Chromatography Forum

I am looking for the possible reasons that I should consistently get double peaks with very consistent retention times when analyzing for Co enzyme A compounds (e.g., acyl Co A, malonyl Co A, acetyl Co A, etc.). We are using a C18 column with sodium phosphate and sodium phosphate with acetonitrile in gradient.

thank you in advance for any input any of you may have.

Lots of possibilities.

First question: do you have more than one (expected) compound in the sample and if so, do all the peaks show the same splitting?

If all peaks show the same symptom, the problem probably occurred when they were together (i.e., from the column inlet upstream). If you are using a guard cartridge, change it. If your column can be run in reverse-flow direction (not all columns can!), then reverse it. Check for air bubbles in the injection. If the autosample has been serviced recently, check to make sure the injection valve was reconnected properly. If your sample was dissolved in something stronger than your initial mobile phase, try diluting with mobile phase before injection.

If only some peaks on the chromatogram are split (other peaks on the same chromatogram look OK), then the problem probably ocurred after the peaks had started to separate. In that case look for chemistry problems. Mobile phase composition (especially pH) would be the first place to look, followed by changing the guard cartridge or trying another column. Also make sure that your column heater is functioning properly and that sufficient preheater tubing is installed to allow the mobile phase to come to column temperature. Finally, are you sure that you don't actually have two peaks?

Tom,

Thank you for all your input. I will delve further into my problem with your leads and will, of course, start with the most basic things first. I'll update the forum with my progress.

I suggest to consider mutarotation around the link between the sugar and the adenine.