What is pI in ion exchange chromatography?

Ion exchange chromatography separates molecu- les according to type and strength of their charge. The isoelectric point (pI) is the pH where a protein or molecule has no net electrical charge. Depending on the pH of the buffer a protein has different surface charges in solution.

How does ion exchange chromatography of proteins work?

Ion exchange chromatography is a process for separating proteins and other molecules in a solution based on differences in net charge. Negatively charged molecules bind to positively charged solid supports and positively charged molecules bind to negatively charged supports.

How does isoelectric point affect ion exchange chromatography?

If the isoelectric point (pI) of your protein is known The pI of the protein determines the buffer pH and the ion exchange column to use. At a buffer pH above its pI, a protein is negatively charged and will bind to an anion exchanger.

What is ion chromatography method?

Ion chromatography (or ion-exchange chromatography) is a chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids.

How does ion chromatography work?

How Does Ion Chromatography Work? Ion chromatography, a form of liquid chromatography, measures concentrations of ionic species by separating them based on their interaction with a resin. Ionic species separate differently depending on species type and size.

What is the purpose of ion exchange chromatography?

Ion exchange chromatography (IEX) is a chromatographic separation method essentially based on the net charge of the protein, and is generally used to follow deamidation and succinimide formation. Positive (cationic) or negative (anionic) charge moieties are directly linked to the chromatographic matrix.

How are proteins separated in ion exchange chromatography?

An impure protein sample is loaded into the ion exchange chromatography column at a particular pH. Charged proteins will bind to the oppositely charged functional groups in the resin. A salt gradient is used to elute separated proteins.

How do you elute protein from ion exchange?

Proteins will elute when the pH gradient reaches their pI, because they will no longer carry a net charge that allows them to interact with the column resin. To elute proteins from an anion exchange resin, a decreasing pH gradient is chosen, while an increasing pH gradient is chosen for elution from cation exchangers.