Ion exchange chromatography

What is ion exchange chromatography?

Ion exchange chromatography (IEX) separates proteins with differences in surface charge to give high-resolution separation with high sample loading capacity. The separation is based on the reversible interaction between a charged protein and an oppositely charged chromatography resin.

Ion exchange chromatography resins can be used at high flow rates, because binding kinetics for IEX are fast, and rigid chromatography particles can be used.

How does ion exchange chromatography work?

The net surface charge of proteins varies according to the surrounding pH. Above its isoelectric point (pI), a protein will bind to a positively charged anion exchanger. Below its pI, a protein will bind to a negatively charged cation exchanger.

Proteins bind as they are loaded onto a column at low ionic strength. The conditions are then altered so that bound substances are desorbed differentially. Elution is usually performed by increasing salt concentration or changing pH in a gradient.


When should I use ion exchange chromatography?

Ion exchange chromatography can be used in any part of a multistep purification procedure: as a first step, in which high binding capacity and high flow rates allow capturing of both target protein and bulk impurities from a large-volume sample, as an intermediate purification step, or as a final step for high-resolution purification to remove the remaining impurities.

IEX is used to bind the target molecule, but it can also be used to bind impurities, letting the target protein pass the column. Ion exchange chromatography can be repeated at different pH values to separate several proteins that have distinctly different charge properties. Alternatively, a purification step using a cation exchanger can be followed by a second purification step using an anion exchanger at the same pH.

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