3. Gel Permeation Chromatography

This type of chromatography is also called as "Molecular Sieve Chromatography" or "Molecular Exclusion Chromatography". When a gel having pores on its matrix is packed in the column and a heterogeneous mixture is pass through it, smaller ions enter the gel and come out of the column phase. The bigger molecules can not enter the gel, they move only with the mobile phase through the inner phases of the gel. In this process, a type of sieving of molecules occur.

Widely used gels are Agarose, Porous Glass, Sephadex, Polyacrylamide, Polystyrene and Silica Granules.

Gel Permeation chromatography can be carried out using the technique of column chromatography. (By determining the distribution coefficient, molecular weight of macro molecules can be determined).

Image Source: American laboratory.

After running a chromatogram, a fraction of macro molecules is collected and volume is known. From this value, distribution coefficient can be calculated.

The distribution coefficient (Kd) is referred to as the the distribution of solute particles between inner and outer solvent in a gel. The value of individual fraction (Ve) depends on the volume of outer solvent (Vo) and volume of inner solvent (Vi).

Ve = Vo+Kd(Vi)

Vi = (a)(Wr) [a = dry weight of the gel and Wr= water regain value.]

Ve = Vo + (Kd x aWr)

Distribution Coefficient (Kd) = (Ve - Vo) / (aWr).

To know the molecular weight of the fractioned compounds, a standard graph with Kd of standards plotted against their molecular weight.

Next Post: Ion-Exchange Chromatography

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