A protein mixture is passed into the column and binds to the adsorbent. By proper choice of the eluting buffer, specific proteins can be eluted from the adsorbent and separated from other proteins in the mixture. By repeating this procedure with several different adsorbents, pure protein can be obtained. The properties of some adsorbents are described below. Ion Exchange Chromatography Ion exchange resins have fixed charges - either positive or negative.
Proteins bind to the resin via electrostatic interactions.
The strength of these interactions depends on the net charge on the protein, which is a function of pH and the nature of the weak acid amino acid side chains, and the salt concentration of the buffer - high salt concentrations reduce the interaction. The higher the net charge on the protein at the pH of the environment on the column, the more tightly it sticks to the oppositely charged resin, and the higher the salt concentration required to elute it from the column. This is a more specific interaction in which a ligand specifically recognized by the protein of interest is attached to the column material. When a mixture of proteins is passed through the column, only those few that bind strongly to the ligand will stick, while the others will pass through the column. By changing the buffer one weakens the interaction between the protein and the ligand, which causes the protein to be eluted from the column.