Hello, inexperienced grad student here. I've been processing samples via HPLC/LCMS (tissue extractions from plants) over the past year or so. We have two mobile phases that we use in a shared HPLC machine: Phase B: Acetonitrile with 0.1%Formic Acid, and Phase A: Water with 0.1%Formic Acid. I recently put on a run and was rushing a bit, didn't fully check the labels of the solvents before leaving and when I came back I realized that the Water that was attached to mobile phase A tubes had 0.1% ammonium acetate added, not FA.
Can I search for the compounds by mass and adjust that way, or are there other considerations I haven't taken into account? We use a 10.5 min run time per sample, and all of the compounds elute within 6 min when formic acid is used.
Our samples are extracted and then suspended in methanol.
Hi Zoe,
So, your water (mobile phase A) contains 0.1 % acetonitrile and no formic acid? And does your acetonitrile (mobile phase B) contain 0.1 % formic acid, or did you also add acetonitrile to that?
I don't expect the 0.1 % acetonitrile will affect your chromatography, I'm more worried about the absence of formic acid, as this is both an ion pairing agent impacting chromatographic resolution as well as an acid that aids ionization of your analytes that is required for proper mass spec analysis.
So, if something is impacted by this little mix up, I think it will be your mass spec data, not your chromatography.
Hope this helps!
Eef Dirksen Thank you so much for your response! so, the plot thickens....it wasn't acetonitrile, it was ammonium acetate that was strangely labelled....yikes! Formic acid was added, although only to the acetonitrile mobile phase B. Mobile phase A (updated): Water+0.1% ammonium acetate. Mobile phase B: Water+0.1%FA. The joys of shared equipment! I'll take a look at the data and try to see if it affected something soon, the peaks looked a little less distinct from one another on an initial glance but I didn't have access to the qual browser at the time
Dear Zoe Economos,
First of all, you can use amonium acetate in the eluent. Other buffers like Phsophate buffer would not be suitable for LC-MS as they leave non-volatile salt deposits in the ESI sprayer.
The important aspect in your case is the pH of the eluent. By replacing formic acid with ammonium acetate, you buffer the eluent and your pH changes from about pH 2.5 -3 to pH 3-4.
This can of course change retention times if your analytes react pH dependent (neutral molecules are mostly not affected so much). Normally buffers in the eluent do not affect the mass spectra of the analytes, they essentially serve, as in the case of formic acid, to protonate the analytes or in the case of a buffer such as amonium acetate to better chromatograph pH-dependent analytes. As long as your analytes elute in the "normal" range this should be ok for you. With the mass spectra as safety, you can safely assign the peaks.
Many greetings
Joachim Horst
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