Elution Of Ion Exchange Chromatography- Welch Materials, Inc.

Elution Of Ion Exchange Chromatography

Elution in ion exchange chromatography can be achieved by linear or stepwise salt gradients. It is generally best to start with a linear salt gradient and maintain the gradient for about 10 column volumes. The gradient operation requires two buffers, an equilibration buffer (Buffer A) and a buffer for loading oppositely charged ions (Buffer B). In most cases, the second half of the gradient is not needed because most proteins can be eluted from conventional ion exchangers at 150-500 nmol/L salt concentrations. So, usually a maximum gradient of 50% is sufficient.

Step elution can also be applied in buffers A and B. But retention times are often affected by salt retention and dead volume in HPLC. Also due to the elution characteristics of the buffer mixer, only a sigmoid curve can be obtained. Therefore, an ideal step-elution process suitable for chromatography columns is not obtained. Depending on the salt concentration chosen in the step elution, the protein will move or move behind the salt peak. Yamamoto et al named it as type I or type II elution. After the concentrated eluate is washed out, the chosen salt concentration must be suitable for Type I elution. The salt concentration at this time may be somewhat higher than the maximum salt concentration achieved with the corresponding linear gradient elution. Type I elution requires less than 1 column volume of buffer.

pH gradient elution is more difficult to optimize. Therefore, it is best to first find a condition based on the pI of the protein – when the protein is not bound to the ion exchanger. If it is lower than pI, an anion exchanger should be used, and if it is higher than pI, a cation exchanger should be used. Therefore the pH must be lowered or raised. The ion exchanger itself acts as an acid or base, and the resulting pH gradient follows a titration curve. It is very difficult to obtain a linear pH gradient, but it can be achieved with borate buffers containing glycols such as mannitol. When eluting with a lower concentration buffer of 10 mmol/L, the pH gradient induced by the application of salt can improve the resolution, and it is also beneficial to the concentration of protein peaks. In this case, several column volumes of buffer are also required.