Gel filtration Chromatography

Gel-filtration chromatography, also known as the size exclusion chromatography, is a versatile technique that permits the separation of proteins and other biological molecules. The gel filtration chromatography separates the proteins solely based on molecular size differences. For this, a porous matrix is used to which the molecules, for steric reasons, have different degrees of access. The matrix is enclosed in a chromatographic column, and the separation is accomplished by passing an aqueous buffer through the column. The molecules, confined outside the matrix beads, sweeps through the column by the mobile phase. An in-line UV monitor detects the separated protein zones and the fractions of the sample are collected for subsequent specific analysis. The gel-filtration chromatography has numerous applications including the fractionation of proteins and other water-soluble polymers, size determination and analysis, desalting, and buffer exchange.

Principle: The gel filtration chromatography is based on the molecular size and the hydrodynamic volume of the components. The molecules are separated by the differential exclusion or inclusion of solutes as they pass through the stationary phase containing hetero-porous cross-linked polymeric gel or beads. Different permeation rates of the solute molecules cause them to sift in the interior of the gel particles. A column of the porous matrix is in equilibrium with the mobile phase for separation of the molecules. Large molecules are entirely excluded from the pores and come first in the effluent. Smaller molecules get distributed between the mobile phase and the outside of the sieve. Then, they pass through the column at a slower rate and appear later in the effluent

• The stationary phase used is a porous polymer matrix whose pores are completely filled with the solvent to be used as the mobile phase.


• The molecules in the sample are pumped through specialized columns containing such microporous packing material (gel).


• The basis of the separation is that molecules above a certain size are totally excluded from the pores, while smaller molecules access the interior of the pores partly or wholly.


• The flow of the mobile phase hence will cause larger molecules to pass through the column unhindered, without penetrating the gel matrix, whereas smaller molecules will be retarded according to their penetration of the gel.