Chromatography Forum

Please could somebody explain to me (if possible) why this situation.
I inject 4 compounds in mobile phase, using Micromass quattro ultima in ESI +
Retention time of my first compound and its internal std is approx 4 mins. The retention time of next analyte is approx 7.5 mins and its internal standard is about 8 mins. The thing is that the injections now seem to show that in each TIC for each compound a peak at about 1.3 mins exactly but no peaks for the other compounds. Whats even stranger is that if I reinject, there will be no peak at 1.3 mins and very poor quality peaks at the proper retention times. Whats going on???

What are your LC conditions? Had anything about your sample or conditions changed when you first noticed the problem? What do you mean by "poor quality" peaks?

My conditions are
Mobile phase flow rate (0.25 ml/min
Binary gradient run with 30% B (95% organic) at time zero linearly risen to 90% B at 8 min. Then, the 90% B and 10% A are held for 0.5 min, decreased to 30% B over 0.5 min which is held again for 3 min until next injection.
Mobile phase composition: A
95:5 Water:ammonium buffer
Mobile phase composition: B
95: 5 Acetonitrile:ammonium buffer

Nothing has changed, last night ran standard 5ng/ml in acetonitrile and it gave the 1.3 min retention time for all four compounds in all channels.
Today I repeated the injection, every single thing the same, no changes and lo and behold the injection was as it was meant to be all retention times where they would be expected. Is it worthwhile taking out the column and doing loop injections with isocratic mobile phase to rule out mass spec. This instrument is used for other assays aswell and it appears to be performing well for these assays, so ??? I am looking at transitions of m/z 208 - 357.
It appears to be an intermittant problem. Has anyone seen anything like this

It doesn't sound like the MS is the problem. Your problem could be caused by injection solvent mismatch. If your samples are dissolved in mobile phase A, then that should not be the problem. It could be that the column was not properly equilibrated to initial conditions, when you saw the short retention times. Try reinjecting your standard about 5 times in a row and observing the retention times. The first injection might have different retention times, but the remaining injections should give reproducible times. If not, then you may not be equilibrating your column for long enough. With gradients, I make it a habit never to use the first injection, because retention times will never be the same.

Ok, sometimes I tie these injections in at the end of another assay which uses the same gradient. So the column is well equilibrated. I have been told to remove the column and do loop injections to identify that the ms is not the issue. I do not expect that to yield anything since other assays are working well.
Thanks for all your help already MG