Testing: HPLC purity and mass spectrometry

An HPLC instrument pumps the dissolved sample, under high pressure, through a steel column filled with fine particles. Peptide work almost always uses the reversed-phase mode: a C18 column, with the solvent changing gradually from water to acetonitrile. Components travel at different speeds, so they emerge one after another and appear on the chromatogram as separate peaks. Detection is by ultraviolet light at 214 to 220 nm, a range the peptide bond absorbs strongly. The purity figure is the main peak's area divided by the area of every peak, expressed as a percentage.

What it records: the main peak's share of the material that absorbs at that wavelength.

What it leaves out: the identity of the main peak. A sequence missing one amino acid can still elute as a single clean peak. Water and salts give no signal at 214 nm, so the figure says nothing about how much of the powder is peptide.

Mass spectrometry weighs molecules. The instrument charges them, then sorts the resulting ions by mass-to-charge ratio (m/z). Peptides are charged either by electrospray (ESI) or by MALDI. On a certificate, look for two numbers side by side: the mass found and the mass expected from the sequence.

A molecule this small usually shows up as the singly charged ion: the monoisotopic mass plus one proton, close to m/z 343.23. A KPV certificate should give a mass consistent with these figures.

What it records: that the sample contains a molecule with the predicted mass.

What it leaves out: that molecule's share of the sample. A batch at 75% purity and a batch at 99% can return the same identity result.

Each result covers the other's blind spot, so a certificate carrying only one of them answers half the question.

• Net peptide content: the peptide's share of the total mass of powder. What is left is mostly water, residual salts and the counter-ion bound during synthesis and purification, usually acetate or trifluoroacetate. It is found by amino acid analysis or nitrogen determination, and it normally reads lower than HPLC purity because it measures a different thing.
• Water content: measured by Karl Fischer titration in most laboratories.
• Counter-ion: which salt the peptide is supplied as; acetate is common.
• Endotoxin: a separate test, usually by the LAL method. Plenty of peptide certificates omit it, and where the line is absent, the test was not done.
• Appearance: a visual description of the powder, such as white and lyophilised.

The manufacturer tests each batch before release, and the certificate is its report. Manufacturer's figures stay under the manufacturer's name. An independent laboratory's report, when a batch has one, is added to that batch's entry in the certificate library as a separate file, under that laboratory's name. Without such a file, we do not use the words third party tested.

1. The mass. Copy the sequence from the product page into the peptide calculator and note the mass it returns. That figure and the certificate's measured mass should be close. A gap that no sodium (+22) or potassium (+38) adduct explains points to another molecule.
2. The chromatogram. Behind every purity percentage is a chart. Where the chart is missing, request it: a clean single peak and a peak carrying shoulders both reduce to one percentage, and only the chart shows which you have.
3. The paperwork. Look for a batch number, a manufacture date, an expiry or retest date and a named laboratory, and compare each with the delivery note.

A laboratory in Iraq with its own HPLC can also run a sample from the delivered batch and set its chromatogram beside the manufacturer's.

The certificate for your batch comes with the goods, and you can ask us for it. For more on the two purity numbers, read peptide purity explained; for a certificate taken from top to bottom, read how to read a certificate of analysis.

This page is written for businesses and institutions in federal Iraq and the Kurdistan Region; private individuals cannot buy from us.