**3. The matrix**

332 Ion Exchange Technologies

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 An ion exchanger consists of an insoluble matrix to which charged groups have been covalently bound. The charged groups are associated with mobile counter ions. These counter-ions can be reversibly exchanged with other ions of the same charge without altering the matrix. It is possible to have both positively and negatively charged exchangers (Fig. 2). Positively charged exchangers have negatively charged counter-ions (anions) available for exchange and are called anion exchangers. Negatively charged exchangers have positively charged counter-ions (cations) and are termed cation exchangers. The matrix may be based on inorganic compounds, synthetic resins or polysaccharides. The characteristics of the matrix determine its chromatographic properties such as efficiency, capacity and recovery as well as its chemical stability, mechanical strength and flow properties.

**Figure 2.** Ion exchanger types.

The nature of the matrix will also affect its behaviour towards biological substances and the maintenance of biological activity. The first ion exchangers were synthetic resins designed for applications such as demineralisation, water treatment, and recovery of ions from wastes. Such ion exchangers consist of hydrophobic polymer matrices highly substituted with ionic groups, and have very high capacities for small ions. Due to their low permeability these matrices have low capacities for proteins and other macromolecules. In addition, the extremely high charge density gives very strong binding and the hydrophobic matrix tends to denature labile biological materials. Thus despite their excellent flow properties and capacities for small ions, these types of ion exchanger are unsuitable for use with biological samples. The first ion exchangers designed for use with biological substances were the cellulose ion exchangers (2) . Because of the hydrophilic nature of cellulose, these exchangers had little tendency to denature proteins. Unfortunately, many cellulose ion exchangers had low capacities (otherwise the cellulose became soluble in water) and had

poor flow properties due to their irregular shape. Ion exchangers based on dextran (Sephadex), followed by those based on agarose (Sepharose CL-6B) and cross-linked cellulose (DEAE Sephacel) were the first ion exchange matrices to combine a spherical form with high porosity, leading to improved flow properties and high capacities for macromolecules.

The presence of charged groups is a fundamental property of an ion exchanger. The type of group determines the type and strength of the ion exchanger; their total number and availability determines the capacity. There is a variety of groups which have been chosen for use in ion exchangers (3); some of these are shown in Table 1


**Table 1.** Functional groups used on ion exchangers.

Sulphonic and quaternary amino groups are used to form strong ion exchangers; the other groups form weak ion exchangers. The terms strong and weak refer to the extent of variation of ionization with pH and not the strength of binding. Strong ion exchangers are completely ionized over a wide pH range. Whereas with weak ion exchangers,

the degree of dissociation and thus exchange capacity varies much more markedly with pH. Some properties of strong ion exchangers are:

