Size Exclusion Chromatography

Size exclusion chromatography (SEC) is a standard separation technique for determining molar mass averages and molar mass distributions of polymers such as whole proteins, protein conjugates, peptides, carbohydrates, large oligonucleotides and other synthetic or natural polymers. Size exclusion chromatography is also known as exclusion chromatography, steric-exclusion chromatography, restricted-diffusion chromatography, liquid-exclusion chromatography, gel permeation chromatography, gel filtration, gel chromatography. Size exclusion or gel permeation chromatography (SEC or GPC) is a useful analytical technique that allows the characterization of polymeric materials by determining their molecular weight distribution. In order to resolve polymeric species, long column lengths and banked column configurations are commonly used. Typical analysis times can range from 30 to 60 minutes but longer analytical test cycle times may be needed in some cases. The term size exclusion chromatography describes the actual separation mechanism the best. In this technique polymer molecules are separated according to their hydrodynamic volumes with the larger size molecules exiting first from the column followed by the smaller. The hydrodynamic volume of each polymer determines the observed retention time for each polymer and can be correlated with the molar mass of the polymer analyzed. Molar masses are determined either from a calibration or using molar mass sensitive detectors. The separation technique is based on the molecular size of the components and separation is achieved by the differential exclusion of the sample polymers from the pores of the packing material. The sample molecules pass through a bed of porous particles and the principle feature of size exclusion chromatography is its gentle non-adsorptive interaction with the sample. In most cases this type of separation does not destroy biomolecular activity. Some times when biomolecules are separated in aqueous or aqueous/organic mobile phases size exclusion chromatography is referred to as gel filtration chromatography (GFC). The separation of organic polymers in non-aqueous mobile phases is also called gel permeation chromatography (GPC). Typically, size exclusion chromatography is used primarily for analytical assays and semi-preparative purifications. It is not commonly used for process scale work due to the low capacity of the size exclusion mode. Size exclusion chromatography columns such as TSKgel GFC columns and others are available for protein analysis, the analysis of water-soluble polymers, or polar organic-soluble polymers, organic-soluble polymers, and, in some case, for the purification of these compounds as well.