What is the principle of size exclusion chromatography?

The principle of size exclusion chromatography (SEC) is based on HPLC techniques which have porous particles in the column to separate macromolecules by their size, geometry, or molecular weight. SEC separates molecules based on their size by filtration through a gel. This gel is composed of spherical beads which contain pores of a specific size. SEC works by capturing and trapping smaller molecules in the pores of the chromatographic column. Separation occurs when molecules of different sizes are incorporated or kept out from the pores within the matrix. Small molecules diffuse into the pores and their flow rate through the column begins to decrease according to their size. In contrast, large molecules are unable to enter the pores and are instead eluted in the column’s empty volume. Molecules separate based on their size, and are eluted in order of decreasing MW. SEC is primarily used for the separation of macromolecules such as proteins, antibodies, enzymes, nucleic acids, and industrial polymers.