What an HPLC purity figure measures
A purity percentage is a ratio of peak areas from one chromatographic run. What the figure covers, what it leaves out, and what to read beside it.
"≥ 99% (HPLC)" is the most quoted figure in this category and the least explained. It is a real measurement with a narrow meaning. This is the meaning.
A ratio of peak areas
High-performance liquid chromatography separates the components of a dissolved sample as they pass through a packed column. For peptides the usual mode is reversed-phase: a component leaves the column sooner or later according to how strongly it holds to a non-polar packing, and a detector records a signal as each one passes. Plotted against time, that record is the chromatogram. Every component the detector can see draws a peak.
The purity figure is arithmetic on that plot. The area under the main peak is divided by the summed area of every integrated peak, and the result is given as a percentage. A figure of 99.0% says that 99.0% of the detected signal belonged to the main peak in that run. It is a statement about a signal.
What the detector can see
Peptide methods usually read ultraviolet absorbance near 214 to 220 nm, where the peptide bond absorbs. A component that absorbs there and separates from the main one shows up as a peak of its own. A component that does not absorb there draws no peak at all.
Water draws none. The salts a peptide powder carries are typically not counted either: they leave the column with the solvent front, outside the window that is integrated. Both are in the vial. Neither is in the percentage.
What the figure leaves out
Three things a purity percentage does not state. Each has a test of its own.
- Identity. A peak shows that something left the column at a given time. It does not show what that something is. Identity is confirmed by mass spectrometry, where a measured mass is set against the mass computed from the molecular formula. A certificate that reports both shows them as separate results.
- How much of the powder is peptide. Lyophilised peptide carries residual water and the counter-ion of its salt form, commonly acetate or trifluoroacetate. The share of the powder's mass that is peptide is a different figure, usually called net peptide content, and it comes from a different method: amino acid analysis or nitrogen determination. A vial can read 99% by HPLC and hold less than 99% peptide by mass. The two figures answer different questions.
- Everything else. Endotoxin, residual solvents and microbial counts are separate tests with separate results. A purity percentage says nothing about them in either direction.
Why two laboratories can disagree
The figure depends on the method that produced it: the column, the solvent gradient, the detection wavelength, and where the software draws the baseline under small peaks. A shallower gradient can split one apparent peak into two. A different wavelength weights the components differently. Two laboratories can report different percentages for the same batch without either having made an error.
That is why a certificate that states its method is worth more than one that states only a number, and why a figure is read together with its date, its batch and the name of the laboratory that produced it.
Reading the figure on this site
A listing states where its purity figure comes from — a specification, a laboratory result, or both — and says on its face whether a batch certificate is published for it. The assay library lists every certificate on file and counts them. The count reads zero while there are none.
What a certificate has to carry before it is published is set out in Quality and methodology, and field by field in Five things a certificate has to state.