Chromatography Forum

Hello, recently i Changed My usual HPLC - ACN solvent for Methanol. I prepared Methanol + 0.1% TFA, and i observed that this mixture absorbe in the UV. Is this right??
At 220 nm
Metahol (Grade HPLC)before filtration, absorb less than 0.2 A
Metahol (Grade HPLC) after filtration, absorb less than 0.2 A
Metahol (Grade HPLC) with 0.1% TFA absorb more than 0.6 A
Water with 0.1% TFA absorbs arround 0.2 A

I don´t kwno what is happening, should i use less TFA %?

Thanks for your answers...

I also prepared the same solution with a Brand New TFA and obtained the same value, and if i Add more more ir absorbs. SO i don´t think the Methanol is the problem.
Should i use a lower percetaje of TFA? How much?
Please help!

Do not use TFA with Methanol at 220 nm.

Unless at a higher wavelength, substitute the appropriate strength of phosphoric acid at the same wavelength .........

It looks like esterification takes place. Methanol reacts with trifluoroacetic acid

Any organic solvent will shift the TFA UV absorption towards lower frequencies/longer wavelengths. Hence the user will observe UV absorption at wavelengths that didn’t pose a problem if it were aqueous solutions/mixtures. So, nothing’s new here.
Some people add something like 0.08 – 0.09 % TFA to the B eluent (the higher organic) in order to reduce the baseline increase in gradient elution.
I’m not that big a fan of that routine, because of pH fluctuations and thus unstable chromatography, but you can try it and if it works for you, then it must be OK.

Best Regards

Any organic solvent will shift the TFA UV absorption towards lower frequencies/longer wavelengths.

The method was used with acetonitryle first and there was no shift. Everything was OK.

Shouldn't TFA be replaced by phosphoric acid? There will be no UV absorption from this acid. There will by only absorption from methanol which is low at 220nm.

The method was used with acetonitryle first and there was no shift. Everything was OK.

Tomasz,

This is one of the few cases in which I’ll have to declare my disbelieve. It might be the case that you didn’t observe the higher absorption due to the degree of zoom you used, but there has been higher absorption in the organic (ACN) containing eluent. At shorter wavelengths (f. ex. 220 nm) anyway.
Regarding utilization of phosphoric acid instead of TFA, I’ll have to disagree even stronger with you. The thing is: TFA is an ion pairing reagent (apart from its acidic properties) and depending on the analyte of course, there are situation in which you won’t even be able to chromatograph the compound – let alone the quality of the chromatography (separation, plate count etc.)

Best Regards

Changing acetonitryle to methanol can also change the selectivity of the method. TFA is a weak ion pairing reagent. Maybe phosphoric acid could be good enough for this analysis. Nobody knows if ion pairing is required here for a satisfactory separation of the analytes. Nevertheless, in this case TFA definitely can not be used with methanol because of too high absorption of this mixture (no matter what is the reason for high absorption).
I also had a problem with changing acetonitryle to methanol in my lab. Methanol gave different selectivity and I had to change the analytical column to get resonable separation of my analytes.

Thanks for all the answers. I will try now to make the mixture with phosporic acid. Should I use the same percetaje that i have with ACN (I used it with 0.08% of TFA)? ANother question, if i have phosporic acid in methanol, can i use the TFA in teh water, or i have to use the same for both?
Thanks again for all your anwers...

Use phosphoric acid with both solvents. Its percentage should be optimized. You can start from 0.08%. Why not? In fact it would be a new method.

same thing happened to me when i was using a uv wavelength of 254. I was observing a decrease in analyte reponse when using TFA
Effect was reduced when i increased uv wavelength to 436 (corresponsing to a second maximum in my analyte's uv spectrum)