Chromatography Forum

Hi there,

I’ve been wondering about something: When I went to school I learnt that TFA absorbs more light/energy the more organic modifier there is in the mixture and fortunately this is true – the typical baseline appearance in TFA/Water/ACN gradient is rising the more ACN is mixed in the flow. But I’ve seen a lot of examples where the baseline drops drastically when the ACN concentration reaches approximately 90 % and above (usually in the rinsing step, before re- equilibration accures) despite of the TFA’s concentration is constant e.g. 0.1 %.
I thought this was a refractive index thing, but then I checked the RI of ACN and water and they are 1.344 and 1.333 respectively.
Does anyone have any thoughts on this subject?

Best Regards

Hi danko,

I've seen this sudden dip from baseline before (I'm sure many others have seen it too), but thought it might be an impurity absorbing differently or that the mobile phase composition at that point in the gradient causes the impurity to adsorb differently. I also thought the sudden negative peak is called an eigen peak...or something like that.

Be interesting to see what others have to say.

Cheers.

Hi again

Actually I didn’t pay that much attention to this phenomenon, until I posted the question.
But the thought has been haunting me the last several hours and it begins to worry me.
I was sure somebody wrote back and offered a perfectly beautiful explanation, but I guess I’m rather impatient.
So I’m thinking: Could it be some kind of normal phase effect – the solvation of TFA drops down dramatically at a point where there is too little water in the flow and the TFA “choosesâ€

I believe that TFA forms a charge-transfer complex with ACN, so that the actual spectrum (not just the absorbance) shifts as a function of the ACN concentration, with an isosbestic point at 214 nm.

There was a brief note on this in LC/GC magazine years ago (like, almost 20), but I can't put my finger on it at the moment.

TFA is somewhat retained on the column. During the gradient the equilibrium concentration in the C18 phase is higher at the exit than at the entrance. The gradient pushes a wave of TFA through the column. You will notice that the amount of baseline shift during the gradient depends on the gradient rate: a faster gradient has more rise than a slow one. When you get to the end of the gradient and switch to the 90% MeCN for washout, first you get a slug of TFA washing off the column, then a drop as the column goes to equilibrium.

Hi guys

And thanks for your responses.
I’ve just conducted an experiment, which confirmed the idea of strong retention of TFA when the ACN concentration is above 80 %. I just ran a “normalâ€

I am not convinced that you get strong TFA retention due to HILIC interactions. You can easily determine it though by plotting the TFA breakthrough curves at high and low concentration of ACN...

so do we know how to explain TFA absorb more energy in the solution with higer percentage of ogainc ?
Is this true for others such as acetic acid, acetone, chloroform, pyridine, and so on

Kostas wrote:

I am not convinced that you get strong TFA retention due to HILIC interactions.

Why do I believe that the mechanism is HILIC rather than RP? Because the high ACN concentration (up to 98 %) surely would’ve “wonâ€

It seems that Mark´s explanation fully accounts for the results.