Size Exclusion Chromatography (SEC) HPLC Columns

About size exclusion chromatography

The mode of separation in Size Exclusion Chromatography (SEC) is considerably different from Reverse Phase (RP) and other traditional modes of chromatographic separation. Where other modes rely mostly on the interaction of the analytes with the stationary phase, SEC differentiates molecules based on the molecule size using columns packed with porous particles. The stationary phase is designed so to minimize—ideally completely remove—any interactions with the analytes. Any secondary interactions between the analyte and column particle could negatively affect retention.

Size exclusion chromatography for proteins and biologics

SEC is routinely used for the analysis of proteins and more specifically often to evaluate the aggregation profile of the protein. Aggregation is a degradation product and must be closely monitored and kept within acceptable limits. This is a Critical Quality Attribute (QCA) for monoclonal antibodies (mAbs) and other antibodies because it can cause an immunogenic response in the patient. Large antibody aggregates are sterically excluded from most pores and therefor flow quickly flow through the column. Smaller proteins spend more time diffusing inside of the particle pores and elute later from the column. The smallest proteins, such as antibody fragments can enter all pores and elute last.

Size exclusion chromatography for water soluble polymers

SEC is also used for water soluble polymers and oligomers. Water soluble polymers are a highly varied family of products developed and used by medical, cosmetic, food, oil, agriculture, environmental, and many other industries. Examples of water soluble polymers include polyethylene glycols (PEGs), polyvinyl alcohols, polyvinyl pyrrolidones, dextrans, polyacrylic acids (PAA), polyacrylamides (PAM), polyamines and polyethyleneimines, quaternary ammonium polymers, and polyvinylpyrrolidone.

How SEC works and why pore size matters

The differential separation is then achieved because of the differential rates of diffusion for different molecules through the pores of the stationary phase. The rate of diffusion is dependent on the analyte’s size, or more specifically, the hydrodynamic radius. This is very closely linked to the mass of the analyte. Larger analytes are sterically excluded from an increased number of pores compared with a smaller analyte and so they pass through the column at a quicker rate with a lower retention time. This is the opposite to what you typically expect for analysis of larger analytes with reversed phase (RP) or hydrophobic Interaction chromatography (HIC) modes. There is a point at which larger analytes will be sterically hindered from all pores and elute in the void volume.

Similarly, there is a point at which smaller molecules will be able to diffuse into all available pores and the resolution power is also lost. This is also quite different to typical chromatographic modes, for example RP, where the retention will increase as the carbon chain length increases over a much wider range (until the molecule become too big for the pore size etc.) Therefore, the pore size is the critical factor that controls the separation mechanism and its efficiency. As a result, SEC columns are manufactured with a variety of pore sizes, for a variety of different applications and analyte sizes. The overall pore volume in the column particles controls the retention time of each species. The pore size of the particle controls the relative separation of proteins.

Special notes about SEC and the HPLC system

Size exclusion chromatography encounters some challenges, because unlike other chromatographic techniques, SEC has no focusing of analytes at the head of the column. So, chromatographic quality is also heavily dependent on dispersion of the HPLC chromatography system. Small particles reduce diffusion times and allow for shorter columns and faster separations. However, small molecules are difficult to manufacture with sufficiently large pores and they are prone to clogging / fouling. Some size exclusion chromatography column particles can be prone to robustness issues, with chromatography system pressure spikes the particle pores can deform, or rust particulates from the system can block pores, reducing column longevity and robustness.