Chromatography Forum

Hi!

I am new to ion chromatography (using strong anion exchange column for protein separation) and I don't know what is the meaning of adding DTT and CaCl2 to mobile phases? Also, is it important, what buffer do you use (ex. phosphate or tris-hcl) if they have the same pH value (pH 7)? And in most cases, elution is achieved with increasing the amount of NaCl in the mobile phase. Is only Cl- suitable for elution?

Thank you so much in advance for these basic explanations...

Well, DTT is a reducing agent and is useful for keeping proteins with a redox center from changing oxidation states on you (preserve functionality). CaCl2 can be necessary for helping your protein bind to the column (heparin column, etc), can also help to keep your protein in solution.

In terms of choosing your buffer, well, it depends on the properties of your protein of interest.. You choose the buffer with the appropriate pKa for your analysis. Since you say you're using anion exchange, I am presuming that the pH of the buffer is above the IP of the protein. So, you may try other buffers you have on hand that are good at that pH to see if there is a difference in separation. Additionally, the buffer you choose can have an effect on the experiments you are running with the purified protein. This has happened to friends of mine, and they had to add an additional washing step at the end of their purification.

As for the counter ion, Cl- is the counter ion for anion exchange. Na+ is the counter ion for cation exchange.

Check out volume 182 of "Methods in Enzymology." It might be in your school library. It's a good one!

Cheers!
kerri