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Chapter: Biochemistry: Biochemical Techniques

Thin Layer chromatography

Partition, adsorption, exclusion chromatography can be carried out in a thin layer mode. In this technique the stationary phase is made in the form of a slurry and applied as a thin coating on the surface of a glass plate.

Thin Layer chromatography

Principle: Partition, adsorption, exclusion chromatography can be carried out in a thin layer mode. In this technique the stationary phase is made in the form of a slurry and applied as a thin coating on the surface of a glass plate.After activating the plate, the sample to be separated is applied at one end of the plate . The plate is kept vertically in a chamber specially designed for this purpose( TLC chamber) and allowed the sample and the mobile phase to raise through the stationary phase by capillary action. The whole procedure consists of.

a. Thin layer preparation : A slurry of the stationary phase, generally applied to a glass, plastic or foil plate as a uniform thin layer by means of a plate spreader starting from one end of the plate and moving progressively to the other. Calcium phosphate is incorporated into the slurry in order to facilitate the adhesion of the adsorbent to the plate. The plate is heated in an oven at 1000 C to activate the adsorbent.

b. Sample application : The sample is applied to the plate by means of a micropipette or syringe as spot or as a band on the stationary phase.

c. Plate development : Separation takes place in a glass tank which contains mobile phase to a depth of about 1.5 cm. This is allowed to stand for atleast an hour with a lid over the top of the tank to ensure that the atmosphere within the tank becomes saturated with solvent vapour.

d. Component detection : The components separated are detected by (i) spraying the plate with 50% sulphuric acid or 25% sulphuric acid in ethanol and heating; (ii) examining the plate under ultraviolet light; (iii) spraying of plates with specific colour reagents, for example ninhydrin for amino acids.

 


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Biochemistry: Biochemical Techniques : Thin Layer chromatography |


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