What we measure, and what the number actually means

HPLC answers one question: how much of this sample is a single component, relative to everything else the instrument can see?

A weighed portion of the lot is dissolved and pumped at high pressure through a column packed with a stationary phase — for peptides, typically a reversed-phase C18 material. A mobile-phase gradient carries the sample through it. Components with different affinities for that stationary phase move at different speeds, so a mixture that entered the column together leaves it separated in time.

A detector at the column outlet records absorbance continuously. For peptides the wavelength is usually 214 or 220 nm, where the peptide backbone itself absorbs. The output is a chromatogram: signal plotted against time, with each separated component appearing as a peak. The time at which a peak appears is its retention time; the area underneath it is proportional to how much of that component was present.

Purity is then reported as area percent — the area of the main peak divided by the total integrated area of all peaks, times one hundred. A result of 99.2% means the principal component accounted for 99.2% of the detected, integrated signal. The remainder was other peaks: process-related impurities, deletion or truncation sequences, oxidation products, residual reagents.

Mass spectrometry answers a different question: is the dominant component the molecule the label names?

The sample is ionised — for peptides, almost always by electrospray, which is gentle enough to leave large molecules intact — and the resulting ions are separated by their mass-to-charge ratio. Electrospray tends to produce a series of multiply charged ions from the same molecule; deconvoluting that series yields the molecular mass.

That measured mass is compared against the theoretical mass calculated from the compound's formula or amino-acid sequence. Agreement within the instrument's tolerance is strong evidence of identity. Disagreement is diagnostic in its own right: a mass low by one residue points to a deletion sequence, a shift of roughly +16 suggests oxidation, and a mass bearing no relation to the expected value means the vial does not contain what the label says.

Where a certificate lists LC-MS, the two techniques were run coupled: the chromatograph separates the mixture and feeds each fraction into the mass spectrometer as it elutes, so identity can be assigned peak by peak rather than to the sample as a whole.

Chromatographic purity is a relative measurement, not an absolute statement about the contents of the vial. Four qualifications matter, and any honest supplier will state them plainly.

It only counts what the method can see

Area percent is calculated across species that dissolve in the mobile phase, elute from the column within the run time, and absorb at the detection wavelength. A contaminant failing any of those three conditions contributes no peak, and therefore no area — it is invisible to the calculation rather than counted as zero. This is why confirming identity by a second, orthogonal method is not optional.

It is not the same as net peptide content

A lyophilized peptide is not pure peptide by mass. Residual water, which these materials readily reabsorb from air, and counterion salt left over from purification both add to the weight in the vial without appearing as chromatographic impurities. A lot can be 99% pure by HPLC and still be meaningfully less than 99% peptide by mass. The two figures answer different questions, and net peptide content requires its own assay. Where a certificate does not list one, none was run.

Decimal places are not a ranking

Integration involves judgement about where a peak begins and ends, and repeat injections of the same sample do not return an identical figure. The difference between a 99.2% and a 99.4% result generally sits inside method variability. Treat a purity figure as a threshold that was cleared, not as a score for sorting suppliers.

It describes one lot, on one day

A certificate is a snapshot of the material sampled at the moment it was sampled. It says nothing about a different lot of the same compound, and nothing about how the material was handled afterwards. That is the entire reason lot codes exist, and the reason ours are printed on the vial and searchable below.

Every certificate we publish carries the same six fields, and the library is built on them, so a lot code taken off a vial label can be traced to its document in a single search.