Gel exclusion chromatography

There is now a considerable choice of materials which can separate proteins on the basis of their molecular size. The original cross-linked dextrans (Sephadex G- series, Pharmacia Ltd.) and polyacrylamides (Bio-Gel P- series, BioRad Ltd.) are still, quite rightly, widely used. Both types are available in a wide range of pore sizes and particle size distributions. However, as the pore size increases, for use with larger enzymes, these gels become progressively less rigid and therefore less suitable for large scale use. Consequently alternative, but generally more costly, rigid gel materials have been developed for the fractionation of proteins of molecular weight greater than about 75,000. These are the cross-linked derivatives of agarose (Sepharose CL and Superose) and dextran (Sephacryl S) made by Pharmacia Ltd., the cross-linked polyacrylamide-agarose mixtures (Ultrogel AcA) made by LKB Instruments Ltd. and the ethylene glycol-methacrylate copolymers (Fractogel HW) made by Toyo Soda Company (TSK). These are available in a range of forms capable of fractionating enzymes, and other materials, with molecular weights up to 10⁸ and at high flow rates. Although these gels are described as 'rigid', it should be appreciated that this is a relative term. The best gels are significantly compressible so scale-up from laboratory sized columns cannot be achieved by producing longer columns. Scale-up is achieved by increasing the diameter of columns (up to about 1 m diameter) but retaining the small depth. Further scale-up is done by connecting such sections in series to produce 'stacks'. Extreme care must be taken in packing all gel columns so as to allow even, well distributed, flow throughout the gel bed. For the same reason, the end pieces of the columns must allow even distribution of material over the whole surface of the column. The newer materials are supplied in a pre-swollen state which enable their rapid and efficient packing using slight pressure.

Gel exclusion chromatography invariably causes dilution of the enzyme which must then be concentrated using one of the methods described earlier.