ABSTRACT
The main applications of size-exclusion chromatography (SEC) are: separation of molecules differing in size, determination of average molecular weights, determination of hydrodynamic diameters, and separation of proteins from small molecules, for example, desalting. The only possibility of further increasing resolution in SEC – as in all types of high-performance liquid chromatography — is to minimize peak dispersion by reduction of particle diameter and optimization of eluent velocity. Crosslinked hydrophilic gels have been used in classical SEC of proteins. These materials swell considerably in the presence of water, thus generating the pore structure required for SEC. To extend the separation capacity in SEC, the use of combined columns packed with silicas with different pore size distributions of exclusion limits is recommended. A paramount advantage of silica-based stationary phases in SEC is the possibility to calibrate the columns with polystyrene standards in organic eluents and to use them in aqueous systems for protein separation.
