Reduction in peak area when using TFA in HPLC-UV

[deborahborg]

I know that with TFA you get ion suppression when you use MS. But why am I getting a drastic decrease in area when im using UV as detector. Conc of TFA was 0.01%

[tom jupille]

What wavelength? What compound? and what was your starting point for comparison?

The easiest way to get an answer is to run UV spectra of your compound with and without the TFA.

[deborahborg]

Hi,

Thanks for replying.

The compound is Emodin and I am analysing it at 254 and 436nm.

I tried running the method first with 0.1% formic acid as the buffer and methanol 10:90. I used this as reference.

Then I substituted the formic acid with 0.1% TFA. I noticed a drastic decrease in area so I tried reducing the concentration to 0.01%TFA. The decrease in peak area was still prominent when using 0.01%TFA yet not as drastic as was observed with 0.1% TFA.

[tom jupille]

Are you seeing the same percentage reduction in peak area at both wavelengths? If you are, then that suggests some loss of the parent compound (I'm not enough of an organic chemist to speculate on a mechanism). If the percentage reduction is not the same, then that suggests that the UV spectrum is changing in the presence of TFA (or as a function of pH). You could check that by running UV spectra under various conditions). My guess is that the spectrum is pH dependent.

On a practical level, that brings up the question: why change from formic acid to TFA?

[deborahborg]

The reduction in peak area is higher at 436 than at 243nm.

So you suggest that I run UV spectra for Emodin at different pHs using TFA? Will that mean that my drug is interacting with TFA causing a change in the spectrum?


I'm performing scouting experiments and I got the best tailing factors using TFA.

Thanks once again

[tom jupille]

So you suggest that I run UV spectra for Emodin at different pHs using TFA? Will that mean that my drug is interacting with TFA causing a change in the spectrum?

That's the best hypothesis I can come up with at this point. You have to compare the spectra to know for sure.