chromatography -Ion exchange

Ion Exchange Chromatography (IEC) is a powerful liquid chromatographic technique used for bio-separation. The separation is done by a reversible interaction between charged molecules of the sample with charged ligands attached to a column. The method offers a sizeable sample-handling capacity, powerful resolving ability, broad applicability, moderate cost, and the ease of handling. These characteristics have made the ion-exchange chromatography one of the most versatile and widely used liquid chromatography techniques. Ion exchange chromatography is frequently used for the separation and purification of polypeptides, proteins, enzymes, antibodies, nucleic acids, polynucleotides, and other charged biomolecules.

There are two types of ion-exchange chromatography: anion-exchange and cation-exchange. Cation-exchange chromatography is used for the positively charged molecules. In this type of chromatography, the stationary phase is negatively charged, which attracts the positively charged molecules of interest. Whereas, in anion-exchange chromatography, the stationary phase is positively charged which attracts the negatively charged molecules. It is prominently used in protein purification, water analysis, and quality control. The bound molecules are eluted and collected using an eluent containing anions and cations by running a higher concentration of ions through the column or changing the pH of the column.

Principle: Ion-exchange chromatography separates the molecules based on their respective charged groups. The ion-exchange chromatography consists of both mobile and stationary phases, the former is usually an aqueous buffer system into which the sample of interest is introduced, and the latter is an inert organic matrix chemically derived with ionizable functional groups carrying an oppositely charged counter ion. The molecules undergo electrostatic interactions with opposite charges present on the stationary phase matrix. For electroneutrality, these inert charges interact with exchangeable counter ions in the solution. Ionizable molecules that are to be resolved compete with these counter ions for binding to the displaceable charges on the stationary phase. These molecules are retained or eluted according to their respective charges.