Size Exclusion Chromatography (SEC), also referred to as gel-filtration chromatography when used with aqueous mobile phases, uses porous particles to separate biological molecules of different sizes. The advantages of this method include good separation of large molecules from the small molecules while preserving the biological activity of the particles to be separated.
Proteins larger than the pore size cannot enter the pores and will thus co-elute as the first peak in the chromatogram. These molecules are said to be totally excluded from the pores. Proteins that at least partially enter the pores will have varying residence times dependent on their hydrodynamic radius. Different proteins will therefore have different residence times when passing through the column. Molecules that are smaller than the pore size are able to enter all of the pores in the resin and will therefore have the longest residence times. These molecules typically elute together as the last peak in a chromatogram.
