I've a problem that is frustrating the heck out of me. Would appreciate input from experienced gradient chromatographers.

Using water/AN gradient I continually get ugly lumpy baselines and broad/ill-formed "peaks" that seem to elute above 40%AN and near the end of a gradient or during re-equilabration. I use 10-15 col volumes for re-equilibration. I have them in both high and low pressure mixing systems and with variety of water/AN grads.

Currently having problems with these "peaks" at 220nm with 5% to 55%AN gradient. Using hplc grade purchased water. Low conc. analytes are giving peaks 0.06au while "ghost peaks/lumps" are 0.02--0.06au. Blank injections and 0uL inj give same results. Analytes are quite pure and dissolved in hplc water. Running gradient with no column, only ZDV union and 0inj shows no problem.

Found some correlation with cleanliness of filteration flask. Scrupously cleaning w/Micro, rinsing with DI water, AN, and drying at 150C in oven helps but for no more than one batch of mobile phase/day. Am now using plastic beaker 1x with no filteration only degassing online while trying to id source of problem. Are these organics in the hplc water that are being eluted with each gradient? Would commercial hplc water be that dirty? Flushing column with AN shows some absorptions flushed off column but seems not to help; first injection after equilibration has larger peaks than next injection suggesting mobile phase contamination. But how do I clean it up?

What should I expect for baseline at 220nm with this gradient? I see published chromatograms of my analytes showing only slight uniform rise in absorption for similar gradients and sensitivities.

Thanks guys/gals.

It would seem you are getting impurities from the mobile phase onto the column and when the AN concentration gets high enough, they are washed off. These typically are from the water though we have had problems with AN as well. Do you use fresh solvents every day or do you refilter or top off a reservoir? I suppose you could try running the water through a solid phase extraction (SPE) cartridge to see if that helps. Any impurities concentrating on your analytical column should be trapped by the SPE cartridge. If you are filtering your solvents, you might check out the filter. The contamination might be coming from the filter. We have received improperly labeled filters. Just a few suggestions, there will probably be more coming.

As was mentioned in the previous message, the cause of problem such as this is almost always contaminated water. In my opinion purchased HPLC water is usually of a lower quality than water produced through a high quality water system. I have seen microbial growth in bottled water which can cause ghost peaks. I also saw microbial growth one time in the aq. solvent line. In regards to solvent filters, some brands and and some filter types can also introduce contamination. Tracking down these types of problems can be a challange, I wish you luck.

If you are using a "high-pressure-mixing" (2-pump) gradient system, you can also put a C18 guard cartridge between the A pump and the mixer to strip out UV absorbing impurities before they get to the column. Check the John Dolan troubleshooting column in March '96 for an example [Dolan et al.,LC/GC 14(3) 202 (1996)].

-- Tom Jupille

I agree with the comment about bottled water. Another caution is exposure of the mobile phase to anything polymeric. Only use glass containers and once you have that container clean don't use any detergents or automatic dishwashers. If you are still having problems, switch to a different instrument and column to help identify the source of the problem. Alltech makes filters to clean-up mobile phases:
http://alltechweb.com/literature/brochure/404p11.pdf

Thanks everyone, you've been a big help and I appreciate your taking the time to respond.