Chromatography Forum

This question may very well been asked before, I would appreciate if anyone can direct me to the right resource.

I have a reverse phase method running a water/ACN/TFA gradient. The baseline gradually goes up as the percentage of Acetonitrile increased in the mobile phase; however, in the last few weeks, the baseline actually goes down as the percentage of Acetonitrile increased.

We have explored different sources of water and ACN to try to solve the problem, but with no success.

Any suggestion to fix the problem?

This is usually a result of a buffer mixing error - is the TFA in both solvents or are you adding via a mixer?

Thanskk for your reply.

The TFA was premixed in both mobile phases.

Can it be a problem associated with online mixing?

What wavelength?

TFA apparently forms a charge transfer complex with ACN such that the actual absorbance spectrum shifts. If I recall, there is an isosbestic point on the spectrum at 214 nm. See this article by Rozing et al:
http://tinyurl.com/7gg8hht

If your wavelength is off a nanometer or so (just enough to move from one side of the isosbestic point to the other) the direction of the drift could change.

Thanks for your reply.
This may explain it.