How to Read a Peptide COA: HPLC Purity and Mass Spec Explained
A Certificate of Analysis is only useful if you know how to read it. This guide walks through HPLC purity, chromatograms, HPLC standards, mass spec identity, lyophilised powder and net peptide content.
What a COA should show
A Certificate of Analysis (COA) is the lab report for a peptide. A useful one includes:
- the peptide name and sequence or molecular formula
- a lot or reference number that matches the vial
- appearance (for example, white lyophilised powder)
- purity by HPLC, as a percentage, with the chromatogram
- identity by mass spectrometry, comparing the measured mass with the expected mass
- the testing lab and date
Every AussiePeptides product is backed by a verified COA showing ≥99% HPLC purity.
How HPLC works
High-performance liquid chromatography (HPLC) pushes a dissolved sample through a packed column under high pressure. Different molecules travel through the column at different speeds, so they come out at different times. A UV detector records each one as a peak.
| Setting | Typical choice for peptides |
|---|---|
| Column | Reverse-phase C18 |
| Mobile phase | Water and acetonitrile, often with 0.1% TFA |
| Elution | Gradient (rising acetonitrile) |
| Detection | UV at about 214 to 220nm, where peptide bonds absorb |
Reading the chromatogram
The result is a chromatogram: a line with one tall main peak (your peptide) and, ideally, only tiny peaks around it (impurities). Purity is the area of the main peak as a percentage of the total peak area.
- Main peptide peak99.2%
- Impurity peak 10.5%
- Impurity peak 20.3%
Illustrative example of how a purity figure is built from peak areas. Always read the actual chromatogram on your COA.
| Purity | What it usually means |
|---|---|
| ≥99% | High purity, suitable for most research |
| 98 to 99% | Good, minor impurities present |
| Below 95% | Significant impurities; results may be affected |
What HPLC standards are
An HPLC standard (or reference standard) is a sample of the compound with a known identity and purity. Running it on the same method shows the exact retention time where the real peptide should appear. Labs use standards to confirm that the main peak in a sample is the right compound and that the method is working properly. When people compare "HPLC standards" between labs, they are usually comparing these reference materials and method settings.
Why mass spec matters too
HPLC tells you how pure a sample is, not what it is. A wrong peptide can still produce one clean peak. Mass spectrometry closes that gap by measuring the molecular mass. If the measured mass matches the theoretical mass of the sequence, within a small tolerance, the identity is confirmed.
Lyophilized powder and net peptide content
Lyophilisation (freeze-drying) works in three stages:
- Freezing: the peptide solution is frozen solid.
- Primary drying: pressure is lowered so the ice turns straight to vapour (sublimation).
- Secondary drying: remaining bound water is removed with gentle heat.
What is left is a dry, stable cake or powder. That powder is not 100% peptide by weight: it also holds a little water and counter-ions such as acetate or TFA from synthesis. So HPLC purity (how much of the peptide material is the right peptide) and net peptide content (how much of the powder's weight is peptide) are different numbers.
Quick COA checklist
- Does the lot number match your vial?
- Is there a chromatogram, not just a percentage?
- Is there a mass spec identity result?
- Is the purity ≥98 to 99% for research use?
- Is the lab and test date shown?
Further reading
For laboratory research use only. Not for human consumption. Not medical or legal advice.
