Chromatography Forum

hi

is there any concept for protein Ion exchnage chromtography, the pH of mobile phase should be + or - one pH of its pI value..??

thanks a lot

In principle yes: you want to be on the correct side of the pI to get retention on the exchanger than you are using. However, what really counts is the surface charge of the protein, and not the pI. You may still get retention even if you are on the wrong side of the pI.

To expand a bit on Uwe's answer: in developing a separation, most people start well away from the pI (below for cation exchange; above for anion exchange) in order to ensure enough ionization to get retention. If you cannot get adequate selectivity any other way, move the pH toward the pI (and in some cases, slightly to the other side). Often, you will get the best selectivity as you get close enough to the pI that retention starts to decrease significantly (of course, you pay the price in poorer robustness).

thanks for the reply...

so from what you are saying if i need to develop a Ion exchange method for a protein lets say of pI 5.6 then to get the best profile my mobile phase must be with pH in range 4.6 or 6.6 (plz correct me if i am wrong)...

Hi Rick1112,

You’re wrong alright. Maybe you should re-read Uwe’s and Tom’s post once again. The answers to your questions are there.
Also, you need to make up your mind whether you you’d like to do anion- or cation- exchange before you move any further.

Best Regards

Rick1112, you are looking for a simple answer to a complex question.

The true answer is that you have no way of knowing the best pH ahead of time (that's why chromatography is an experiment-based technique). To the extent that there is a "cookbook", it would go something like this:

1. (per danko's advice) decide if you want to do anion or cation exchange chromatography.

2. Start with a pH 2 or more units away from the pI on the appropriate side (below for cation exchange, above for anion exchange). That will ensure that your protein has a net charge appropriate for retention. Select an appropriate buffer for that pH. Usually the buffer will be in the 25mM or so range.

3. Run a gradient from 0 - 1N NaCl and see what happens. Trim the gradient range as appropriate.

4. If you need different selectivity, move the pH closer to the pI and try again. (note that a selectivity change may make the separation worse!).

5. Other ways of changing selectivity:
- changing the salt (either the driving ion or the counterion)
- using a different ion exchange packing
- changing the temperature
- adding small amounts of organic modifier.

The above assumes that you are using strong anion or cation exchange. If you are using weak exchangers, then you have to take the pKa of the stationary phase into account, and the situation gets even more complex.