How Peptide Purity Is Tested: HPLC and Mass Spectrometry Explained

HPLC: how clean is the main component?

In reversed-phase HPLC, a dissolved sample is pushed through a column packed with a water-repelling material while the solvent mixture is gradually changed, typically from mostly water to mostly acetonitrile. Different molecules leave the column at different times, depending on how strongly they cling to it. A detector, usually ultraviolet light at around 214 to 220 nanometres where the peptide bond absorbs, records a peak for each component as it emerges.

From chromatogram to purity percentage

Software measures the area under each peak. Purity is the area of the main peak divided by the total area of all peaks, expressed as a percentage. A result of 99% means that, of the material the detector saw, 99% of the signal came from the main component.

What HPLC purity cannot tell you

• It only counts components the detector responds to. Salts, water and some other substances are largely invisible at these wavelengths.
• Two different compounds can emerge at nearly the same time and appear as a single peak.
• It says nothing, on its own, about what the main peak is. That is the job of mass spectrometry.
• Area percent is not weight percent. See Purity vs Quantity.

Mass spectrometry: what is the main component?

A mass spectrometer measures the mass-to-charge ratio of ions. For peptides, electrospray ionisation usually produces ions carrying several charges, so a report may list peaks such as [M+2H]2+ or [M+3H]3+. Software works back from these to the molecular mass of the peptide, which is compared with the theoretical mass calculated from its sequence. Agreement supports the identity of the compound.

What mass spectrometry cannot tell you

• How much of the sample is the compound. It is not a reliable measure of quantity.
• Differences between peptides with the same composition in a different order, which can have identical masses.
• That every impurity is absent. A matching mass confirms the main species, not the absence of everything else.

Other tests you may see

• Amino acid analysis or similar: determines how much peptide is actually present by weight.
• Karl Fischer titration: measures water content.
• Bacterial endotoxin (LAL) and sterility testing: relevant to contamination, not to purity.
• Residual solvent, trifluoroacetic acid (TFA) or heavy-metal testing.

Used together, HPLC and MS give a good picture of identity and relative purity. Neither, alone or together, is a complete inventory of everything in a vial.