Types Of Column Chromatography

Gel filtration, ion-exchange, and affinity chromatography are the common types of column chromatography used to extract pure compounds from a mixture, such as a protein suspension.

Gel filtration chromatography uses an inert matrix of polymeric gel beads to create specific pore sizes.

In the case of a protein suspension, the proteins larger than the pore size elute first, while the smaller ones are trapped in the gel beads. These trapped proteins can then be eluted with multiple solvent washes.

In contrast, an ion-exchange column is packed with matrix beads coated with weak acids or bases that give them a net positive or negative charge at a certain pH.

This helps separate proteins based on their surface charge by forming ionic bonds with the oppositely charged proteins.

Lastly, affinity chromatography uses a matrix tagged with ligands, such as antibodies, to pull out a target protein from a mixture based on specific binding interactions.

In affinity and ion-exchange chromatography, the target protein is finally eluted by adding an elution solvent that weakens the column-protein interactions due to a change in conditions such as the pH, ionic strength, or introduction of a competitive molecule.