ABSTRACT
Size exclusion chromatography (SEC, earlier referred to as gel filtration chromatography) has several uses in the development of biopharmaceuticals. It is used as a preparative tool to isolate biologically active species, often in concert with other chromatographic techniques in a multistage purification process. It is also used as an analytical tool to obtain information about analyte molecular sizes or shapes and aggregation states, or to determine the extent or kinetics of ligand-biopolymer binding. Preparative SEC often employs soft gels such as dextrans, agarose, or polyacrylamide.1-3 These are compressible and only compatible with elution by the use of gravity or low-pressure pumps. They may be stabilized by cross-linking, in which case they can be eluted at higher flow rates using modest pressure. Analytical SEC is most often performed using rigid supports such as inorganic silica or cross-linked organic polymers. These materials are mechanically stable at high flow rates and pressures, and are used with HPLC systems.4 This chapter will focus on analytical applications of SEC.
