What does size exclusion chromatography primarily utilize to separate molecules?

Size exclusion chromatography primarily utilizes the pore size of the substrate to separate molecules. This technique operates on the principle of size differences among molecules. In a size exclusion chromatography setup, a column is packed with porous beads. When a mixture of molecules is injected into the column, smaller molecules can penetrate the pores of the beads, while larger molecules are excluded and travel along the outside of the beads. As a result, larger molecules elute from the column first, followed by smaller molecules that take longer to pass through due to their ability to enter the pores.

This method is particularly useful for separating proteins, polymers, and other macromolecules based on their size rather than their charge, affinity, or the specific characteristics of the solvent or temperature used during the process. Thus, focusing on the pore size of the substrate is crucial for determining the separation efficiency and resolution in size exclusion chromatography.