**4. Resolution in ion exchange chromatography**

The result of an ion exchange experiment, as with any other chromatographic separation, is often expressed as the resolution between the peaks of interest. The resolution is defined as the distance between peak maxima compared with average base width of the two peaks. Elution volumes and peak widths should be measured with the same units to give a dimensionless value to the resolution (4, 5). The resolution (Rs) is determined from the chromatogram as shown in Figure 3.

**Figure 3.** Determination of the resolution (Rs) between two peaks.

Rs is a measure of the relative separation between two peaks and can be used to determine if further optimization of the chromatographic procedure is necessary.

### **4.1. Capacity**

334 Ion Exchange Technologies

macromolecules.

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

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

Diethylaminoethyl (DEAE) -O-CH2-CH2-N+H(CH2CH3)2

Quaternary aminoethyl (QAE) -O-CH2-CH2-N+(C2H5)2-CH2-CHOH-CH3

Sulphopropyl (SP) -O-CH2-CHOH-CH2-O-CH2-CH2-CH2SO3 Methyl sulphonate (S) -O-CH2-CHOH-CH2-O-CH2-CHOH-CH2SO3

completely ionized over a wide pH range. Whereas with weak ion exchangers,

Quaternary ammonium (Q) -O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N+(CH3)3

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

the degree of dissociation and thus exchange capacity varies much more markedly with pH.

Sample loading capacity does not decrease at high or low pH values due to loss of

A very simple mechanism of interaction exists between the ion exchanger and the

Ion exchange experiments are more controllable since the charge characteristics of the

The result of an ion exchange experiment, as with any other chromatographic separation, is often expressed as the resolution between the peaks of interest. The resolution is defined as the distance between peak maxima compared with average base width of the two peaks. Elution volumes and peak widths should be measured with the same units to give a dimensionless value to the resolution (4, 5). The resolution (Rs) is determined from the

**Anion exchangers Functional group**

**Cation exchangers Functional group** Carboxymethyl (CM) - O-CH2-COO

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

Some properties of strong ion exchangers are:

media do not change with changes in pH.

**4. Resolution in ion exchange chromatography** 

charge from the ion exchanger.

chromatogram as shown in Figure 3.

solute.

The capacity of an ion exchanger is a quantitative measure of its ability to take up exchangeable counter-ions and is therefore of major importance. The capacity may be expressed as total ionic capacity, available capacity or dynamic capacity. The total ionic capacity is the number of charged substituent groups per gram dry ion exchanger or per ml swollen gel. Total capacity can be measured by titration with a strong acid or base. The actual amount of protein which can be bound to an ion exchanger, under defined experimental conditions, is referred to as the available capacity for the gel. If the defined conditions include the flow rate at which the gel was operated, the amount bound is referred to as the dynamic capacity for the ion exchanger. Available and dynamic capacities depend upon: The properties of the protein. The properties of the ion exchanger. The chosen experimental conditions. The properties of the protein which determine the available or dynamic capacity on a particular ion exchange matrix are its molecular size and its charge/pH relationship. The capacity of an ion exchanger is thus different for different protein.

### **5. Choice of exchanger group**

Substances are bound to ion exchangers when they carry a net charge opposite to that of the ion exchanger. This binding is electrostatic and reversible. In the case of substances which carry only one type of charged group the choice of ion exchanger is clear-cut. Substances which carry both positively and negatively charged groups, however, are termed amphoteric and the net charge which they carry depends on pH (Fig. 4). Consequently at a certain pH value an amphoteric substance will have zero net charge. This value is termed the isoelectric point (pI) and at this point substances will bind to neither anion or cation exchangers (6). The pH ranges in which the protein is bound to anion or cation exchangers and an arbitrary range of stability are shown in Figure 4.

**Figure 4.** The net charge of protein as a function of pH.

The pH of the buffer thus determines the charge on amphoteric molecules during the experiment. In principle therefore, one could use either an anion or a cation exchanger to bind amphoteric samples by selecting the appropriate pH. In practice however, the choice is based on which exchanger type and pH give the best separation of the molecules of interest, within the constraints of their pH stability (7, 8).

Many biological macromolecules become denatured or lose activity outside a certain pH range and thus the choice of ion exchanger may be limited by the stability of the sample. This is illustrated in Figure 4. Below its isoelectric point a protein has a net positive charge and can therefore adsorb to cation exchangers. Above its pI the protein has a net negative charge and can be adsorbed to anion exchangers. However, it is only stable in the range pH 5-8 and so an anion exchanger has to be used.

### **5.1. In summary**

