separeted into individual
spots. In the second direction, either the same solvent or different solvent system
can be used for development.

8. Detecting agent

After the development of chromatogram, the spots should be visualised.

Detecting coloured spots can be done visually, But for detecting colourless spots,
any one of the following techniques can be used.

a.

Non specific method: Where the number of spots can be detected but not exact
nature of compound
Example
i. Iodine Chamber Method: Where brown or amber spots are observed when
the paper is kept tank with few iodine crystals at the bottom

ii. UV Chamber for flourescent compounds: When compounds are viewed

under UV chamber at 245 nm or at 365 nm flourescent compounds can be
detected.

Detecting agent
b. Specific methods: Specific spray reagents or detecting agents visualizing agents are
used to find out the nature of compounds for identification purposes
Examples
i.

For phenolic compounds and tannins

ii. Ninhydrin in acetone

For amino acids

iii. Dragendroffs reagent

For alkaloids

iv. 3,5-Dinitro benzoic acid

For cardiac glycosides

v.

Ferric chloride

2,4-Dinitrophenyl hydrazine -

For aldehydes and ketones

Rf VALUE
In thin layer chromatography the results are represented by Rf value
which represent the movement or migration of solute relative to the
solvent front. This is indicating position of migrated spots on
chromatogram.

The Rf value is calculated as:-

Factors affecting Rf value

It depends on following factors:
Nature adsorbent
Mobile phase
Activity
Thickness of layer
Temperature
Equilibrium
Loading
Dipping zone
Chromatographic techniques

APPLICATIONS OF TLC
Purity of sample
Examination of reaction
Identification of compounds
Biochemical analysis
In pharmaceutical industry
Separation of multicomponent pharmaceutical formulations
In food and cosmetic industry