Guide
Peptide counterions: TFA, acetate and hydrochloride salts
A synthetic peptide is almost always supplied as a salt. Reversed-phase HPLC purification commonly uses trifluoroacetic acid (TFA), so the freeze-dried peptide carries trifluoroacetate counterions on its basic groups. Those counterions add weight that is not peptide, which is one reason net peptide content is lower than the fill weight. Exchanging to acetate or hydrochloride is a separate processing step.
Where the counterion comes from
Peptides are cleaved from the synthesis resin with TFA and purified by reversed-phase HPLC, where TFA is the usual ion-pairing additive in the mobile phase. Every positively charged site on the peptide — the free N-terminal amine and the side chains of lysine, arginine and histidine — is paired with a trifluoroacetate anion when the purified fractions are freeze-dried.
The result is a TFA salt. How many counterions it carries depends on how many basic groups the sequence has, so a peptide rich in lysine and arginine carries proportionally more.
Why it changes the numbers
Counterions, together with residual water, are part of the solid but not part of the peptide. That is why the net peptide content on a certificate — the share of the powder that is peptide — is lower than the weight of powder in the vial, often by a margin well beyond rounding.
It is also why a molecular weight quoted for the free peptide and the formula weight of the salt are different numbers. The compound references here state the free-peptide formula from the cited record.
Acetate and hydrochloride forms
A peptide can be converted from its TFA salt to an acetate or hydrochloride salt by ion exchange or by repeated freeze-drying from a solution of the replacement acid. The exchange is an extra processing step with its own yield loss, and it is usually specified by laboratories whose assays are sensitive to residual TFA.
Which salt form a batch is supplied as is a manufacturing fact that belongs in its specification. It does not change the peptide's sequence or its identity.
How residual TFA is measured
Trifluoroacetate is quantified by ion chromatography, among other methods, which separates it from the other anions present and reports it as a proportion of the material. Counterion content, water content and peptide purity together account for what is in the vial.
Common questions
What is a TFA salt peptide?
A peptide supplied with trifluoroacetate as the counterion on its positively charged groups, a consequence of TFA being used in cleavage and HPLC purification.
Does the counterion change the peptide's identity?
No. The sequence and the mass of the peptide ion observed by mass spectrometry are the same whichever salt form it is supplied as. The counterion changes the weight of the solid and therefore the net peptide content.
Why is net peptide content lower than the fill weight?
Because the powder also contains counterions and residual water. Net peptide content measures the peptide alone.
References
- Determination of residual trifluoroacetate in protein purification buffers and peptide preparations by ion chromatography — Kaiser E, Rohrer J, Journal of Chromatography A, 2004
- Related impurities in peptide medicines — D'Hondt M et al., Journal of Pharmaceutical and Biomedical Analysis, 2014
- Advances in Fmoc solid-phase peptide synthesis — Behrendt R et al., Journal of Peptide Science, 2016
From the catalog
Compounds referenced on this page
For laboratory research use only. Not for human or veterinary consumption.