Acidic additives only in aqueuous eluent or in both eluent ?

[Nat21]

Hello Everybody,

I’m in charge of the purifications of synthetic compounds by reversed phase Prep-LC in a Pharmaceutical Research laboratory. The purified compounds can be acidic, basic or neutral. Mainly the purifications are made by C18 columns with a gradient of H2O with 0.1% TFA and ACN (or MeOH) with 0.1% TFA in 10 or 20min ( 10% organic at the injection to 95% at the end of the run). For multiple reason (time and cost saving, for example), I wonder if we can have the same results (resolution, loading..) ,if we only add TFA in the water part. Do you add your TFA (or any other acidic) in the both eluents ? Do you know some literature in this area?

Thank you in advance for your reply,
And sorry for my bad English !

[mardexis]

I don't think you need TFA in your organic for good chromatography. You need TFA in your organic to keep a flat baseline in the UV, depending upon what wavelength you monitor.

I would have guessed you were born in the US. Didn't see any flaws in your English!

[tom jupille]

There are actually three questions here:

1. Do you have to add TFA in both the aqueous and the organic?

I agree with mardexis on this one; I don't think its required, but by adding appropriate concentrations of TFA to each solution and choosing the wavelength wisely, you can minimize baseline drift. A good explanation can be found here.


2. If you do , does it have to be at the same concentration in both the aqueous and the organic?

Most people do it that way; but it's not necessary, and it may not be the best way. In addition to the changes in the absorbance spectrum of TFA as a function of ACN concentration (per the article I cited above), the amount of TFA that "sticks" to the stationary phase will also change. If the goal is to maintain a constant TFA concentration on the stationary phase, then keeping a constant concentration in the mobile phase is *not* the right way to do it.


3. If you have a method that adds TFA to both, and you add TFA only to the aqueous, will you get the same results?

Probably not, but the only way to tell is to try. The amount of TFA on the stationary phase changes as a function of ACN concentration, so you will have a different "profile" in the two cases. You might get lucky and find that the changes are not significant to your separation.



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[Uwe Neue]

In preparative chromatography, a high concentration of modifier that controls the pH is a good thing. Essentially, your acid concentration limits how much you can load. I would not bother with TFA, I would use massive amounts of formic acid in both A and B.

[Nat21]

Thanks a lot for your quick and useful answers !!