[Sam Tiktin]

HPLC and mass spectrometry are the two tests you’ll see on nearly every peptide COA, and they get discussed almost interchangeably even though they measure completely different things. This post breaks down what each test actually tells you.

HPLC: Measuring Purity

High-performance liquid chromatography (HPLC) works by pushing a liquid sample through a column packed with material that separates its components based on chemical properties, most commonly reversed-phase separation for peptides. As each separated component exits the column, a detector (typically UV absorbance around 210–220 nm for peptide bonds) records a peak. The area under each peak, as a percentage of the total area of all peaks, is reported as the purity percentage.

What HPLC tells you: how much of the sample is the target peptide versus other substances (synthesis byproducts, degradation products, or unrelated impurities) that separate out as distinct peaks. What it doesn’t tell you on its own: whether the largest peak is actually the peptide you think it is — HPLC identifies purity relative to whatever peaks are present, not the chemical identity of any of them.

Mass Spectrometry: Confirming Identity

Mass spectrometry (MS) measures the mass-to-charge ratio of ionized molecules in the sample, producing a spectrum with a peak (commonly reported as [M+H]+ for a singly protonated peptide) at the observed molecular mass. That observed mass is compared against the theoretical mass calculated from the peptide’s amino acid sequence.

What MS tells you: whether the substance in the sample actually has the molecular weight expected for the labeled peptide. A close match (ideally within a small fraction of a percent on modern instruments) supports that the right molecule is present. What it doesn’t tell you on its own: how much of the sample is that molecule versus other substances — that’s HPLC’s job.

Why You Need Both

A high HPLC purity number with no mass spectrometry confirmation could describe a very pure sample of the wrong molecule. Conversely, a mass spectrometry result confirming the right molecular weight says nothing about how much unrelated material is mixed in. A trustworthy COA reports both tests together, ideally with the underlying chromatogram and spectrum available, not just summary percentages.

Common Misunderstandings

“99% pure” from HPLC alone is sometimes treated as a complete quality guarantee — it isn’t, without a matching identity confirmation. Similarly, a mass spec result confirming identity doesn’t say anything about purity level. Both numbers answer different questions, and neither substitutes for the other.

Related Reading

See our guide to how to read a peptide Certificate of Analysis for how these tests fit into a full COA, and what “99% purity” actually means for a deeper look at that specific number. Browse COAs, Lab Testing & Documentation for related discussion.

Questions for Discussion

Have you seen COAs that report only one of these two tests, and how did that affect your confidence in the product? What third-party lab services have members used to independently run HPLC or MS on a purchased batch? What other analytical methods (NMR, amino acid analysis) would you want to see on a more thorough COA?

This post is educational information about analytical chemistry methods and does not constitute a recommendation to purchase or use any research compound.

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