Ion chromatography

Ion chromatography (or ion-exchange chromatography) is a form of chromatography that separates ions and ionizable polar molecules based on their affinity to the ion exchanger.^[1] It works on almost any kind of charged molecule—including small inorganic anions,^[2] large proteins,^[3] small nucleotides,^[4] and amino acids. However, ion chromatography must be done in conditions that are one pH unit away from the isoelectric point of a protein.^[5]

The two types of ion chromatography are anion-exchange and cation-exchange. Cation-exchange chromatography is used when the molecule of interest is positively charged. The molecule is positively charged because the pH for chromatography is less than the pI (also known as pH(I)).^[6] In this type of chromatography, the stationary phase is negatively charged and positively charged molecules are loaded to be attracted to it. Anion-exchange chromatography is when the stationary phase is positively charged and negatively charged molecules (meaning that pH for chromatography is greater than the pI) are loaded to be attracted to it.^[7] It is often used in protein purification, water analysis,^[8][9] and quality control. The water-soluble and charged molecules such as proteins, amino acids, and peptides bind to moieties which are oppositely charged by forming ionic bonds to the insoluble stationary phase.^[10] The equilibrated stationary phase consists of an ionizable functional group where the targeted molecules of a mixture to be separated and quantified can bind while passing through the column—a cationic stationary phase is used to separate anions and an anionic stationary phase is used to separate cations. Cation exchange chromatography is used when the desired molecules to separate are cations and anion exchange chromatography is used to separate anions.^[11] The bound molecules then can be eluted and collected using an eluant which contains anions and cations by running a higher concentration of ions through the column or by changing the pH of the column.

One of the primary advantages for the use of ion chromatography is that only one interaction is involved the separation, as opposed to other separation techniques; therefore, ion chromatography may have higher matrix tolerance. Another advantage of ion exchange is the predictability of elution patterns (based on the presence of the ionizable group).^[12] For example, when cation exchange chromatography is used, certain cations will elute out first and others later. A local charge balance is always maintained. However, there are also disadvantages involved when performing ion-exchange chromatography, such as constant evolution of the technique which leads to the inconsistency from column to column.^[13] A major limitation to this purification technique is that it is limited to ionizable group.^[6]