HPLC vs mass spectrometry: what each peptide test tells you
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Two tests appear on almost every peptide certificate of analysis: HPLC and mass spectrometry (MS). They answer different questions. HPLC tells you how pure the sample is. Mass spectrometry tells you whether it is the right molecule at all. Here is how each one works and how to read the results.
HPLC: how pure is it?
HPLC stands for high-performance liquid chromatography. A small amount of the dissolved sample is pushed at high pressure through a column packed with fine particles. Different molecules travel through the column at different speeds, so they come out at different times.
A detector at the end records each one as a peak on a graph called a chromatogram. The target peptide should produce one large peak; impurities, such as shorter or incomplete sequences left over from synthesis, show up as small peaks either side.
The purity figure is the area of the main peak as a share of all the peaks. A result of 99.40%, as on our TB-500 certificate, means 99.40% of what the detector saw was the target peptide.
What HPLC can't tell you
- It doesn't confirm identity. A sample of the wrong peptide can still give one clean peak and a high purity figure.
- It doesn't see water or salts. That is why peptide content and water content are measured separately.
Mass spectrometry: is it the right molecule?
Mass spectrometry weighs molecules. The sample is turned into charged particles, most often by electrospray ionisation (the "ESI" in "MS (ESI)" on a certificate), and the instrument measures their mass.
Every peptide has a known molecular weight worked out from its sequence. TB-500 (thymosin β4), for example, has a molecular weight of about 4,963 g/mol; CJC-1295 without DAC is about 3,368 g/mol. If the measured mass matches the expected one, the certificate records the identity test as "Conforms".
What mass spectrometry can't tell you
- On its own, it doesn't give a precise purity figure.
- Two molecules with the same mass but a different structure can be hard to tell apart without further tests.
Why a good certificate shows both
| Test | Question it answers | How it appears on a COA |
|---|---|---|
| HPLC | How pure is the sample? | A percentage, e.g. 99.40%, against a specification such as ≥ 98.0% |
| Mass spectrometry | Is it the right molecule? | "Conforms" against the expected molecular weight |
Together, they show the sample is both the right compound and highly pure. Purity without identity, or identity without purity, leaves a gap.
Where to see both on our certificates
Each product page on our site shows its lab results, including HPLC purity and, where the certificate includes it, identity confirmed by mass spectrometry. The full certificate is there to download. For a line-by-line walkthrough of everything else on a certificate, read our guide on how to read a peptide certificate of analysis, or browse our research peptides.
All products sold by Absolute Peptides are for laboratory research use only. Not for human or veterinary use.