Size Exclusion (Gel Filtration) Chromatography

Size exclusion chromatography is used for semi-preparative purifications and various analytical assays. It is a separation technique which takes the advantage of the difference in size and geometry of the molecules. The molecules are separated based on their size. Grant Henry Lathe and Colin R Ruthven was the pioneer of size exclusion chromatography who started this technique for separation of analytes of different size with starch gels as the matrix, later Jerker Porath and Per Flodin introduced dextran gels. Other gel filtration matrices include agarose and polyacrylamide.

Note: Unlike ion exchange chromatography, g el filtration does not depend on any chemical interaction with protein, rather it is based on a physical property of the protein - that being the effective molecular radius (which relates to mass for most globular proteins).

Principle: Size exclusion chromatography (SEC) is the separation of mixtures based on the molecular size (more correctly, their hydrodynamic volume) of the components.  Separation is achieved by the differential exclusion or inclusion of solutes as they pass through stationary phase consisting of heteroporous (pores of different sizes) cross linked polymeric gels or beads. The process is based upon different permeation rates of each solute molecule into the interior of gel particles. Size exclusion chromatography involves gentle interaction with the sample, enabling high retention of biomolecular activity. For the separation of biomolecules in aqueous systems, SEC is referred to as gel filtration chromatography (GFC), while the separation of organic polymers in non-aqueous systems is called gel permeation chromatography (GPC).