HPLC asks: of the material my detector can see, how much is the right molecule? It is a ratio within the peptide fraction, and it is the number almost always quoted.
Net peptide content asks a different question: of everything in this vial by weight, how much is peptide? Water absorbed during handling, residual counter-ion from purification and any salts all add mass without adding peptide, and none of them appears in the HPLC figure.
Peptides made by solid-phase synthesis are usually purified in a system containing trifluoroacetic acid, and basic residues retain TFA as a counter-ion. A sequence rich in lysine or arginine carries proportionally more.
Lyophilised powder is also hygroscopic; it takes up water from the air whenever a vial is open. That is why water content is reported separately on a good certificate, and why vials are not left standing open.
For work where the exact quantity matters, net peptide content is the figure that matters, and a certificate reporting purity alone is not giving enough to calculate from.
It is also why two lots at identical stated purity are not necessarily interchangeable by mass. Reading water content and residual solvent alongside the purity figure is the whole of the discipline here.
HPLC asks: of the material my detector can see, how much is the right molecule? It is a ratio within the peptide fraction, and it is the number almost always quoted.
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