how to remove residual TFA from peptides after HPLC

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

21 posts Page 1 of 2
hello, all

I am purifying peptide using TFA in Water/Acetonitrile. After lyophilization, the peptide is in TFA salt form. As I have to use the peptide for some cell experiment, I would like to change the peptide from TFA salt form to some cell friendly salt forms (Cl-, Acetate).

Someone told me I can dissolve peptide in 0.1M HCl and lyophilize to remove TFA. However I was also warned I may have to do the process several times to completely remove TFA, which I am afraid will result in loss of my peptides.

Did anyone do this before? What is the yield? Or could anyone give me some other suggestions?

It may be possible to purify these peptides using a different mode of separation (RP+IEX OR Normal Phase).
Can you tell us more about the peptide?

ammonium acetate is an acceptable for your purification, correct?

Thank you, Bryan. The peptide is basic (composed primarily of Arg and Lys, ~2k M.W.). Ammonium acetate is ok. Unfortunately, the only HPLC column I have is RP.

What I concern most is that i do not want to lose too much peptide druing TFA-->Cl (or acetate) transformation.

Bryan is correct: you could use the same column that you have used for purifying the peptide, equilibrate it with acetic acid or formic acid, and then inject the purified peptide. Now, the TFA will run through the column nearly unretained, and the peptide will elute in the form of the acetate or formate salt. You do not need TFA to elute peptides from a column. Most of the time, TFA gives better peak shapes, but since you have done the purification already, you do not need the high resolution any more.

thank you so much! how much acetic acid should I use? 0.1% ok?

I assume the acetic acid/HPLC method is better than the HCl method for removing TFA from the peptide. Is that correct?

With the chromatography method, you will end up with the salt of the peptide and either formic acid or acetic acid, whaitever you pick. You can use a somewhat higher concentration than 0.1% for the acid, maybe 0.5% or 1%. The only disadvantage of the HPLC method is that you dilute the analyte, and you have to reconcentrate.

Thank you so much, Uwe.

Just out of curiosity: acetic acid is a weak acid while TFA is a strong acid, why acetate can replace TFA? Simply because there are much more acetic acid than TFA during HPLC?

The proper use of a chromatography method assumes that the compound of interest is retained. While sitting on the column, it will exchange its counterion to the one in the mobile phase. The original counterion will move through the column with no (or little) retention. The compound of interest will move through the column with the new counterion.

Thank you very much!
There are some scavenger for TFA. If you like to try it, let me know
Charles Levesque M.Sc.
Product Manager--Analytical Chemistry
SiliCycle Inc

Thank you, Charles. This is the first time I hear of TFA scavenger. Yes, I very much want to know about it. Could you give me some reference?


I used couple times the SiliaBond Carbonate (R66030B). SiliaBond Carbonate is the silica bound equivalent of tetramethyl ammonium carbonate. It be used as a general base to quench a reaction, to free base amines. Org. Lett. 4(7) 2002, 1167 and Org. Lett. 5(24) 2003, 4721.
The only limitation, is if you have acidic peptides, this will not work perfectly.
Good Luck
Charles Levesque M.Sc.

Product Manager--Analytical Chemistry

SiliCycle Inc

One can get rid of TFA with ultrafiltration (washing and filtering several times), at the end you add whatever you want and how much of it you want to solvate the protein.

I am wondering if Uwe's suggestion worked? I usually remove TFA using ion-exchange resin.
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