Chromatography Forum

I had different problems with the following:

mobile phase: methanol/water/glacial acetic acid (HPLC grade)
inj volume: 100µl
flow: 0.8ml/min
fluo: exc: 330
em: 460
When I injected an Ochratoxin standard of 1 or 10ppb dissolved in pure methanol, the baseline was not stable and after the dead volume there was a ghost peak. No one could help me, but one person told me that it was due to the pure methanol in the standard solution. I could solve the problem by dissolving in mobile phase, but that's not a very good option. Does anyone have an idea/link/prove of methanol being fluorescent? Another lab uses the same method but doesn't have the problem of the unstable baseline, how can i solve the issue (without having to use mobile phase to dilute samples and standards).

Thanks for your help!!
Olivier

First of all, test this hypothesis by injecting 100µL of your methanol and seeing if the problem happens in the blank.

What is your baseline offset? Zero your detector with pure water or acetonitrile, then switch to your mobile phase. If the baseline increases significantly, you have a problem with dirty mobile phase. In this situation, the "dirt" is in equilibrium with the column, and the injection of pure methanol upsets that equilibrium, with a bad baseline as the result.

Actually, using mobile phase for the injection solvent is the preferred practice unless there is some specific reason not to.

Methanol quality is variable between vendors since fluorescence is not usually a specification. Even reliable vendors have the occasional bad lot. (Personally I like Fisher Optima, but there are other good brands too.)

I already tested it with pure methanol, and indeed, this was the case...but I also tested with acetonitrile and it gave the same result. Offset is about 0.2 LU. Thanks for the suggestion, I will try to zero the detector with water tomorrow!
I also injected the mobile phase itself, and this gave no problem, only a constant offset from zero, but no significant peaks. So, my next question: does that tells me something about the purity of my mobile phase or is a constant offset possible for a pure phase also?
I will also try other methanol...the same method works well in another lab, and the only thing different is probably the brand of solvents!
Thanks in advance!

The baseline rose and stayed up after you injected mobile phase? There could be two obvious things causing this: You injected enormous amounts of "dirt" (fluoresces or scatters light) which slowly comes off the column, or you injeted air. Here a suggestion analogous to Mark´s: Inject different amount of air.......