I am having an issue with ion exchange chromatography. The material has been eluting later into the gradient and in some cases fully into the 100% high salt wash. Some things that have been studied,
Buffer conductivity has not change
instrument pumps are flowing and mixing as intended
pH has not changed
molecule has not changed
Only item that has changed is a new lot of the ion exchange media. I have contacted the vendor but have received the usual "nothing has changed on our end"
What is a way where I can test the media and the retention of the resin since it seems like that is the only variable in this case? An older media lot was used and the material eluted as expected.
Proteins are binding with ion exchanger too strongly. Check if buffers are in the correct containers. If you are using an anion exchanger decrease buffer pH and if cation exchanger increase buffer pH
It is weird that you are indicating only the batch/lot changed and you are obtaining the expected chromatogram using an older one. Are you applying the recommendations based on regeneration, initial conditioning, and storage of the resin that is supplied by the vendor?
If everything same (analyte, buffer system, method, sample composition, sample preparation etc.) it is reasonable to accuse the column but sharing the chromatograms, brief info about the method, and resin properties may enlighten the way of troubleshooting of this tough problem.
Dear Julie Wold
A good way to prevent hiccups like this is to perform the test procedure suggested by the manufacturer. This will allow you to either identify issues with the resin performance or help you to focus on other issues.
In one case - a very long time ago - we were able to run separations on older columns, whereas the same resin in a new column didn't work to our expectations. It turned out that we "aged" the older column running different samples and matrix components changed the selectivity... Unfortunately, we could not reproduce the aging process and had to start developing a method from scratch using a brand-new column.
Hope this helps
Good luck with your research
Detlef.
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