CHAPTER 3 - CHROMATOGRAPHIC FRACTIONATION MEDIA

Abstract

This chapter lists the different types of media used for the major types of chromatography. Chromatography has been one of the most important methods used for the separation of both large and small molecules. There are four main types of chromatographic separations: (1) separations dependent on molecular size, (2) separations based on the overall charge or charge distribution on molecules, (3) reversed-phase separations, and (4) affinity separations in which the chromatographic medium has a particular affinity for a molecule. In the first case, separations are based on the use of media with the pores of specific sizes. The pore size is chosen such that small molecules pass in and out of the pores essentially without hindrance, while large molecules, which do not enter the pores, elute from the column first. Separations may be made under denaturing conditions or nondenaturing conditions. A range of media are available for the separation of molecules on the basis of their charge, with media for both low-pressure and high-pressure liquid chromatography (HPLC) separations. The separation proceeds because ions of opposite charge are retained to different extents. The third type of separation, reversed-phase HPLC, is a rapid and powerful separation technique of unusual specificity. The typical conditions such as low pH and high concentrations of organic solvents favor denaturation. This technique is used in many circumstances in which denaturation may be unimportant, such as in structural studies, or where structure and activity are retained despite the high concentrations of organic solvents used. In the case of affinity separations, the molecule to be purified is specifically and reversibly adsorbed by a complementary binding substance immobilized on an insoluble support. The choice of medium is usually very dependent on the type of molecule being separated.