I'm sure the solution to this problem is simple, but I'm baffled: I'm detecting at 198 nm. The run I've programmed is 60 min at 65% B, 5 minute gradient to 90% B, 5 minute hold at 90%, then 10 minute reequilibration at 65%B. Everything was fine with just A=H2O and B=ACN, but I wanted to improve the resolution of a few compounds so I went to A=H2O+0.1%TFA and B=ACN+0.1%TFA. Resolution is improved, but the baseline is a little noisier and it PLUMMETS during the acetonitrile cleanup, and then returns during the reequilibration. I reprepped both the aqueous and organic and still had the same drop. Suspecting an impurity in the aqueous, I reprepped it from a different water source and expected a drop in absorbance at 65% B, but there was none. Previous work on another method using ACN:H2O:0.1%TFA showed a continuously INCREASING baseline with increasing organic, so this is the opposite of what I would have expected. It shouldn't be the TFA because the H2O and ACN have the same amount. Does anyone have an idea of what's going on? Thanks.

TFA forms a charge-transfer complex with ACN with the result that the UV absorbance spectrum varies with concentration of ACN. Therefore, even if TFA concentration stays the same, you will see absorbance changes as the ACN concentration changes during the gradient. See LCGC, 5(12),1044(1987) for more details.

-- Tom Jupille / LC Resources Inc.

Its also related to your UV wavelength given the concentrations of organic and TFA. At the wavelength that you're using, you may try putting more TFA in your B phase (i.e. 0.1% TFA in A and 0.2% TFA in B). This may make your baseline rise instead of fall.