The short version
When labs say a peptide is “verified,” they usually mean some combination of:
- HPLC (How pure is it? What else is in the vial?)
- MS / LC–MS (Is it the right molecule? Are there near-matches or degradants?)
Understanding what each technique can—and can’t—tell you will help you interpret COAs, compare suppliers, and troubleshoot odd experimental results.
What HPLC actually tells you (and what “% purity” usually means)
Analytical HPLC separates components and reports a chromatogram: peaks over time.
Most “purity %” numbers are based on peak area (often UV detection). In practice, that means:
- If one main peak dominates, you’ll see a high purity percentage.
- Small impurities can hide under shoulders, overlap peaks, or have weak UV response.
- Purity is method-dependent: change the gradient/column and the peak profile can change.
Good signs on an HPLC report
- A clear chromatogram image (not just a number)
- Stated method basics (column type, detection wavelength, gradient notes)
- A retention time for the main peak (useful for lot-to-lot comparison)
Why RP-HPLC is common for peptides
RP-HPLC has become a workhorse because it’s broadly applicable to peptides from many sources and sizes—synthetic and biological.
What MS tells you (and why LC–MS is even better)
Mass spectrometry is excellent for confirming identity because it measures molecular mass with high specificity. But peptides can still be tricky:
- Some impurities have nearly identical masses
- Adducts (salts/solvents) can shift observed mass
- Isomers can share the same mass
That’s why many workflows pair chromatography and MS:
- LC separates
- MS identifies
When fragmentation (MS/MS) is used, it can provide even more confidence in sequence-related questions.
A practical way to read a COA (in 60 seconds)
When you open a COA, scan in this order:
- Lot number & sample ID — can you trace it later?
- HPLC chromatogram — is it shown? does it look clean?
- Purity statement — is it tied to a method?
- MS result — is the expected mass shown clearly?
- Notes on handling/storage — do they acknowledge peptide-specific stability issues?
If you only get a purity number with no chromatogram and no MS detail, you’re flying blind.
Why this matters for reproducibility
Peptides can fail “silently.” You don’t always get a dramatic problem—often you get:
- shifted dose-response curves
- inconsistent replicates
- unexpected baseline activity
- lot-to-lot drift
When that happens, the fastest way to narrow the problem is to re-check identity and impurity profile using these same two tools.
