"99%+ HPLC purity" appears on almost every research peptide listing, but the method behind that number is worth understanding rather than taking on faith. Reverse-phase high-performance liquid chromatography (RP-HPLC) is the industry-standard technique for separating and quantifying the components of a peptide sample, and it's what most Certificates of Analysis are built on.
How the separation works
In RP-HPLC, a liquid sample is pushed under pressure through a column packed with a hydrophobic stationary phase. Different molecules in the sample — the target peptide plus any related impurities or truncated sequences left over from synthesis — interact with that stationary phase to different degrees, based on their size and hydrophobicity.
As a result, each component travels through the column at a different rate and exits at a different time, producing a series of peaks on a chromatogram rather than one blended signal. A detector, typically measuring UV absorbance, records each peak as the sample elutes.
Where the purity percentage comes from
Purity by RP-HPLC is generally reported as the target peptide's peak area as a percentage of the total peak area across the whole chromatogram. A 99%+ result means the target compound accounts for at least 99% of everything the detector registered — the remaining fraction is residual impurity from synthesis, not a separate unrelated contaminant.
This is a relative measurement, not an absolute mass balance, which is a useful nuance to understand: it tells you how clean the peptide fraction is relative to itself, rather than measuring against an external reference standard the way some other assays do.
Why it's paired with mass spectrometry
HPLC purity confirms how clean a sample is, but it doesn't independently confirm what the compound is. That's why many CoAs pair an HPLC purity result with mass spectrometry (MS), which measures the mass-to-charge ratio of the molecule to confirm its identity matches the expected sequence.
Together, the two methods answer two different questions a researcher needs answered before use: is this the right compound, and is it clean enough to trust in a controlled protocol.