all of a sudden, no elution

[seraphin]

Hi, guys
I am a newbie to UPLC/HPLC (<3 wk experience). Starting to run a new assay to quantitate a compound using fluorescence detector (exc: 370nm, em: 460nm). Using 80:20 (0.2% formic acid, 100% methanol) solvent system.
My compound used to elute at 6 min run (for week 1). All of a sudden, it stopped coming out throughout the 12 min run cycle during the past 4 days.
I thought I screwed up the system, but when I ran a standard compound (exc: 405nm, em:619nm), the standard ran fine (under 92:8 (ammonium acetate, acetonitrile)) so I assumed the fluorescence detector/lamp is fine.
I might have prepared a 2% formic acid, instead of the 0.2% formic acid solvent. Is it possible that 2% formic acid could screw up my elution? If not, what could other possibilities be that all of a sudden I am getting no elution.

terrified as the samples are precious and I could not keep testing the system using the samples. Any suggestion will be greatly appreciated. Thank you very much.

[tom jupille]

I might have prepared a 2% formic acid, instead of the 0.2% formic acid solvent.

That should be easy to determine. Check the pH of your mobile phase then prepare a fresh batch with 0.2% formic acid and check the pH of that.

[danko]

If the mobile phase contain 2% formic acid instead of 0.2%, it is possible that your analyte is being destroyed upon injection. Provided the compound is pH sensitive anyway.
Besides, one should remember that fluorescence is highly pH dependant.
Drastic pH changes can quench the fluorescence or at least cause wavelength shift - either of the excitation or emission wavelengths. Or both.

Best Regards