Testing: HPLC purity and identity

High-performance liquid chromatography (HPLC) pushes a dissolved sample through a packed column. For peptides the usual method is reversed-phase HPLC: a C18 column and a gradient of water and acetonitrile. Each component reaches the detector at its own time and is drawn as a peak; the UV detector reads at 214 or 220 nm, the wavelengths where the peptide bond absorbs. Purity is reported as area per cent: the main peak's area divided by the total area of all peaks.

Answers: what share of the UV-absorbing material comes off the column as the main peak.

Does not answer: which molecule the main peak is. A peptide with one residue missing or swapped can elute as a single sharp peak. Water and salts are invisible to the detector, so HPLC purity is not the share of the powder that is peptide.

A mass spectrometer turns molecules into ions and measures their mass-to-charge ratio. Electrospray (ESI) and MALDI are the two usual ways of ionising a peptide. The figure that matters is the gap between the measured mass and the mass calculated from the sequence.

A certificate for this peptide should report a measured mass that agrees with one of those two figures, depending on how the instrument reports it. Electrospray spectra often also show the doubly charged ion, at half the mass plus one proton: about m/z 710.4 here.

Answers: whether the molecule has the expected mass.

Does not answer: how much of the sample that molecule makes up. Read this way, mass spectrometry gives a yes or a no, and a sample at 70% purity can give the same yes as one at 99%.

So a certificate needs both lines. A purity figure with no identity result, or an identity result with no purity figure, is half a certificate.

• Net peptide content: the share of the powder's mass that is peptide. The rest is water, counter-ions left from synthesis and purification (acetate or trifluoroacetate) and residual salts. It is measured by amino acid analysis or nitrogen content, and it normally comes out lower than the HPLC purity. The two numbers do not contradict each other; they answer different questions.
• Water content: usually by Karl Fischer titration.
• Counter-ion: the salt form, for example acetate.
• Endotoxin: a separate test, usually by the LAL method, that many peptide certificates do not include. No line on the certificate means no test.
• Appearance: the colour and form of the powder, checked by eye.

Testing is done by the manufacturer, batch by batch, before release; the certificate you receive is its report. We never relabel a manufacturer's result as independent. When an independent laboratory has also tested a batch, its report is published next to the manufacturer's certificate, under the laboratory's name. A product without such a report is not described here as third party tested.

1. Calculated mass. Enter the sequence from the product page into the peptide calculator. The result and the measured mass on the certificate should agree closely; a gap of tens or hundreds of daltons means a different molecule.
2. Chromatogram. The purity figure is read from a chart. If the certificate shows only the number, ask for the chart: one sharp peak on a flat baseline and a main peak with shoulders can both be printed as a percentage.
3. Paperwork. Batch number, dates and the testing laboratory's name should all be present and should agree with your delivery note.

The certificate for your batch comes with the goods. Peptide purity explained goes further into purity and net peptide content, and how to read a certificate of analysis takes a certificate line by line.

We supply businesses and institutions in Egypt only, never individuals.