**14. Regeneration**

340 Ion Exchange Technologies

**11. Flow rate** 

separated during the

**12. Elution** 

The maximum flow rate that can be applied in any particular ion exchange chromatography step will differ between different parts of the chromatographic cycle. Since low molecular weight substances show high diffusion rates, i.e. are transported rapidly between the mobile phase and stationary phase, the flow rate during equilibration, washing and regeneration procedures is limited primarily by the rigidity of the chromatography media and by system constraints regarding pressure specification. Larger molecules, i.e. the substances to be

Chromatographic run, show a lower diffusion rate which will limit the flow rate that can be applied during sample adsorption and desorption. In a typical capture situation, the flow rate during sample application has to be controlled so that the residence time in the column allows for a complete binding without leakage in the flow through fraction. Maximum flow rate is defined by running the frontal analysis test (break-through) referred to above at a number of different flow rates. Optimal conditions will depend on the requirements for speed and capacity in the system. If speed, i.e. sample application time, is critical due to proteolysis or other detrimental effects in the feed material, a higher flow rate may have to be used on the expense of the binding capacity in terms of amount of sample that can be applied per volume of media. If speed is not a big issue, binding capacity can be increased on the expense of flow

If starting conditions are chosen such that only unwanted substances in the sample are adsorbed, then no change in elution conditions is required since the substance of interest passes straight through the column. Similarly no changes are required if sample components are differentially retarded and separated under starting conditions. This procedure is termed isocratic elution, and the column is said to be developed under starting conditions. Isocratic elution can be useful since no gradient apparatus is required for the run and, if all retarded substances elute, regeneration is not required. Normally, however, separation and elution are achieved by selectively decreasing the affinity of the solute molecules for the charged groups on the gel by continuously changing either buffer pH or ionic strength or

The net charge on a molecule depends on pH. Thus altering the pH towards the isoelectric point of a substance causes it to lose its net charge, desorb, and elute from the ion exchanger (13).

At low ionic strengths, competition for charged groups on the ion exchanger is at a minimum and substances are bound strongly. Increasing the ionic strength increases competition and reduces the interaction between the ion exchanger and the sample

rate which will reduce the scale of work in the final production process (12).

possibly both. This procedure is termed gradient elution.

**13. Change of pH and ionic strength** 

substances, resulting in their elution.

After each cycle, bound substances must be washed out from the column to restore the original function of the media. Ion exchange adsorbents can normally be regenerated after each run by washing with a salt solution until an ionic strength of about 2 M has been reached. This should remove any substances bound by ionic forces. The salt should contain the counter-ion to the ion exchanger to facilitate equilibration. To prevent a slow build up of contaminants on the column over time, more rigorous cleaning protocols may have to be applied on a regular basis
