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Gel, Affinity chromatography:- Hand written notes


Gel chromatography– Introduction, theory, instrumentation and applications
Affinity chromatography– Introduction, theory, instrumentation and applications

The different separation methods used in chromatography are based on different
principles. However, in many cliromatographic methods it is rather difficult to define the variables which govern the method of separation. In 1954 “Mould and Synge” showed that the separations based on molecular sieving could be performed on uncharged substances during the migration through gels. This formed the basis for separations.based on the relative sizes of the molecules. The systematic use of this principle was introduced in 1954 by “Porth and Flodin” who used the term gel filtration for their method of separating large molecules of biological origin by means of polysaccharide gel Gephade x). Moore used the term “gel permeation chromatography”. In 1964, “Determann” suggested that the “gel chromatography” is the most appropriate term for this technique. This method is mainly based
on the differences in molecular dimensions and has different names like gel filtration, molecular sieve filtration, restricted diffusion chromatography, exclusion chrornatography, molecular sieve chromatography etc. Gel chromatography method separates different substances depending on their molecular size. This technique differs from other partition chromatographic techniques. In this technique the particles which come from stationary phase in the column arc an uncharged gel. The gel swells in the same solvent which percolates through the bed.

The stationary phase is a porous polymer matrix where the pores are completely frlled with the solvent to be used as the mobile phase. The pore size is very very important. The basis of the separation is the molecules above a certain size are totally excluded from entering and occupying the pores and the interior of the pores is accessible partly or wholly to smaller molecules. The flow of mobile phase will cause larger molecules to pass through the column unhindered without penetrating the gel matrix, whereas small molecules will be retarded because of their penetration in the gel

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  • Semester:- sevenths sem, sem 7
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