method development problem

[HW Mueller]

If the two chroms were run with the same moph and really on the same flow rate the area of the analyte should be the same, with and without the column, unless you are coinjecting air or the solvent deviates from mobile phase (or other system interference is present without the column in place). Also the peaks with the column seem to be at to, so also might be system peaks.
What I am trying to say is that such experiments are seldomly taken care of by making single injections, one has to play around a bit, for instance with the length and inner diameter of the capillary which is present when the column is removed.
Nevertheless one can already see that something is crossly wrong here judging from the baseline of the "with column" run. .....Didn´t Uwe suggest that you may have the problem of too much, not too little retention?
One more hint: The peak obtained with the column, if it is at a rt where system peaks are unlikely to hide under it, can not have a higher area than the "without column" peak.

[yanyan]

Just some thoughts - two things I would try if I was you:

1. To extend the PDA wavelength scan further down to 190nm or at least 200nm (in your original post, you went down to 220 nm.) The λ max of ester is 205nm, so I suspect that you might not able to see it at higher than 220nm.

2. To make a run at H2O/ACN 10:90 to compare the resulting chromatogram with the one you ran at 100% ACN. If they are identical, than it means the peaks showed in your chromatogram might just a void peak(s) that from a solvent front; if you see another peak come out later, than that might be what you are looking for.

[Uwe Neue]

You also have two wide and rather flat peaks in your chromatogram, which could come from a previous injection. Anyway, it seems that you have good retention.

[lube chemist]

I have been continuing to work on this problem. I think that the column is OK. My issue now that I would like to resolve is sensitivity. According to a GC-MS that someone else ran, there is about 5% of one of my staring materials remaining in this ester - a long chain carboxylic acid. I can actually see this material easily if I run it alone or intentionally spike a sample with it. It comes out at around 3 minutes with 50:50 ACN/water and a 2ml/min flow rate. I can see it with both the PDA and the RI detectors. However, I cannot see it in the unspiked sample, even though according to the GC-MS it is in there at 5%.

I'm pretty sure that the people who ran the GC-MS did not run the actual acid compound as a comparison and are just going by the MS, so it is possible that there is an error in interpretation. I should be able to detect an impurity at the 5% level, right?

FWIW, I also still can't see the ester for sure. I also keep having random peaks with lambda max at around 260 - I think it may be some leftover fluorinated xylene that I was using as a GPC solvent before installing the HPLC column that may be stuck in the lines or pump heads.