Chromatography Forum

I am trying to resolve a persistent set of ghost peaks in a simple reverse phase method. I am using a Water 2695 with dual wavelength detector at 215nm. Mobile phase A is UHQ, 0.1% TFA, mobile phase B is Acetonitrile, 0.1% TFA. The equilibration phase is 80% mobile phase A, and immediately after injection it starts and up to 50 minutes it ramps up to 100% mobile phase B, then drops back for 10 minutes to 80% A, then back to injection.

The ghost peaks although small (0.004-5 AU) are persistent and unfortunately coelute with a minor component of a drug product I am analysing. I know if I equlibrate for longer durations than specified I can elevate the levels of these ghost peaks on the first injection, on subsequent injection that are consistently present but at a much lower level. When I condition the column (ie: no injection but run gradient) I still get these peaks so my assumption is a minor contaminant in the mobile phase collecting on the column during equlibration then washing off at a sweet spot of organic during the gradient, then building up margainally on subsequent cycles.

I have however, sourced 4 different water sources, 5 different column batches, 4 different acetonitrile sources, attempting different glassware cleaning regimes, purchased in pre-made mobile phases, etc and I cannot shift the appearence of these peaks. HELP !

There are a couple of suggestions in the mini-tutorial on gradient artifact peaks:
viewtopic.php?f=31&t=19085
Look around the 8-minute mark.

Thanks Tom. Bar the suggestion of an in line filter or mobile phase clean up I think I've tried everything. I cannot understand why mutiple sources of water, acetonitrile, TFA, columns and HPLC systems don't seem to effect the appearence of these peaks. One curious observation that I haven't developed a theory on yet, is that when I run the column conditioning with no TFA present, these peaks sharpen rather than flaten, I'd expect the opposite.

I've seen them come from the pH electrode, from residual detergent left behind when glassware was washed, from latex residue when someone got in the bad habit of picking up beakers with their thumb on the inside. Sometimes, it's just easier to trap them out rather than trying to keep them from getting there in the first place.