Chromatography Forum

I'm looking for any and all ideas, cuz I'm out of them.
Instrument: Agilent 1260 with ELS-D

Runs one method for one product 99% of the time, a standard 40-95 gradient of ACN and Water, both 0.1% formic acid modified. Reverse phase, C18 column.

Product we test is a standard mixture of 7-12 peaks, all identified via pure standards and quantified via calibration curves. Method has run with great repeatability and reproducibility for over 1 year.

~3 weeks ago, all retention times shifted ~5.5 minutes downfield with no explanation. No change to column, plumbing, operating pressure, solvent, etc... Fine, I rerun calibration curves based on new retention times, method runs under new retention times until today, when the peaks shifted BACK 4.5 minutes, nearly to where they were originally. I am at a complete loss. I'm looking for any and all ideas, no matter how crazy. Ghosts? Sabotage? anything,

This should be easy to solve. The two most likely reasons for the retention time to change are:
(1) Your mobile phase composition has changed (IOW: It is not what you think it is due to programming, hardware defect, evaporation etc) OR (2) your flow rate has changed (always measure it using a timer and graduated cylinder. Do not rely on the instrument's displayed values).

Note: A common crossflow leak where solvent composition is mixed might be the most likely reason. As the seals dry out from lack of use (always use ALL of the channels in your HPLC system over time), a leak can generate from one channel to another changing the % composition. It can close back up too, depending on the solvents which are in the lines and in contact with the valve seals.

Could you share with us exactly which HPLC pump you have ? Agilent 1260 is just a series #, not a model #.