ABSTRACT
Size-exclusion chromatography (SEC) has been a primary tool used by investigators in the life sciences for purification of proteins. The goal of high-performance size-exclusion chromatography (HPSEC) is to obtain maximum resolution of components of a mixture by separating them according to size. It is therefore imperative in SEC that elution be due strictly to size and all partition mechanisms be eliminated. Silica-based HPSEC supports meet most of the criteria; however, their pH stability is generally limited to pH 3 to 8 and they have a slight ionic character. Another important physical property of an HPSEC support is the pore volume, which must be adequately large to have a high peak capacity. The column efficiency or plate count is more important in HPSEC than in interactive modes of chromatography because the molecules are eluted isocratically instead of during a gradient in a bind/release mechanism. SEC has the lowest loading capacity in terms of both sample weight and volume.
