Thin layer chromatography

 Principles, Procedure and Applications of Thin Layer Chromatography (TLC), Paper Chromatography

Thin Layer Chromatography was discovered by Izmailov and Shraiber in 1938 by using plant extract.

What Is Thin Layer Chromatography?

Chromatography is an important biophysical technique that enables the separation, 

identification, and purification of the components of a mixture for qualitative and quantitative analysis.

 Thin Layer Chromatography is used to isolate non-volatile mixtures. The experiment is 

conducted on a sheet of aluminium foil, plastic, or glass which is coated with a thin layer of adsorbent material. The material usually used is aluminium oxide, cellulose, or silica gel.

 Separation of complex mixtures (known as chromatography) is an used to identify and compare samples of drugs, explosives, inks and biological samples such as saliva, urine, blood and other.

Principle

The process begins here by moving the mobile phase over the stationary phase’s surface. During this movement, the higher affinity compounds gain less speed as compared to the lower affinity compounds. This results in their separation.

Procedure

TLC Plates: These are used for applying the thin layer of stationary phase, material like silica gel,

Mobile Phase: This comprises a solvent (or solvent mixture). The taken solvent needs to be chemically inert, of the highest possible purity,

 Petroleum ether < n-hexane < carbon tetrachloride < toluene < benzene <chloroform < dichloromethane < diethyl ether < n-butanol < isopropanol < acetone < methanol < water

TLC Chamber: This is where the thin layer chromatography procedure takes place. It keeps the dust particles away from the process and does not let the solvent evaporate.

Filter Paper: This gets placed inside the chamber after being moistened with the mobile phase solution. It ensures that the mobile phase rises uniformly throughout the TLC plate’s length.

Thin Layer Chromatography Plates: ready-made plates, and Filter Paper thin (0.25mm) layer of some inert material such as Al2O3, MgO, or SiO2 is used.

 The process starts by making a thin mark on the TLC plate’s bottom with a pencil. It helps in the application of sample spots. These spots are kept at equal distances.

 The sample is then applied to these spots made on the line.Then the TLC chamber is filled with the mobile phase (solvent 10-20 ml) up to a few centimeters of its bottom.

 After pouring the mobile phase, the TLC plate or moistened filter paper is placed along with 

the inside of the chamber wall. 

 Finally, the prepared stationary phase plate is put inside the chamber. At this point, the sample spots are kept on the mobile phase’s side.

 The chamber is then closed after placing the plate into it.Once enough time has elapsed for the process, the plate is taken out and allowed to dry.

 At last, the sample spots get analyzed through a suitable method for the sample, such as UV light, KMnO4 stain, and iodine staining.

Calculation:

On completion of the separation, each component appears as spots separated vertically. Each spot has a retention factor (Rf) expressed as:

 

 Rf = dist. travelled by sample / dist. travelled by solvent

Thin Layer Chromatography Applications

 The qualitative testing of Various medicines such as sedatives, local anaesthetics, 

anticonvulsant tranquilisers, analgesics, antihistamines, steroids, hypnotics is done by TLC.

 TLC is extremely useful in Biochemical analysis such as separation or isolation of biochemical Metabolites from its blood plasma, urine, body fluids, serum, etc.

 Thin layer chromatography can be used to identify natural products like essential oils or volatile oil, fixed oil, glycosides, waxes, alkaloids, etc.

It is widely used in separating multicomponent pharmaceutical formulations.It is used to purify of any sample and direct comparison is done between the sample and the authentic sample.

 It is used in the food industry, to separate and identify colours, sweetening agent, and 

preservatives It is used in the cosmetic industry.

Disadvantages Of Thin Layer Chromatography:

1. Thin Layer Chromatography plates do not have longer stationary phase.

2. When compared to other chromatographic techniques the length of separation is limited.

3. The detection limit is high and therefore if you want a lower detection limit, you cannot use TLC.

4. It is only a qualitative analysis technique and not quantitative.