Chromatography Forum

Hi all,
I am running a method to analyse simultaneously two compounds and using two analogues as respective internal standards (ISs). I tried four different C18 columns (same types/brands) and found out that retention times of one analyte and 2 ISs are similar when using different columns and very stable between days. The other analyte behaves very weirdly: different retention times among different columns and with one column, it also show different retention times on different days. My mobile phase is gradient 0-100% B within 2 mins with A 0.1% formic acid in 5% ACN/water and B 0.1% formic acid in 95% ACN/water. One analyte and its IS always have retention time round 1.7 mins; the other IS elutes at 1.4 mins and the weird analyte elutes from 1.4 to 2.1 mins when it supposes to elute around 1.4 mins as its IS.
Highly appreciate if you have any suggestion to fix this retention time changing.
Thank you!

Those are short retention times times for a gradient method, that's for sure. Maybe you left out details like UPLC or something. By chance is this a USP procedure?

Anyway, easiest thing to try is to lengthen the equilibration time like 5 minutes and see if that helps.

Thank you for your reply.
Sorry for not providing full info, yes my run last until 4 mins with 2-2.5 mins at 100%B then 2.6-4mins equlibration at 0%B before new run. Looking at column pressure, I believe that column is equilibrated enough and also the other 3 compounds elute consistently, only the trouble one keeps moving.....

You did not provide enough details about the column (perhaps one of the most important parameters in HPLC). What are the exact column dimensions? What is the flow rate? These basic values are needed to understand any HPLC method (not "UPLC" which is the wrong term to us).

Some reasons for your issue might be: poor equilibration (too short); inappropriate column wash procedure; not enough retention (K prime too small); poor mixing or degassing; temperature; pH problem or just a bad method. Any one of these could effect one peak or all of them.

Here is an article which may help you. "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it"; https://hplctips.blogspot.com/2015/11/h ... hange.html

Those are short retention times times for a gradient method, that's for sure. Maybe you left out details like UPLC or something. By chance is this a USP procedure?

Anyway, easiest thing to try is to lengthen the equilibration time like 5 minutes and see if that helps.

You don't state what your flow rate and column dimensions are. So better to estimate equilibration using 10 column volumes, not time. Pressure stability is not a good way to determine equilibration.

There is not enough information to give accurate advice as posters above me noted. It's helpful to post a chromatogram and name the compounds you're working with.

I'll add a guess: it's possible that the compounds that are shifting are phrone to slight changes in pH while the others are not. In that case you should move towards using a buffered mobile phase instead of simply adding formic acid.

Thanks all for your suggestion. I am going to have a couple of tests with buffering mobile phase and extending the equilibration time. I will keep you posted!