TFA concetration

[GT]

Dear all,
I'm using a HPLC / DAD method for peptides analysis. This method works with H2O + 0.1% v/v TFA and ACN + 0.088% v/v TFA, with gradient elution from about 50% B to 65% B in 20 min.
With this condition at 215 nm I've got a visible negative drift.
Is it normal?
Why in some articles is the % of TFA in ACN equal to 0.1% and sometimes 0.085% or 0.088%? Are these differences really due to different kind of samples?

Thanks a lot!

[tom jupille]

Is it normal?

unfortunately, yes.

Why in some articles is the % of TFA in ACN equal to 0.1% and sometimes 0.085% or 0.088%? Are these differences really due to different kind of samples?

TFA is, among other things, a weak ion pairing reagent; the amount that sticks to the stationary phase varies with the %ACN. Just to make things more complicated, TFA and ACN form a charge-transfer complex, and the absorbance spectrum of TFA shifts with changes in ACN concentration. The relative amounts of TFA in the A and B solvents can, in principle, be "tweaked" to minimize the baseline drift, but it's hard to get rid of it entirely. How much drift (and in which direction) can vary with the exact wavelength (i seem to recall that the TFA spectra have an isosbestic point at 214) and on the bandpass of the detector.

There was a fairly thorough write-up in LC-GC Europe a few years ago. I think it's still accessible on their web site:

http://www.lcgceurope.com/lcgceurope/ar ... p?id=45019

[peptidemetdev]

Tom did a fine job answering your question, but I had a suggestion for you.

If you have access to a UV-Vis spectrophotometer, do a scan from ~200-400nm or so of 0.1%TFA/H2O, 0.1%TFA/ACN, and a 50/50 mixture of the two.

The interesting thing is that the first two are more or less identical, just shifted a bit due to the different absorbancy of ACN versus H2O. The 50/50 mixture, though, has a "peak" that begins around 205 or so, and extends to about 240 or so. The mixture has to have at least ~40% H2O in order to see that hump, though. This knowledge helped me fix a detector problem once, when the system was selecting something like 213nm when we I had it set to 215nm. It doesn't seem like it's much, but it was enough to make chromatograms using TFA mobile phase look terrible, particularly at high ACN.