Peptide purity explained

In reversed-phase HPLC the dissolved sample is pushed through a packed column. Components leave the column at different times, and a UV detector draws each one as a peak. Peptide bonds absorb strongly at around 220 nm, so peptides are usually measured there. Bachem defines HPLC purity as the percentage of the requested peptide relative to all material absorbing at 220 nm: the area of the main peak as a share of the total.

Most of the smaller peaks are close relatives of the target, made during synthesis:

• chains missing one amino acid (deletion sequences);
• chains that stopped growing early;
• chains still carrying a protecting group;
• chains in which one residue has changed, for example by oxidation.

Water and most counter-ions do not appear on the trace, so the purity figure says nothing about them.

Two compounds that leave the column at the same time show as one peak, and the purity figure counts both as the target. A second method, based on a different physical principle, is the usual answer. For a buyer the practical version is simple: an HPLC figure is strongest when a mass spectrum confirms that the main peak is the right molecule.

The same logic explains a gap in testing in Iraq. Medicines are tested by the National Center for Drug Control and Research before registration and, usually, batch by batch before release. Research materials and cosmetic raw materials are not. For them, the certificate and the laboratory behind it are the whole of the evidence.

A freeze-dried peptide is a salt. During purification it takes up counter-ions, usually trifluoroacetate (TFA) or acetate, on its charged groups, and the powder holds some water. Net peptide content is the share of the powder's weight that is peptide. Certificates print it as "net peptide content", "peptide content", "NPC" or "assay".

Bachem determines it by elemental analysis for nitrogen, since counter-ions and water contain none, or by amino acid analysis. GenScript notes that it counts all peptide in the powder: the target and its peptidic impurities together.

GenScript's rule: the amount of target peptide is the gross weight multiplied by the net peptide content and by the HPLC purity.

Bachem publishes an example for bradykinin acetate with an HPLC purity of 98.7% and a net peptide content of 85.0%. Multiply the two and you get 0.839. Weigh out 10 mg of that powder and you hold about 8.39 mg of bradykinin, not 10 mg: some 16% less than the balance shows. The peptide calculator does this arithmetic for concentration and molarity.

A complete set of results accounts for the whole vial. GenScript writes it as: target peptide + peptidic impurities + counter-ion + water = 100%. When a certificate gives all four, do the sum. A large gap means something was not measured.

Counter-ions sit on the peptide's charged groups: the free N-terminus and basic side chains, those of lysine, arginine and histidine. Bachem notes that a relatively low peptide content is to be expected when a peptide contains a large proportion of basic amino acids, especially arginines. A short, arginine-rich peptide can carry noticeably more counter-ion by weight than a long neutral one, and nothing is wrong with the batch.

Bachem supplies its peptides as trifluoroacetate salts unless stated otherwise. The counter-ion changes how much salt you weigh for a given amount of peptide, and some experiments and formulations are sensitive to it. If your work depends on the salt form, say so when you ask for a quotation and check it on the certificate.

There is no single pass mark. Synthesis companies sell the same sequence at several purity grades, and the higher grades cost more because every extra purification step loses material. Purity matters most where a result depends on the exact molecule or on exact quantities, because the impurities are close copies of the target and can behave like it.

A purity figure means something only when it belongs to a batch, a method and a named laboratory, and when a mass spectrum confirms the identity. "99% pure" on a web page is a claim. A certificate for your batch, with its chromatogram, is a record.

Every manufacturer tests its batches before release; that is the manufacturer's certificate. Some suppliers also send samples to an independent laboratory. Both are useful. When a supplier says "lab tested", ask which laboratory, which tests, which batch, and whether that laboratory will confirm the report number. Testing sets out how our batches are tested.