Evaporation of TFA in an aqueous mobile phase (semi-prep)

[Jackus]

My compound is already dissolved in ~3% acetonitrile/97% 0.1%TFA. Are you suggesting mixing this with 50% ethanol and then evaporating?

I'm sorry. I forgot that your sample is diluted in mobile phase. Mentioned procedure was applicable for evaporated product.
In this case I can recommend procedure by Uwe Neue. I used it several times and works well. But I always added water into collected efluent (with desired product) to reduce compound mobility in chromatographic column.

Regards

[Bruce Hamilton]

Another possibility could be to adjust the pH of your solution with a buffer or alkali before concentrating it. It would depend what happens to your compound.

Please keep having fun,

Bruce Hamilton

[Uwe Neue]

Considering the large quantities that you are dealing with, this may not be a trivial enterprise. I don't think that anion exchange will work, since it will enrich both the sialic acids and the TFA. I still think that reversed-phase will work, but you need to work out a functional procedure. Precipitation may be the best approach, with large amounts of acetonitrile. Is the concentration of the sialic acids high enough to do a precipitation?

[Faith]

Uwe- I think your right, anion exchange would not work, I had came to the same conclusion.
Reverse phase is difficult because as mentioned previously, I run the sample in a very low percentage of acetonitrile, mostly none at all- it practically comes out at the void! This also makes the separation difficult. The compounds are highly polar and acidic-highly water soluble- but that is another issue altogether.
With regard to precipitation, the concentration of the sialic acid generally is not concentrated enough.

Bruce-That was my first thought to neutralize, and then concentrate, but the chemists told me it would form a salt and they do not want that.

At this stage we are looking into another azeotrope, and will play with it without the compound in the solution. I will keep you informed.

[SIELC_Tech]

I am not sure what is the problem here (if any). TFA is one of the most common additives in prep chromatography and people in pharma are doing it every day even with basic drugs which trap TFA. If you are bound to use TFA then I would add any solvent with higher boiling point and do vacuum distillation. Vacuum will narrow boiling point at because you only have a fraction of a percent you should not have any problems. If you worry about stability of amide bond in your compound then use a weaker acid Acetic acid might have a higher boiling point but it creates higher pH (compare to TFA). Problem with AcOH that it will keep your compounds (pKa below 3) in ionized state which will reduce retention time even further.

In case of TFA you can use vacuum to concentrate your sample and then lyophilization-you will remove the acid from your compound. Your biggest problem is not in solvent/TFA but rather the approach you are trying to use to separate sialic acids (but this is a different story)

You also can try to use ammonium acetate or formate (pH 3-6). It is considered as a volatile buffer that is why people are using it LC/MS and ELSD.

Regards,

Vlad

[Serg]

Boronate affinity chromatography? Should bind your derivatives, assuming that hydroxyls are still intact...

Good luck.
Serg.

[Uwe Neue]

Something along the lines of a precipitation/extraction may still do the job. I am trying to think of something that has a good solubility for the TFA, close to no solubility for a sugar (your sialic acids), and at the same time removes at least some of the water to concentrate your analytes to make the dry-down step easier. How about a mixture of acetonitrile and ethylacetate for extraction? You should get two solvent layers, one predominantly ethylacetate and acetonitrile, and the other predominantly water and some acetonitrile. The TFA should go into the organic layer, and the sialic acid into the aqueous layer. You could then manipulate the ratios of acetonitrile to ethylacetate to get to a spot that delivers the best results.

Never tried this... May just be a crazy idea...

[Faith]

Hi Vlad, I also thought that TFA is a common acid used in chromatography and the solution should be simple. However, some of our compounds decompose, it is a fact! Formic acid and ammonium acetate are out of the question. It is not the stability of the amide bond that we are worried about because even the compounds that do not have it go black.
In my pass life we freezed dried the fraction after we rotavapped off a lot of the solvent, however the TFA was not good for the freeze dryer.
I read a paper once where they would rotavap most of the solvent off, then add more water and evaporate off most of the solvent again, and do this three times, each time evaporating off some TFA, and then lyophilizing.

Being able to get on to the vacuum distillation would be a challenge here and we do not have much control of the vacuum. One of our rotavaps is great though where you can set the pressure, and you can set the temperature of the water bath.
Tomorrow I am going to perform a few experiments and see what other solvent OR combination of solvents (ethanol and toluene for example) I can add to the eluent to help pull the TFA off!
Thank you so much for your feed back

[Faith]

Uwe- That is a great idea, I will talk it over with one of the chemists tomorrow. I do not think it is that crazy.

[Faith]

Thank you everyone,
I think the problem the two chemists had previously with their compounds decomposing when they were rota-vapping their solvent off is they let it go to dryness. Their compounds were sensitive to acid.
What they have tried now is to leave some liquid in the flask, add some more water and rotavap that off and do this three times. They leave the last 5-10 mL in at the end and then freeze dry it.
They have not had a compound decompose since.

[mbreslav]

I am using lyophilization. There is no need to concentrate before. However if you want your compound in a nice small vial at the end, then re-dissolve lyophilate in a small volume of water, or water/ACN and repeat freeze-drying in a small pre-weighed vial. I am using 20 ml vial. Freeze your solution, cover vial opening with Kimwipe secured by a rubber band and place frozen material in a larger flask supplied with your freeze-drier.

[Bryan Evans]

An alternate approach would be to try a different stationary phase.

Sialic acid is well retained on our Unison UK-Amino column
(and has a higher % organic in the mobile phase):
http://www.imtakt.com/TecInfo/TI333E.pdf

[Bryan Evans]

In addition, 3'- and 6'- sialyllactose are easily separated using Unison UK-Amino (under highly organic conditions and using a volatile buffer):

http://www.imtakt.com/TecInfo/TI342E.pdf