THIN LAYER

CHROMATOGRAPHY

PRASHANT PANDEY
M.PHARM.
 THIN LAYER CHROMATOGRAPHY

TLC (Thin Layer Chromatography)

Principle:
Thin layer chromatography is a method of analysis in which the stationary phase (a finely
divided solid) is spread as a thin layer on a solid rigid supporting plate; and the mobile phase: a
liquid is allowed to migrate across the surface of the plate by the capillary action and separation
takes places due to adsorption phenomenon and gives different R f values for each sample
compound.
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒 (𝑠𝑝𝑜𝑡)
𝑅𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡

Types and Techniques of TLC

S. No. Technique Stationary Phase Mobile
Phase
1. Adsorption Silica gel, alumi na, charcoal, Nonpolar/Polar organic
polyamide solvents
2. Partition Cellulose, silica gel Mixed aqueous, organic
solvents
3. Reversed Phase ODS silica gel, coated silica, Mixed aqueous, polar
acetylated cellulose solvents
4. Ion exchange Ion exchange resins: CM cellulose, Buffered aqueous solutions
Diethyl amino ethyl
5. Size exclusion Dextran gels Buffered aqueous solutions

Stationary Phase:
Details of stationary phase are already given above under selection of stationary phase. Silica
gel, the most commonly used stationary phase, has the empirical formula SiO 2. However, at the
surface of the silica gel particles, the dangling oxygen atoms are bound to protons. The presence
of these hydroxyl groups renders the surface of silica gel highly polar. Thus, polar functionality
in the organic analyte interacts strongly with the surface of the gel particle and nonpolar
functionality interacts only weakly.

Mobile Phase:
 The mobile phase is the solvent or solvent mixture moving through the stationary phase
on the TLC/HPTLC plate during development.

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 Mobile phase should be chosen taking into consideration chemical properties of analytes
& sorbent layers.
 Use of mobile phase containing more than three or four components should be avoided as
it is often difficult to get reproducible ratios of different components.
 Mobile phase evaporates before derivatization.
 Does not interfere with determination of the position of solute spots/ bands
 Smaller volume of mobile phase required.
Solvent Polarity
1. n-Hexane 0.0
2. Carbon tetrachloride 1.7
3. Toluene 2.3
4. Xylene 2.5
5. Benzene 2.7
6. Diethyl ether 2.8
7. Dichloroethane 3.7
8. 1-Butanol 3.9
9. n-propanol 4.0
10. 2-Propanol 4.3
11. Ethyl acetate 4.3
12. Chloroform 4.4
13. Methanol 5.1
14. Ethanol 5.2
15. Acetone 5.4
16. Acetonitrile 6.2
17. Acetic Acid 6.2
18. Dimethylsulphoxide 7.2
19. Water 9.0
Solvent System Development
 First Level: Neat Solvent Selection.
 Second Level: Decreasing Hexane or Increasing Water Strength.
 Third level: Try mixtures, consider the addition of modifiers (acids, bases)
 Fourth Level: Selection of optimal mixture.
 If the analytical goal is not achieved, repeat the procedure on a different stationary phase.

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Preparation of TLC plate

 Suspend 100 g of silica gel G in 200-250 ml of water, mix with a stirrer to get homogeneous
slurry.
 Take the air dried TLC glass plates or dried in oven at 110 0C and pour the silica gel G slurry
into the glass plate. (Thickness should be around 250 μm) Slurry should be used within
2 minute of preparation otherwise slurry will dry and needs more water to maintain the
fluidity.
 TLC Plate is prepared by using four methods:
o Pouring
o Dipping
o Spraying
o Spreading
 Dry the plate in a TLC chamber until complete drying occurs.
 Dried TLC plates are activated in oven at 1100C for 30 minutes and immediately used for
development after cooling.
 Thickness of preparative TLC plate is 0.5-2mm (500-2000μm). Preparative TLC plate is used
for isolation of the compounds.

Sample Preparation
For TLC on silica gel, the use of least polar solvent which allows quantitative dissolving and
spotting of sample and there is no preliminary development and separation within the initial spot
at the origin, is recommended.
Application of sample
 Sample application is most critical step for obtaining good resolution. The sample should
be completely transferred to the layer, however, under no circumstances; the application
process should damage the layer, as damaged layer results in unevenly shaped spots.
 Wherever possible, use of automatic application device is recommended. The sample should
be applied through clean smaller diameter capillary.
Preconditioning
Chamber saturation has pronounced influence on the separation profile. When the plate is
introduced into an unsaturated chamber, during the course of development, the solvent
evaporates from the plate mainly at the solvent front. Therefore larger quantity of the solvent
shall be required for a given distance; hence resulting is increase in Rf values. If the tank is
saturated (by lining with filter paper) prior to development, solvent vapours soon get uniformly
distributed throughout the chamber. As soon as the plate is placed in such a saturated chamber, it
soon gets preloaded with solvent vapours, hence less solvent shall be required to travel a
particular distance, resulting in lower Rf values.

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Development and drying

Develop the chromatogram in twin trough chamber or other TLC development chamber until
and unless solvent reaches the three fourth distance of the plate. Dry the developed plate in
chromatographic drying chamber.

Evaluation of thin layer chromatogram

First of all spots of TLC are detected by using suitable detecting reagent or physical methods.
The evaluation depends on the purpose of a chromatographic analysis. For quantitative
determination often localization of substances is sufficient. This can be easily achieved by parallel
runs with reference substances.
Rf values:
A parameter often used for qualitative evaluation is the Rf value (retention factor). The Rf value
is defined as follows:

𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒 (𝑠𝑝𝑜𝑡)

𝑅𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡

i.e. Rf values are between 0 and 1, best between 0.1 and 0.8.
Diagrammatic representation:

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Applications of TLC:
1. Purity of any sample : Purity of sample can be carried out with TLC. Direct comparison is done
between the sample and the standard or authentic sample; if any impurity is detected, then it shows
extra spots and this can be detected easily.
2. Identification of compounds: Thin layer chromatography can be employed in purification,
isolation and identification of natural products like volatile oil or essential oil, fixed oil, waxes,
terpenes, alkaloids, glycosides, steriods etc.
3. Examination of reactions: Reaction mixture can be examined by Thin layer chromatography to
access whether the reaction is complete or not. This method is also used in checking other
separational processes and purification processes like distillation, molecular distillation etc.
4. Biochemical analysis: TLC is extremely useful in isolation or separation of biochemical
metabolites or constituent from its body fluids, blood plasma, serum, urine etc.
5. In chemistry: TLC methodology is increasingly used in chemistry for the separation and
identification of compounds which are closely related to each other. It is also used for
identification of cations and anions in inorganic chemistry.
6. In pharmaceutical industry: Various pharmacopoeias have adopted TLC technique for detection
of impurity in a pharmacopoeial chemical.
7. Various medicines like hypnotics, sedatives, anticonvulsant tranquillisers, antihistaminics,
analgesics, local anaesthetics, steroida have been tested qualitatively by TLC method.
8. One of the most important application of TLC is in separation of multicomponent
pharmaceutical formulations.
9. In food and cosmetic industry: TLC method is used for separation and identification of colours,
preservatives, sweetening agent, and various cosmetic products.