Certificates of Analysis usually report two distinct numbers: an HPLC purity percentage and a mass spectrometry (MS) result. They measure different things, and understanding the difference helps you read a COA correctly instead of treating "99% pure" as the entire story.
What HPLC actually measures
High-Performance Liquid Chromatography separates the components of a sample as they pass through a column, producing a chromatogram where each compound appears as a peak at a specific retention time. Purity is calculated as the area of the target peak relative to the total area of all peaks — in other words, HPLC tells you how much of the sample is "the main thing" versus impurities or byproducts, not what that main thing actually is.
What MS actually measures
Mass spectrometry ionizes the sample and measures the mass-to-charge ratio of the resulting ions, which reveals the molecular weight of the compound present. This confirms identity — that the peptide is structurally the sequence it claims to be — rather than quantity.
Why you need both
A sample could show 99% HPLC purity for the wrong compound entirely, if that compound simply happens to dominate the chromatogram. Conversely, MS could confirm the correct molecular weight while HPLC reveals meaningful impurity levels alongside it. Only together do the two tests confirm "this is the right molecule, and it's present in high purity."
Reading a typical report
- Retention time: Where the main peak appears on the chromatogram — useful mainly for comparing consistency across batches from the same method.
- Peak area %: The HPLC purity figure, usually the headline number on a COA.
- Observed vs. expected mass: The MS result, ideally within a small margin of the compound's theoretical molecular weight.
A note on limitations
Neither test tells you about biological activity or functional performance — they characterize chemical identity and purity only. For research purposes, that's exactly what these tests are designed to do, and it's worth not over-reading them as answering questions they weren't designed to answer.