Chromatography Forum

Hi!

I am fairly new HPLC and very new Ion Exchange HPLC. I am attempting to develop an IEX method for a protein (about 64kD) at a pI of 4.0. I first went with a weak anion exchange phenomenex column (Shodex IEC DEAE-825 8mmx75mm) and for the mobile phase I was using 20mM piperazine pH 5.0 and buffer b was the same thing plus 1M NaCl. For the gradient I started with going from 100% A to 100% B for 60 minutes at a flow rate of 0.5mL/min. And I got a peak at 3 minutes. I have tried changing a few things such as going to a strong anion exchange (also by phenomenex IEC QA-825 same dimensions) and tried going down to 5mM piperazine, and the peak moved alittle to about 5 minutes I also tried bis-tris and that didnt work either. The bottom line is that my boss is not comfortable with an assay where the peak comes off in 5 minutes, he would prefer to see it come off at about 10-15. Is this a common retention time for ion exchange? Is there something else I can do increase the retention time? Thanks in advance for your help!

How about increasing the pH of your mobile phase so that you will further charge negatively your protein?

I tried bumping the pH to 6.5 with the bis-tris, should I go higher than that. I read for anion exchange that the pH for the mobile phase should be 1 pH unit above of the pI of the protein. Is this not true?

Did you try the 6.5 buffer? In general we are working with mixtures of proteins so we are can not optimize the conditions for just one protein.

KAR10 wrote/asked:

I read for anion exchange that the pH for the mobile phase should be 1 pH unit above of the pI of the protein. Is this not true?

It is definitely not true. Maybe you read at least 1 pH.…… and this should be more acceptable. Actually I recommend (when asked) 2 - 3 pH units. And yes you can try pH 7.0 (maybe phosphate buffer) if 6.5 wasn’t high enough.
Another optimization parameter I will suggest to you is reducing the gradient steepness. You can either run a gradient from 0 % to 50 – 60 % B in 60 min or maybe reduce the NaCl concentration to, let’s say 0.5 M.
The latter would be my preferred option. You see, the more salt (especially chloride) you introduce to the HPLC system/the hardware the faster corrosion of especially the pumps you will experience.

Good luck to you

I will try both of those suggestions. Thank you for clearing up my misconception!