Ion-exchange chromatography

Proteins contain charged groups on their surfaces that enhance their interactions with solvent water and hence their solubility. Charged residues can be cationic (positively charged, e.g., lysine) or anionic (negatively charged, e.g., aspartate) and it is noteworthy that even polar residues can also be charged under certain pH conditions. These charged and polar groups are responsible for maintaining the protein in solution at physiological pH. Because proteins have unique amino acid sequences, the net charge on a protein at physiological pH is determined ultimately by the balance between these charges (i.e., negatively charged proteins possess more negatively charged residues than positively charged groups). This also underlies differing isoelectric points (pIs) of proteins. Ion-exchange chromatography (1) separates proteins first on the basis of their charge type and, second, on the basis of relative charge strength (e.g., strongly anionic from weakly anionic). The basis of ion-exchange chromatography (Fig. 40.1) is that charged ions can freely exchange with ions of the same type. In this context, the mass of the ion is irrelevant. Therefore it is possible for a bulky anion like a negatively charged protein to exchange with chloride ions. This process can be later reversed by washing with chloride ions in the form of a NaCl or KCl solution. Such washing removes weakly bound proteins first, followed by more strongly bound proteins with greater net negative charge. Like most column chromatography techniques, ion-exchange chromatography requires a stationary phase, which is usually composed of insoluble, hydrated polymers, such as cellulose, dextran or Sephadex (2). The ion exchange group is immobilized on this stationary phase, and some of the chemical structures of commonly used groups are shown in Table 40.1. In this chapter, the use of microgranular diethylaminoethyl (DEAE) cellulose manufactured by Whatman (Maidstone, UK) is described. As described in Section 3.5, novel formats for ion-exchange chromatography are provided by the immobilization of ion-exchange groups on membrane or filter formats (3) and in perfusion chromatography (4). The separations can be performed in low pressure or high pressure systems (e.g., FPLC).