This is a small question with big implications for quantity calculations. If I purify a peptide with lysines or other compounds with free amines with preparative HPLC using 0.1% formic acid and freeze dry, do I end up with a free amine or a formate salt in the powder? And would I expect to see the formate on NMR? Any references to this would be highly appreciated as it appears to be quite controversial. Thank you very much!
FA is volatile and can be removed by F.drying and amine compounds washed out along with salts during RP peptide purification. No need to worry about residues.
Dear Erik Hebels
In my opinion, your peptide will end up as a formate salt. This is because formic acid will bind to amine to counteract the positive charge. I do not know any references, but from practice, taking the mass of such counter ion (in my case, trifluoroacetic acid) works well when I cross-check the concentration with the other method (e.g., UV spectra, potentiometry, or metal ion titration). Another indication that it is a formate salt is that after dissolving such peptide in pure water, the pH of the solution is clearly acidic.
Regards
Dear Tomasz Fraczyk, many thanks for your answer.
The compound is only mildly acidic in pure water. To further investigate, we went ahead and did an NMR using formic acid and an excess of butylamine in deuterated DMSO, and found the formate proton signal to be clearly present. Unfortunately, this signal is not present in my compound of interest (N-N' bis-cysteinyl-ethylendiamine). Since this compound was obtained by deprotection with TFA, we will next perform a fluorine NMR to check whether TF-acetate can be detected. Since the compound eluted very early (essentially injection peak) with prep HPLC (0.1% FA), this could still be envisioned as a possibility. I will post the result here once we have it.
An update to this: We found indeed that TF-acetate was still the counterion present, by use of fluorine NMR and trifluoroethanol (TFE) as internal standard, calculating back exactly the amount of TF-acetate expected per amine 1:1. In this particular instance, the very short retention time of the compound certainly did not allow any separation from the salt. I highly recommend to also look at this paper that highlights the challenge further and suggests solutions: Article Elimination and exchange of trifluoroacetate counter-ion fro...
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