Question

Describe the basis for separating proteins by ion exchange, hydrophobic interaction, gel filtration, and affinity chromatography.

Short answer

• Ion exchange chromatopgraphy is a type of liquid chromatography for separating anions and cations.
• Hydrophobic interaction chromatography separates proteins according to differences in their surface hydrophobicity.
• Gel filteration chromatography separates molecules according to their size and shape. It is also called size exclusion or molecular sieve chromatography.
• Affinity chromatography utilizes specific binding interaction between an immobilized ligand and its binding partner.

Step-by-step solution

Ion exchange chromatography

In this chromatography, the charged ion binds to oppositely charged groups that are chemically linked to a matrix called cellulose and agarose.The anion exchanger binds to cationic groups and the cation exchanger binds to anionic groups. DEAE (diethylaminoethyl) is used for the anion exchanger matrix and CM (carboxymethyl) is used for the cation exchanger matrix.

Hydrophobic interaction chromatography

This chromatography is useful for purifying proteins while maintaining their biological activity.It works on the high salt concentration that enhances the interaction. Lowers salt concentration weakens the interaction.

Gel filtration

In this chromatography, the solutes pass through the stationary phase containing heterosporous cross-linked polymeric gel and beads. In this, the large molecules are excluded from all the pores and hence elute first and the small molecules can enter the intraarticular pore space and elute last.

Affinity chromatography

In this technique, an impure protein solution is passed through chromatographic material, and the desired protein binds to the immobilized ligand, whereas other substances are washed through the column with the buffer.