we are using reverse phase to separate our goodies. when done in isocratic fashion (25/75 h2o/THF) we find a strong uv/ir peak indicating the presence of our samples. however, when we run a gradient from 50/50 h2o/thf to 100% thf over 15 min we lose the uv peak but still observe the ir peak at an extended retention time. where did the uv absorbance go?

thanx for your help

more details, please:
- what wavelength UV?
- does ir = infrared (with water?) or ir = index of refraction (with a gradient)?

we are detecting at 2 wavelengths: 254nm in the uv and 930nm for ir, infrared to answer your question. we still get ir detection, but the uv peak that we observe in our isocratic method seems to disappear when the gradient method is installed.
still stumped
thanx for the help

I also need more information if you don't mind:

What are the retention times in isocratic and gradient modes?

Are you talking about a single peak or more than one?

What is the primary functional group(s) on your compound which contribute to UV absorbance at 254.

More an aside than anything else, but why use THF/water for reverse phase? THF is pretty hazardous stuff and also not real UV transparent-- its cutoff is around 220 but it still contributes UV background at 254 and will therefore cause baseline shift in your gradient runs.

Fred Klink

I believe Fred may have hit square on the head. A 100% THF MP will give rise to a background at 254nm. What are the concentration's of your analytes? If they are fairly low, the background absorbance from the THF may drown out your signal. Check the reference and sample energy of the cell as you run your gradient. Also, do you see a solvent front? If you don't, you may have your answer.