Ion Exchange Chromatography (IEC) is a technique based on the difference in the strength of the interaction between a sample ion and an oppositely charged functional group on the support. The sample ion competes for the functional group with a counter ion that has been added to the mobile phase as a salt. Elution is most often accomplished by increasing the salt concentration over time.
Ion exchange chromatography is the most common separation mode for protein purification schemes. Biomolecules generally have charged groups on their surfaces, which change with the pH of the solution.
Anion Exchange Chromatography is performed with either a strong anion exchange column, containing a quaternary ammonium ion, or with a weak anion exchanger, having either a tertiary or secondary amine functional group, such as DEAE (diethylaminoethyl). A counter ion, often Cl-, maintains electroneutrality.
Cation Exchange Chromatography is performed with either a strong cation exchanger, containing a bonded sulfonic acid group, such as sulfopropyl (SP), or with a weak cation exchanger, containing a weak acid such as carboxymethyl (CM). A counter ion, often Na+, maintains electroneutrality. The advantage of strong vs. weak ion exchangers is that the first are charged over a wider pH range. Weak ion exchangers often provide slightly different selectivity from strong exchangers.
In ion exchange chromatography, the pH of the mobile phase buffer must be between the pI or pKa of the charged molecule and the pKa of the charged groups on the solid support. For example, a molecule with a pI of 8.2 is run in a mobile phase buffer at pH 6.0 with the solid support pKa at 1.2 in cation exchange chromatography. In anion exchange chromatography a molecule with a pI of 6.8 is run in a mobile phase buffer at pH 8.0 with the solid support pKa at 10.3.
