Peptide purity explained

Reversed-phase HPLC pushes the dissolved sample 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 that is where peptides are usually measured. Bachem defines HPLC purity as the percentage of the requested peptide relative to all the material absorbing at 220 nm: the main peak's area as a share of the total.

The other peaks are mostly relatives of the target made during synthesis:

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

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

Two compounds that leave the column together appear as one peak, and the purity figure counts both as the target. The Egyptian Drug Authority (EDA) deals with this directly in its guide to synthetic peptide medicines (version 4, October 2025). For a medicine it asks for:

• a purity control strategy built on orthogonal analytical methods, meaning methods based on different physical principles, to reduce the risk of an impurity hiding under the main peak;
• any new peptide-related impurity, compared with the reference medicine, to stay at or below 0.5% of the drug substance;
• peptide mapping as an identity test for longer peptides, above about 20 amino acids.

Those rules are for registered medicines. A research material is not held to them. The reasoning still applies to any batch: an HPLC figure is strongest when a mass spectrum confirms that the main peak is the right molecule.

A freeze-dried peptide is a salt. During purification it picks 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 measures it by nitrogen analysis, because counter-ions and water contain no nitrogen, or by amino acid analysis. GenScript points out that it counts all peptide in the powder, the target and its peptide 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's published example is bradykinin acetate with an HPLC purity of 98.7% and a net peptide content of 85.0%. Weigh out 5 mg and you hold 5 × 0.850 × 0.987 = 4.195 mg of bradykinin. Make that up to 5 mL expecting 1 mg/mL and the true concentration is about 0.84 mg/mL, some 16% short. The peptide calculator does this arithmetic for concentration and molarity.

A full set of results accounts for the whole vial. GenScript writes it as: target peptide + peptide 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 such as 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. A short peptide rich in arginine 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 the weight of salt you need 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 quote and check it on the certificate.

There is no single pass mark. Synthesis companies sell the same sequence at several purity grades, and 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 only means something when it is tied to a batch, a method and a laboratory, and when a mass spectrum confirms the identity. "99% purity" on a web page is a claim. A certificate for your batch, with the chromatogram, is evidence.

Every manufacturer tests its batches before release, and 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 the report number can be confirmed with that laboratory. Testing sets out how our batches are tested.