Lab Testing · 12 min read
How to Verify Peptide Purity: Reading an HPLC Certificate of Analysis
Step-by-step reference on how to read an HPLC Certificate of Analysis, what purity percentages mean, how mass spectrometry confirms identity, and the red flags that indicate a low-quality vendor.
How to Verify Peptide Purity
For research use only. Not for human consumption.
A Certificate of Analysis (CoA) is the document that proves what is actually inside a research peptide vial. This guide walks through every section of a credible HPLC CoA, what the numbers mean, and the red flags that distinguish a research-grade supplier from a hobbyist re-packer.
1. What a CoA is
A CoA is a third-party or in-house analytical report that ties a specific physical lot of material to its measured characterization. The lot number on the CoA must match the lot stamped on the vial. Without that match, the certificate is unverifiable.
2. The HPLC chromatogram
Reverse-phase High-Performance Liquid Chromatography (HPLC) separates the target peptide from its synthesis by-products by running the sample through a C18 column with a gradient of water and acetonitrile. The detector (typically UV at 214 nm) records absorbance over time, producing a chromatogram of peaks.
What to look for:
- A single dominant peak carrying ≥99% of the total area
- Symmetric peak shape — a sharp leading edge with a clean tail
- Stable baseline with no large unresolved humps
- Retention time listed in minutes and reproducible across runs
3. The purity percentage
Purity is reported as the percentage of total chromatogram area that belongs to the target peak. Research-grade material should be ≥99.0%. Anything published in the 95–98% range is acceptable for some applications but is not premium.
4. Mass spectrometry confirmation
HPLC tells you how much of one thing is present; it does not tell you what that thing is. Mass spectrometry — usually ESI-MS or MALDI-TOF — measures the molecular mass of the dominant peak. The reported mass must match the theoretical monoisotopic or average mass of the intended sequence within instrument tolerance.
A CoA without mass-spec confirmation is incomplete.
5. Required CoA fields
A credible CoA includes all of:
- Vendor name and address
- Analyzing laboratory name
- Peptide name and amino-acid sequence
- Molecular formula and theoretical molecular weight
- Lot number
- Manufacture date and retest / expiration date
- HPLC method (column, gradient, detector, flow rate)
- HPLC chromatogram with purity %
- Mass spectrum with observed vs theoretical mass
- Analyst name and signature
6. Red flags
- The same CoA shown for every lot of every product
- Purity given as "≥98%" with no chromatogram attached
- Missing mass-spec data
- No manufacture date or no retest date
- No analyst signature or unsigned PDF
- Lot number on the CoA does not match the vial
- CoA is a photo, not a PDF
7. Related references
Educational reference. All products supplied for research use only.
Frequently Asked Questions
What HPLC purity is considered research grade?+
99% or higher by area under the HPLC chromatogram, with mass spectrometry confirming the correct molecular mass, is the standard for serious research-grade peptides.
Does a CoA need to match the specific lot I receive?+
Yes. A CoA is only meaningful when its lot number matches the lot printed on the vial you receive. A generic CoA without a lot match is not traceable evidence.
What red flags indicate a low-quality peptide vendor?+
No published CoA, generic CoA not tied to your lot, no mass-spec confirmation, missing analyst signature, no manufacture or retest date, and purity values that look identical across every lot.