A lyophilised peptide vial is not pure peptide, and the largest non-peptide component is usually the counter-ion. Understanding what it is explains most of the gap between label mass and peptide mass.
Where counter-ions come from
Peptides carry charged groups — the N-terminus, the C-terminus, and the side chains of residues such as lysine, arginine, aspartate and glutamate. Those charges are balanced by ions of opposite charge from the environment the peptide was last purified in.
Reverse-phase HPLC purification almost universally uses trifluoroacetic acid as a mobile-phase additive, because it improves peak shape. The peptide comes out of that process as a trifluoroacetate salt. Acetate is the common alternative, produced by an ion-exchange step after purification.
How much mass they account for
It depends on how many basic residues the sequence carries, since each one can hold a counter-ion. For a peptide with several basic residues, TFA can account for 10 to 25 percent of the powder mass. Combined with residual water — typically another few percent — this is why peptide content figures of 75 to 90 percent are normal rather than a sign of a problem.
A vial labelled 10 mg at 82% peptide content holds about 8.2 mg of peptide. The missing 1.8 mg is not absent; it is counter-ion and water, and it was always going to be.
TFA versus acetate: when the choice matters
- Cell-based work. Residual TFA is the more common concern here. Where an assay is sensitive to it, an acetate salt is the usual request.
- Spectroscopy. TFA absorbs in regions that can interfere with certain measurements, particularly circular dichroism in the far UV.
- Most other applications. The salt form is immaterial as long as peptide content is known and accounted for.
Acetate salts generally cost more, because the ion exchange is an extra step. If your protocol does not require it, TFA is not a deficiency.
What to ask for
The certificate of analysis should state peptide content, and a complete one names the salt form. If neither is present, ask before ordering rather than after — without them, any calculation planned in peptide mass carries an unknown error in the region of 10 to 25 percent, which is far larger than the purity differences people spend time comparing.