In size-exclusion chromatography, how are molecules separated?

In size-exclusion chromatography, molecules are separated primarily by their size. This technique employs a column filled with porous beads, which allows smaller molecules to enter the pores while larger molecules cannot. As a sample mixture passes through the column, the larger molecules travel through the spaces between the beads and therefore elute from the column faster than the smaller molecules. The smaller molecules take longer to elute because they are temporarily trapped in the pores of the beads, resulting in a size-based separation.

The separation process is purely physical, meaning that it does not rely on the charge, hydrophobicity, or binding affinity of the molecules involved. In this manner, size-exclusion chromatography effectively distinguishes molecules solely based on their relative sizes.