Chromatography Forum

For purification of the compound/s (derivatives of sialic acids) using HPLC, the mobile phase is ~97% of 0.1% TFA and 3% acetonitrile. While collecting the peak/s of interest we end up with ~200 mL of eluent. We then rotavap the solvent off. The dilemma is: The boiling point of TFA is: "74C", however, mixed with water it becomes an azeotrope and the boiling point changes to: 105C. What happens is the water evaporates off -concentrating the acid. Sometimes this can decompose the sample. Someone mentioned that I could add to the eluent (sample collected in the mobile phase) some toulene and that would help evaporate off the TFA.
Has anyone got any suggestions, as TFA is commonly used in chromatography, and in preparative work, so I imagine there must be a solution?

Hello Faith,

I'm not sure if our procedure is applicable for your compound. I have some experience since we tried to remove acetic acid from substance.

We dissolved our product in ethanol, then added ultrapure water (approx. 1:1) and using stainless steel stripping capillary under surface with slow stream of nitrogen evaporate on rotavap. It is necessary to repeat this procedure until required acid level.

In the absence of concrete advice it might be helpful to mention that I have seen a paper where touene was indeed used to remove TFA from water via a new azeotrope. No further details given. Since toluene is only very little soluble in water (maybe with TFA present it´s a bit better?) it will take a lot of fiddling to get this going. My guess is that they had small samples which they swamped with touene prior to evaporation.
Surely would like to hear about it if you successfully applied this.

Maybe benzene or cyclohexane would be a better choice judging from the azeotopes of acetic acid. The azeotope of toluene + acetic acid boils at 105 (28% acetic acid). Both benzene and cyclohexane azeotopes with acetic acid are around 80 (only 2% acetic acid).

May be the easiest way is to pass your mobile phase through anion-exchanger and trap TFA. Another approach is to use acetic or formic acid instead of TFA. Vaccume distillation is another option.
I cdid not find azeotropes for TFA but here are ratios for acetic acid. All of them are negative azeotropes (boiling point is higher then both components):
AcOH/1,4-dioxane ratio 80/20 BP 119*C
AcOH/heptane ratio 33/67 BP 92*C
AcOH/toluene ratio 28/72 BP 101*C
AcOH/benzene ratio 2/98 BP 80*C

Regards,

Vlad

Another approach that could make your life easier from several standpoints is to readsorb the collected TFA fraction on a C18 (or an Oasis cartridge) that is equilibrated with water and acetic acid, wash the TFA out with water and acetic acid, then elute with 100% acetonitrile. You may get a more concentrated fraction to start with, and acetonitrile is easier to evaporate.

The question is if the retention will be large enough to do this. Needs to be tried out.

It seems that Polymer Labs has anion-exchange SPE for TFA removal.

Good luck

I would like to thank everyone for there responses.

Hi Sadilek,
I am not sure if I understand.
My compound is already dissolved in ~3% acetonitrile/97% 0.1%TFA. Are you suggesting mixing this with 50% ethanol and then evaporating?

Hi HW Mueller,
Yes, I to have seen a paper mentioning using toulene as a new azeotrope, but that was with (8 mL) TFA and X mL dichloromethane, and they added ~30 mL toulene x3.
I have been told it does not matter that toulene is not soluble in these solutions, infact that it can inprove it??? I will probably end up throwing in some toulene, however it would e nice to know the exact quantities as I do not want my compound to decompose in the process. I suppose I can work with small volumes. I will let you know what happens!

Hello Sassman,
Before I posted my question yesterday I looked up in the literature other alternatives to azeotrope TFA, the CRC handbook if I can remember correctly, did not have any, it did mention the one for acetic acid. However, not being a chemist would acetic acid behave the same way as trifluroacetic acid.
Cyclohexane sounds good, Benzene is nasty. I will discuss it with the chemists tomorrow when I am at work, Thankyou for that.

Maybe it´s a steam distillation rather than azeotropic? Incidentally, you can possibly work this out by evaporating solutions which contain all the components except for the compound.

Hi Vlad,
Thankyou for your suggestions. and input.
Acetic acid is no good because it's boiling point is 118C, formic acid may be an alternative as its boiling point is 100.8C.
Or I may end up using some exchanger to bind the TFA. as has been suggested.

Hi HW Mueller,
I am pretty sure it is an azeotropic effect, well that is what the literature says . But you are right I could try what you suggest. And yes you are right again, I do not have to include my compund/s.

Thanks Serg,
I will look on the internet now for anion exchange SPE cartriges from Ploymer Labs. for the removal of TFA.

Hi Serg,
I just looked at the Polymer labs site. I think that the problem with the SPE cartriges is that they are designed for smaller volumes than what I am looking at. I am looking at volumes between 80-400 mL of eluent. However, I could rotavap a lot of the solvent off.

Well it is all food for thought, and I cannot thank everyone enough for all the different suggestions. I will let you know what I end up doing.