Hi Núria,

if you do not want to derivatize the TFA into its methyl ester form as Wolfram suggested (which may be tricky at high water content), then you'll have the problem that you can never evaporate TFA-Anions (salt) from the liquid, even with freeze-drying - You produce TFA salt of your product. TFA as its protonated CF3COOH form is highly volatile. You need to find a stronger acid to transfer TFA to its protonated species. Its a competition reaction - the stronger acid displaces the weaker acid from its salt.

What we always do to remove cell-toxic TFA from our products (e.g. peptides) - we add appropriate amounts of 1 M HCl to the collected fractions of prep. HPLC. HCl is a ~7-10x stronger acid then TFA. Whenever TFA is protonated, if evaporates really easy from your sample. You just need to add HCl with an amount of 1/10th of the moles of TFA in your mobile phase.

An example: using 0.1% (vol/vol, 1 ml per liter solvent) TFA in the mobile phase means 1.49 g/l (density of TFA =1,49 g·cm−3) and therefore 0.01306 mol/l = 0.1306 mmol/ 10 ml (Mw of TFA = 114.02 g/mol). With that, you need to add 13.06 µl 1 M HCl to every 10 ml of the collected fraction (which is 1/10th of the moles of TFA in the 10ml liquid, 0.01306 mmol). The volume of the collected fractions can be calculated from the collection time and the flow rate in the gradient program.

After additon of HCl, we evaporate (rotary evaporator) the sample nearly until dry and re-dissolve with 50% Acetonitrile/50% water mixture and evaporate again (I guess you can use Methanol instead of AcN in your case). We do that 2-3 times. As the last step, dissolve in water and freeze-Dry for at least 24h.

With that processing, you tranfer the sample into its HCl salt and kick out TFA.

If your sample is sensitive towards acidic conditions below pH 2-3, that will be a problem, for sure.

Dear Núria:

When you concentrate a sample by evaporation, the residue contains ions; only the vapour will be free of ions.

Best,

Thanks Aurea Andrade-Eiroa for your answer.  Then, what do you suggest to remove the TFA?

You are welcome, Núria.

I do not know which kind of analytical technique you want to apply or which instrumental determination you will accomplish after the concentration step but in most of analytical methodologies (overall HPLC separations), TFA is not a problem.

Could you please provide me with more details about your aims and your analytical procedure?

Thanks, Núria.

Best,

Hi Núria,

Why did you use TFA? And, Why do you want to know that TFA is totally removed or, You actually do not want to remove TFA. Please, Clarify.

First, in LC usually TFA has been used as a peak modifier, i.e. It maintains individual peak's sharp symmetry, helps in separation between adjacent peaks,  as well as better resolution. TFA is volatile, if you use TFA in diluting solvent or in mobile phase it slowly lose its presence, you will understand the fact to see your peak's shape, symmetry and sometimes changes of your peak retention time.

Hope it helps :)

With Regards.

Nawaz

Thanks Md. Saddam Nawaz for your answer.

I use TFA for the reason that you explained: to obtain better sharp pekas and separate them better. I want to remove the TFA to have pure substances and analyse them after that by LC-MS and NMR. ( I work with flavolignans)

I was asking that because by evaporation of the solvents ( in my case 50% MeOh and 50% H20 with TFA) the aspect of my substance changes and I was wondering that TFA could be remaining in the solvent and affecting the substances.

Best regards

Núria

Well, if you use MS detector, then TFA will detect with its Mass (m/Z). As well as, UV cut off response of Trifluoroacetic Acid is 210 nm. In your case avoid TFA first (I mean without TFA) or use other peak modifier (e.g. Sodium 1-heptanesulfonate). or, You can avoid MeOH and TFA with 50% of Acetonitrile.

Dear Núria:

As I said to you, ions remain into solution/residue after evaporation;  in addition to this, trifluoroacetic acid is much more stronger than other organic acids such as acetic acid; so, it should be dissociated into aqueous medium. However, I was thinking that if you concentrate until dryness, after removing all the water molecules, trifluoroacetic acid might remain as neutral form, and in this case it is easily removed because trifluoroacetic acid (not solved) is volatile. See the following link:

https://www.lifetechnologies.com/order/catalog/product/28904

Best,

Auréa.

Dear Nuria,

1) Lypholysation whole fraction to isolate the impurity free from TFA as per my practical experience.

2) Extract your aqueous fraction with non polar solvent where your product will extract in solvent while all ionic compounds will be in aqueous phase i.e. TFA.

3) Acid Base purification can remove TFA by salt formation i.e. Treatment with NaOH will result in its sodium salt and them extraction in non polar solvent where TFA salt will remain in aqueous phase.

If you want to remove TFA through evaporisation I would recommend you add MeOH to your sample of combined collected fractions prior to putting the flask on the rotavap.  Reduce the volume to about 1/3, add another portion of MeOH and put on the rotavap again.  This should remove virtually all of the TFA as its (volatile) methyl ester.  Be sure to have a cryotrap between rotavap and vacuum pump.  Do not reduce to dryness but add pure water to the reduced volume and connect the flask with the now aqueous solution (after immersion in liquid N2 to shock freeze its contents) to a freeze dryer.  As an added precaution, when removing the flask from the freeze dryer, use N2 or Ar to vent.

Your problem is that after the evaporation of the solvent your substance change aspect. Right? I just have a guess, so double check that. I would say that you evaporate all, or almost all, solvent you have, but this can change the final pH of your compound/s. This can now led to a changed conformation of your molecule that results in a different aspect. If you have the possibility I would run an MS on your sample and verify if there are still undesired molecules in your extract. I worked with prodiginine molecules and they change greatly the appearance (colour) according with the pH, or with the solvent used to dissolve them.

I agree with Wolfram. Adding methanol is a good idea because you will achieve a more efficient evaporation, because you will get a lesser dissociation of TFA. In fact in organic solvents like methanol, TFA is not dissociated and even in water/methanol mixtures, their dissociation should be small.

thanks all for your answers!

Hi Núria,

if you do not want to derivatize the TFA into its methyl ester form as Wolfram suggested (which may be tricky at high water content), then you'll have the problem that you can never evaporate TFA-Anions (salt) from the liquid, even with freeze-drying - You produce TFA salt of your product. TFA as its protonated CF3COOH form is highly volatile. You need to find a stronger acid to transfer TFA to its protonated species. Its a competition reaction - the stronger acid displaces the weaker acid from its salt.

What we always do to remove cell-toxic TFA from our products (e.g. peptides) - we add appropriate amounts of 1 M HCl to the collected fractions of prep. HPLC. HCl is a ~7-10x stronger acid then TFA. Whenever TFA is protonated, if evaporates really easy from your sample. You just need to add HCl with an amount of 1/10th of the moles of TFA in your mobile phase.

An example: using 0.1% (vol/vol, 1 ml per liter solvent) TFA in the mobile phase means 1.49 g/l (density of TFA =1,49 g·cm−3) and therefore 0.01306 mol/l = 0.1306 mmol/ 10 ml (Mw of TFA = 114.02 g/mol). With that, you need to add 13.06 µl 1 M HCl to every 10 ml of the collected fraction (which is 1/10th of the moles of TFA in the 10ml liquid, 0.01306 mmol). The volume of the collected fractions can be calculated from the collection time and the flow rate in the gradient program.

After additon of HCl, we evaporate (rotary evaporator) the sample nearly until dry and re-dissolve with 50% Acetonitrile/50% water mixture and evaporate again (I guess you can use Methanol instead of AcN in your case). We do that 2-3 times. As the last step, dissolve in water and freeze-Dry for at least 24h.

With that processing, you tranfer the sample into its HCl salt and kick out TFA.

If your sample is sensitive towards acidic conditions below pH 2-3, that will be a problem, for sure.

You may can also try using certain % of Ammonium bicarbonate and followed by Lyophilisation. Common practice in Peptide purification

Review this;

http://www.waters.com/waters/library.htm?cid=511436&lid=10122759&xcid=ext5325&utm_content=sf54187503&utm_medium=spredfast&utm_source=linkedin&utm_campaign=Waters+Corporation&xcid=sfwaters-linkedin-b-CBU+p-CBU+Columns+Field+Marketing+promo-sf54187503&sf54187503=1&locale=101