**2. The theory of ion exchange**

Purification using ion exchange chromatography depends upon the reversible adsorption of charged solute molecules to immobilized ion exchange groups of opposite charge. Most ion exchange experiments are performed in five main stages. These steps are illustrated schematically.

The first stage is equilibration in which the ion exchanger is brought to a starting state, in terms of pH and ionic strength, which allows the binding of the desired solute molecules. The exchanger groups are associated at this time with exchangeable counter-ions (usually simple anions or cations, such as chloride or sodium). The second stage is sample application and adsorption, in which solute molecules carrying the appropriate charge displace counter-ions and bind reversibly to the gel. Unbound substances can be washed out

from the exchanger bed using starting buffer. In the third stage, substances are removed from the column by changing to elution conditions unfavourable for ionic bonding of the solute molecules. This normally involves increasing the ionic strength of the eluting buffer or changing its pH. In Figure 1 desorption is achieved by the introduction of an increasing salt concentration gradient and solute molecules are released from the column in the order of their strengths of binding, the most weakly bound substances being eluted first.

**Figure 1.** The principle of ion exchange chromatography (salt gradient elution).

The fourth and fifth stages are the removal from the column of substances not eluted under the previous experimental conditions and re-equilibration at the starting conditions for the next purification. Separation is obtained since different substances have different degrees of interaction with the ion exchanger due to differences in their charges, charge densities and distribution of charge on their surfaces. These interactions can be controlled by varying conditions such as ionic strength and pH. The differences in charge properties of biological compounds are often considerable, and since ion exchange chromatography is capable of separating species with very minor differences in properties, e.g. two proteins differing by only one charged amino acid, it is a very powerful separation technique. In ion exchange chromatography one can choose whether to bind the substances of interest and allow the contaminants to pass through the column, or to bind the contaminants and allow the substance of interest to pass through. Generally, the first method is more useful since it allows a greater degree of fractionation and concentrates the substances of interest. In addition to the ion exchange effect, other types of binding may occur. These effects are small and are mainly due to van der Waals forces and non-polar interactions. Ion exchange separations may be carried out in a column, by a batch procedure or by expanded bed adsorption. All three methodologies are performed in the stages of equilibration, sample adsorption etc. described previously.
