**6.1. The isoelectric point**

336 Ion Exchange Technologies

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

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,

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

1. If the sample components are most stable below their pI's, a cation exchanger should be

3. If stability is high over a wide pH range on both sides of pI, either type of ion exchanger

2. If they are most stable above their pI's, an anion exchanger should be used.

and an arbitrary range of stability are shown in Figure 4.

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

within the constraints of their pH stability (7, 8).

5-8 and so an anion exchanger has to be used.

**5.1. In summary** 

can be used.

used.

The starting buffer pH is chosen so that substances to be bound to the exchanger are charged. The starting pH should be at least 1 pH unit above the isoelectric point for anion exchangers or at least 1 pH unit below the isoelectric point for cation exchangers to facilitate adequate binding ( 6). Substances begin to dissociate from ion exchangers about 0.5 pH units from their isoelectric points at ionic strength 0.1 M (15). If the isoelectric point of the sample is unknown, a simple test can be performed to determine which starting pH can be used. range of pH 5-9 for anion and pH 4-8 for cation exchangers, with 0.5 pH unit intervals between tubes.
