Chromatography Forum

Hello there!
I have a question regarding stereoisomers that coelute in conventional reverse phase HPLC. We usually see one peak, but one of my injections actually show a split peak corresponding to a stereoisomer and I was wondering what conditions in the HPLC system could have caused such slight separation. I have a pH regulated mobile phase (pH=2.6 with sodium phosphate buffer). This seems to be an isolated case because the previous and following injection (of a standard that also contains the stereoisomer) do not show the split peak. Actually, this is the first time we see this situation. The peak elutes about 2.8 min.
Any ideas or suggestions?
Thanks,
MOchoa

How can you be sure that the split peak is actually stereoisomers? Are you doing LC-MS to confirm this? It is unlikely that the first injection gives one peak, then the next injection is split, then it is back to one again. Is it possible that you have a contaminant in one vial?

Hello and thank you for your reply. No, we didnt do MS on it. It's just that this component has a stereoisomer and I thought something in the chromatographic conditions could have made the peaks slightly to separate. It can be also a contamination but we'd never know because the vial could not be re-injected to try reproducing the peak. I was trying to think in all possibilities. Any other idea?

Unless this problem can be reproduced it is probably a contaminant - look at the vial, glassware, and anything else in contact with the solutions.

If the problem comes back, then we can look at the pattern and figure it out, but a one time failure is almost impossible to troubleshoot.

based on your short retention (although I don't know length of the column and flow rate) I would suspect that you have something in your sample or mismatch between mobile phase and your sample diluent. Sometimes it is possible to separate stereoisomers on mixed-mode column:

quinine/quinidine http://www.sielc.com/compound_163.html
isoleucine http://www.sielc.com/application_009.html

Regards,

Vlad

If your compound has more than one chiral center, it's very possible that conventional RP-HPLC can give resolution to diastereomers, but for enantiomers, I didn't find a case though I have done a lot of chiral separations, besides, Vlad's reference supports this point.