**6.2. Choice of buffer**

As with the choice of ion exchanger, there are a number of variables which have to be considered. These include:


It should be pointed out, however, that in many applications the optimum separation may be achieved by choosing conditions so that major and troublesome contaminants are bound to the exchanger while the substance of interest is eluted during the wash phase (9). This procedure is sometimes referred to as "starting state elution".

*Note:* Concentration of sample does not occur with starting state elution.

The highest ionic strength which permits binding of the selected substances and the lowest ionic strength that causes their elution should normally be used as the starting and final ionic strengths in subsequent column experiments (i.e. the starting and limiting buffers for gradient elution). A third and higher ionic strength buffer is frequently employed as a wash step before column regeneration and re-use.

The required concentration of the start buffer will vary depending on the nature of the buffering substance. In the majority of cases a starting ionic strength of at least 10 mM is required to ensure adequate buffering capacity. Salts also play a role in stabilizing protein structures in solution and so it is important that the ionic strength should not be so low that protein denaturation or precipitation occurs.

### **6.3. Choice of buffer substance**

If the buffering ions carry a charge opposite to that of the functional groups of the ion exchanger they will take part in the ion exchange process and cause local disturbances in pH. It is preferable, therefore, to use buffering ions with the same charge sign as the substituent groups on the ion exchanger. There are of course exceptions to this rule as

illustrated by the frequency with which phosphate buffers are cited in the literature in connection with anion exchangers. In those instances when a buffering ion which interacts with the ionic groups on the matrixnis used, extra care must be taken to ensure that the system has come to equilibrium before application of sample.
