High-performance liquid chromatography is the method behind almost every purity figure printed on a peptide certificate of analysis. Understanding what that number is a measurement of is the difference between reading a certificate and trusting one.
How the separation works. A dissolved sample is pushed under pressure through a column packed with a stationary phase. Components in the sample interact with that packing to different degrees, so they travel through at different speeds. What entered the column as one mixed band leaves it as a series of separated bands, and a detector at the outlet records each one as it emerges.
Why “reverse-phase”. In reverse-phase HPLC the stationary phase is non-polar and the mobile phase is polar — the reverse of the older arrangement, hence the name. A gradient makes the mobile phase progressively less polar during the run, so components are released roughly in order of how hydrophobic they are. For peptides this separates the intended sequence from truncated sequences, deletion products and oxidised variants, which is precisely the population of impurities peptide synthesis tends to produce.
How the peptide is detected. Detection is usually ultraviolet absorbance at a wavelength around 210–220 nm, where the peptide bond itself absorbs. Detecting the bond rather than a particular side chain means the method responds to peptides generally rather than only to sequences containing an aromatic residue.
What “98.6% by RP-HPLC” is actually saying. Almost always: area percent. The integrated area beneath the main peak, divided by the total integrated area of every peak in the run, as a percentage. It is a statement about proportion within what the detector recorded.
The caveat that follows from that. Area percent assumes every component responds to the detector in proportion to how much of it is present, and that assumption is imperfect. A component that absorbs weakly at the detection wavelength is under-counted. A component that does not absorb at all is not in the calculation, because the detector never saw it — and that category includes water, residual solvents and counter-ions, which in a lyophilized peptide can be a significant fraction of the vial by mass. A peptide legitimately reported at 98.6% purity by RP-HPLC may still be a minority of the powder by weight.
Which is why net peptide content is a separate question. How pure the peptide fraction is, and how much peptide is in the vial, are two different determinations requiring two different methods. A certificate reporting only the first has not answered the second, and neither figure is wrong — they are answers to different questions.
What can hide inside a good-looking number. Two components that co-elute — leave the column at the same time — are integrated as one peak, so a shoulder on the main peak may be a separate substance being counted as product. A trace cropped to a window around the main peak can exclude late-eluting impurities from view without excluding them from the material. And a purity figure quoted with no method, no wavelength, no run length and no chromatogram cannot be checked at all.
What to ask of a certificate. The method and its parameters, the detection wavelength, the full uncropped trace, the integration table behind the percentage, the acceptance criterion the result was judged against, and the identity of the laboratory that produced it. Each of those is checkable. A number on its own is not.
Why a retention time is not an identity. A peak appearing at the expected retention time is consistent with the expected compound; it is not proof of molecular identity the way a mass measurement is. Retention time is a property of a compound under one specific method, not a fingerprint. That is one reason identity and purity are tested and reported separately.