ABSTRACT
Ion-exchange chromatography (IEC) is an adsorptive technique by which molecules are separated on the basis of differences in their surface charge distribution (Yamamoto et al., 1988; Sofer, 1995). The molecular forces involved in chromatography have been studied in detail by Forgacs and Cserhati (1997) and include Coulombic (electrostatic) interaction between oppositely charged ions, ion-dipole interactions, hydrogen bonds, and hydrophobic forces such as van der Waals and London dispersion forces. The mechanism of IEC and its adsorption isotherms is based on co-ion adsorption as described by Kokotov (2000). In IEC the binding is generally enthalpy-driven with ∆G<0 and ∆H<0 and is primarily due to the interaction energy of electrostatic and van der Waals forces. Binding energy is typically higher than for entropy-driven processes, and for proteins, ∆G values are typically <−10 kJ/mol. ∆H values of −6 to −13 kJ/mol have been reported for electrostatic binding of β-lactoglobulin A (Chen, 2000; Lin, 2001). Proteins possess either a net positive or net negative charge at pH values away from their pI and will bind to an oppositely charged ion exchanger. As such, IEC is one of the most widely used techniques for protein separation and is typically found in all bio-processes. Proteins are generally adsorbed at low salt concentrations (0.01− 0.05 M) and eluted with a step or gradient increase in salt concentration, typically up to 1.0 M (Yamamoto et al., 1987b). Typical ligand chemistries and their charged pH range include, for cation exchange, carboxymethyl (CM, pH 6-10) and sulfyl-propyl (SP, pH 2-10), and for anion exchange, diethylamino-ethyl (DEAE, pH 2-9) and quaternary amine (Q, pH 2-10) (Scopes, 1994).
