I am using a chiral CD (cyclodextrin) column to analyze 1,1'-Binaphthyl-2,2'-diyl hydrogen phosphate (BNP) enantiomers with the mobile phase composed of ammonium formate buffered water and acetonitrile. The retention time of both enantiomers was consistent over the last several weeks.
After my colleague ran his dye samples (Rhodamine B) through another column last week, since he couldn't see the rhodamine B peak in the chromatogram, I suspect at least a small amount of rhodamine B has got into the chiral CD column because my sequence was queued right after his.
From then on, the retention time of the BNP enantiomers is very inconsistent while the other initial injection volume peaks are always consistent. For example, let's say I run 30 samples, the elution time moves downward for the first 5-10 samples and then suddenly moves upward for 5-10 samples and then again move downward. SO, I'd say the elution time change is not completely random but keep moving back and forth. I tried regenerating the column and also using reverse flow to remove any particulates for 2-3 days, but I couldn't fix this problem.
Here are some reasons that I suspect may be causing this issue:
1. Intermittent interaction of BNP with rhodamine B dye remaining inside the column
2. Sample matrix interference or impurity in my samples (not likely because it never happened before)
3. Build-up of the sample matrix over the sequence (not likely because pressure doesn't increase or change. and no noisy baseline and ghost peaks found)
4. Inconsistent pressure or mobile phase composition due to the instrument malfunction (not likely because the initial injection peaks except the BNP enantiomer peaks stay the same)
Could you provide your opinion on this and maybe the most suspicious reason?
You should proceed a very good rinsing of an opened column starting with warm ultrapure water for at least 30 min after that with Acitonitrile also for 30 min
and you should check the pressure of the apparatus it should be steady during the injection , if not you should check for air bubbles ( usually if there is air bubbles in the injector the retention time won't be steady ...)
and you should check the mobile phase , either the buffer solution is not well prepared or the acitonitrile was evaporated during the preparation ( in very small amount but it is important ) ; or the fraction of the mobile phase you prepared should be checked ( either you put Acitonitrile more then it should be or the buffer the solution more then it should be )
Hope my answer would help you , wish you much of luck
Best regards
If your peak symmetry is fine but your retention time is increase and decrease, you may have a problem with your injector. Injector should be checked, and the line should be purge.
The answer will be found through proper troubleshooting of the complete LC method and system. Here is a link to a free article which addresses your question;
- "HPLC Retention Time Drift, Change, Area Variability or Poor Reproducibility. Common Reasons for it"; https://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
Most likely, the column is fouled/damaged (note: "pressure changes" are not a requirement to indicate this). Cyclodextrin columns are fragile. However, there are many other commons reasons for your observations too. Please have someone local who is experienced in using your specific HPLC and has many years of method development experience assist you through the troubleshooting steps to find the problem(s).
Here is another article link here on RG for the same article; Research Common Reasons for HPLC Retention Time Drift, Variation or Change
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