Question

briefly explain how ion exchange chromatography works. Why is the pH of the elutent changed during the seperation?

Answer 1

Principle - Ion exchange chromatography is a separation technique that is based on the net charge developed on the surface of molecules; specially biomolecules. Depending on the pH of their environment, biomolecules may carry a net positive charge, a net negative charge, or no charge.

Procedure -

1. After loading an impure sample onto an ion exchange column, the column is rinsed thoroughly to remove impurities. The sample is eluted using either a salt gradient or a change in pH.

2. The charged salt ions will compete with sample molecules for the charged resion ions. Sample molecules with few charged groups will elute at low salt concentrations, whereas those with many charged groups will elute at high salt concentrations.

Using pH gradient - Sometimes a pH gradient is also chosen as a method of elution. Sample molecules, specially proteins will elute when the pH gradient reaches their pI, because at this point, they will no longer carry a net charge that will allow them to interact with the resin molecules of the column.

To elute proteins from an anion exchange resin, a decreasing pH gradient is chosen, while to elute proteins from a cation exchange resin, an increasing pH gradient is chosen.

Altering the pH of the elution buffer can affect the resolution of the method.

Advantage - This technique is very sensitive and can separate molecules with a tiny difference of charge; as tiny as a single charged amino acid.