Principle of Ion Exchange Chromatography: Ion exchange chromatography separates proteins or other molecules based on differences in their accessible surface charges. In ion exchange chromatography the analyte molecules are retained on the column based on coulombic (ionic) interactions. The stationary phase surface contains ionic functional groups of opposite charge that interact with analyte ions. The elution is done by increasing salt gradient. Most commonly used salt is NaCl, exists in equilibrium with Na⁺ (cation) and Cl^- (anion) in aqueous solution. As the concentration of salt increases concentration of Na⁺ (cation) and Cl ^- (anion) also increases. The basic principle of ion exchange chromatography is the reversible exchange of analyte ions bound to solid support with similar ions generated from salt in liquid phase. Many biological molecules such as proteins, amino acids, nucleotides and other ions have ionisable groups which carries a net charge (positive or negative) dependent on their pKa and on the pH of the solution, which can be utilised in separating mixture of such molecules as explained in box. Ion exchange chromatography experiments are carried out mainly in columns packed with ion exchangers. On the basis of type of exchanger used for separation this chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography.

Many biological molecules, especially proteins, are stable within a narrow pH range so the type of exchanger selected must operate within this range. Suppose if protein is most stable below its isolecteic point (pI), there will be net positive charge on the protein surface, so for separation of this protein cation exchanger should be used (experimental pH value should be between lowest pH wehere protein is stable and pI value). If protein is most stable above its pI, there will be net negative charge on the protein surface and anion exchanger should be used (experimental pH value should be between highest pH where protein is stable and pI value). If protein is stable over a wide range of pH, it can be separated by either type of ion exchanger (experimental pH value may be decided considering lowest and highest pH value stability of the protein). Weak electrolyte requires very high or very low pH for ionisation so it can only be separated on strong exchanger, as they only operate over a wide pH range, whereas in case of strong electrolytes, weak exchangers are preferred.