Chromatography is a technique used to separate mixtures into individual components. Thin layer chromatography (TLC) is a common form of liquid chromatography that uses a thin layer of adsorbent material, like silica gel, coated onto a flat surface. To perform TLC, a sample is applied to the plate and then placed into a developing chamber containing a mobile phase solvent. As the solvent moves up the plate, it carries the different components of the sample at different rates based on their interactions with the stationary and mobile phases, separating them into individual spots. The separated components can then be analyzed and identified based on their retention factor values and visualization methods like ultraviolet light. TLC is widely used in applications like pharmaceutical analysis

1. Chromatography
History:
Michael Tswett is credited as being the father of liquid chromatography. Tswett developed
his ideas in the early 1900’s.
1938: Izmailov & shraiber described basic principle and used it for separation of plant
extracts.
1944: Consden, Gorden & Martin started using filter papers for separation of amino acid.
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.
Introduction:
Chromatography is a technique that can be used to separate color in inks, food dyes and
other mixtures of color. A medium, such as blotting or filter paper, containing a spot of
the mixture is placed in contact with a solvent (e.g. water). The different colors move
throughout the medium at different rates and so are separated along it.
Thin layer chromatography
Introduction:
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.
Chromatography is used to separate mixtures of substances into their components.
Similar to P.C, except that a thin layer of some inert material, i.e. Aluminium
oxide, mag.oxid. , sili.oxide is used instead of paper.
• A layer of any one of these oxide is made from a slurry of power in a suitable inert
solvent.
• Slurry is spread over a flat surface ( glass, metal or rigid plastic ) & dried.

2. Fig: TLC technics
Glass plate:
Specific dimensions-
20cm Х 20cm, 20cm Х 10cm, 20cm Х 5cm
Microscopic slides can also be used
Plates should be of good quality & withstand high temperatures
PREPARATION OF TLC PLATES:
♦ Pouring ( simplest methods )
♦ Dipping (used for small plates )
♦Spraying ( difficult to get uniform layers )
♦ Spreading ( best technique ) TLC Spreader
Activation of Plates:
 After spreading → Air dry (5 to 10 minutes)
 Activated by heating at about 100˚C for 30 min.
 Then plates may be kept in desiccators.

3. Application of sample:
 With a pencil etch two small notches into the adsorbent about 2-2.5cm from the bottom
of the plate.
 The notches should be on the edges of the plate & each notch should be same distance up
from the bottom of the plate.
 The notches must be farther from the bottom of the plate than the depth of the solvent in
the jar.
 Using a drawn out capillary tube spot the samples on the plate so that they line up with
the notches you etched.
Developmenttank:
 In TLC the plate is placed in a development chamber at an angle of 45.
 The bottom of chamber is covered up to 1 mm by the solvent.
Mobile phase:
 The ability of mobile phase to move up is the ability of mobile phase to move up is
depend on the polarity itself depend on the polarity itself.
 Volatile organic solvents is preferably used as volatile organic solvents is preferably
used as mobile phase mobile phase.
Analysis:
As the chemicals being separated may be colorless, several methods exist to visualize the
spots:
 fluorescent analytes like quinine may be detected under blacklight (366 nm)
 Often a small amount of a fluorescent compound, usually manganese-activated zinc
silicate, is added to the adsorbent that allows the visualization of spots under UV-C light
(254 nm). The adsorbent layer will thus fluoresce light-green by itself, but spots of
analyte quench this fluorescence.
 Iodine vapors are a general unspecific color reagent
 Specific color reagents into which the TLC plate is dipped or which are sprayed onto the
plate exist.
 Potassium permanganate - oxidation
 Bromine
Interpreting the data:
Rf=
distance from start to center of substance spot
distance from start to solvent front

4. 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
References:
 A.I. Vogel; A.R. Tatchell; B.S. Furnis; A.J. Hannaford & P.W.G. Smith. Vogel's
Textbook of Practical Organic Chemistry (5th ed.). ISBN 0-582-46236-3
 Justus G. Kirchner (1978): Thin-layer chromatography, 2nd edition,
 WileyJoseph Sherma, Bernard Fried (1991): Handbook of Thin-Layer
Chromatography (= Chromatographic Science. Bd. 55). Marcel Dekker, New York
NY, ISBN 0-8247-8335-2.