I am seeing three fairly sizable peaks in my gradient chromatography, which are also present in my blank injections. My first gradient system contained acetic acid, the second acetic acid + TEA, and the third phosphate buffer + TEA, and these peaks appear in all 3 systems. We are using water from a Milli-Q system and have not seen this problem before. Could this be some kind of soap residue left in the mobile phase reservoirs when they went down for cleaning? If so, how can I get rid of it? If this isn't it, what are some other likely sources, and what would be a good approach for eliminating these peaks? I'll take any suggestions!! Thanks--JV

What is your blank injections, mobile phase or sample solvent? What is your sample solvent? Have you tried to use bottled water (Fisher)? Contaminated water would be my first suspect.

What kind of column are you using?

My blank injections are sample solvent, which is 70/30 MeOH/water, and my column is a Zorbax Eclipse XDB-C18. I haven't tried bottled water yet, but that is the next thing I intend to look at, since it's the only common component in the three mobile phase systems that I mentioned.

Have you thought about your vials as a source of ghost peaks?

A couple of quick-and-dirty diagnostic tests may help here:

1. If it's possible on your system, run a "dummy" gradient (no injection at all). If the garbage peaks disappear, then vials or injector are the probable source.

2. If the garbage peaks still appear, you have exonerated the vials and autosampler, and so must focus on your mobile phase and system. Run a series of 3 dummy gradients separated by increasing equilibration times in between (e.g., gradient, 10-min equilibration, gradient, 30-min equilibration, gradient) and compare the size of the garbage peaks in the last two gradients. If the garbage peaks are larger after longer equilibration, then you have pinned the source down to the A solvent.

3. Run another dummy gradient with no buffer or TEA in the A solvent. If the garbage peaks disappear, then those represent your source of contamination.

3a. Also consider the pH electrode; if you are adjusting pH with the electrode actually in the mobile phase try again pulling an aliquot of the mobile phase for pH measurement and then discarding it.

4. After you've eliminated all of those, the remaining possibilities are the water system, residual detergent in your reservoirs (as already suggested), or dust floating in from the atmosphere (don't laugh, it's happened!). To pin down, first try a bottle of HPLC grade water, the try another gradient with super-well-rinsed glassware, and make sure you have a dust cap on your reservoir.

And let us know what you find so that we can all learn from it!

A problem that we encountered at one point was the plasticizer(s) in a piece of Tygon tubing at our water outlet. Since that time, this is the first thing I check, when strange peaks occur.