Size-exclusion chromatography (SEC), also referred to as gel-filtration chromatography, uses porous particles to separate biological molecules of different sizes. Molecules that are smaller than the pore size can enter the particles and therefore have a longer path length and longer residence time than larger molecules that cannot enter the particles.

Proteins larger than the pore size can not 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 can at least partially enter the pores will have varying residence times dependent on the formula weight and hydrodynamic radius of each individual protein. Different proteins will therefore have different residence times when passing through the column. Proteins that elute in this portion of a chromatogram are said to be selectively permeable to the pores. 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 and are said to be totally permeable to the pores. 

The main application of size exclusion chromatography is the fractionation and purification of synthetic and biological polymers, such as proteins, polysaccharides and nucleic acids. 

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. The technique is generally combined with others that further separate molecules by other characteristics, such as pI, charge, and affinity for certain compounds and is typically used as a polishing step.