We have an HPLC pump capable of quaternary mixing, but premix the mobile phase and pump through one channel. We performed ten injections of a three component test mix containing uracil, phenol and benzene. The uracil (unretained) peak's retention time gets slightly shorter with each run, but the phenol and benzene peaks don't move. HELP!

Stephanie,

I have found exactly the same thing. I think your results are real. Are you just using organic solvent/water mixes- I find the problem occurs with these. I think there can be other difficulties when you use buffers. I do find however, that the retention time of uracil does tend to stabilise after a few HOURS of equilibration with mobile phase-much longer tha is required for the equilibration of things like benzene and phenol. These things make the whole dead volume consideration even more problematic because they are in addition to the conceptual problem of what you are actually measuring with uracil under a given set of conditions

I know there is some controversy about measuring t-zero and doing k' calcs. This makes me question doing that. I am using a polymer column, and I know that uracil can have some retention on these types of columns, but I would think it would be reproducible.