Size-Exclusion Chromatography

In size-exclusion chromatography (SEC), also known as molecular-exclusion or gel-permeation chromatography, molecules are separated based on their sizes. This technique is important for separating large molecules such as polymers and biomolecules. The two classes of micron-sized stationary phases encountered in SEC are silica particles and cross-linked polymer resin beads. Both materials are porous, but their pore sizes vary significantly.

Silica particles offer advantages such as rigidity, stability, and compatibility with various solvents. In contrast, polymer beads come in different pore sizes and can be hydrophilic (water-loving) or hydrophobic (water-repelling). Hydrophilic gels like agar and polyacrylamide are used for aqueous separations, while hydrophobic gels such as polystyrene-divinylbenzene are preferred for non-polar solvents. Silica particles used for size exclusion are deactivated to prevent unwanted interactions, while polymer resins are synthesized without exchange sites.

Smaller solutes spend more time within the pores of the stationary phase, resulting in a longer elution time from the column. The retention volume for a solute depends on its distribution ratio, which ranges from 0 at the exclusion limit to 1 at the inclusion limit. This reflects how well the solute penetrates the pores.

SEC is a notable application in the analysis of protein mixtures and the determination of formula weights. Calibration curves can be prepared between the exclusion and inclusion limits, but accurate formula weight determinations require carefully chosen standards to minimize the effect of shape.

This technique can be carried out using conventional HPLC instrumentation by replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is typically used to obtain chromatograms.