A peptide analysis has to answer two separate questions. Is it the right molecule? That's identity. How much of the sample is that molecule? That's purity and content. Different instruments answer each one.
HPLC: measuring purity
Analytical high-performance liquid chromatography pushes a dissolved sample through a packed column. Each component leaves the column at a characteristic retention time and is detected by UV absorbance, usually around 214–220 nm, where peptide bonds absorb strongly.
The output is a chromatogram: a trace with a peak for each component. Purity is normally reported as the target peak's area as a percentage of the total peak area. A result of "99.1% (HPLC)" means 99.1% of the UV-absorbing material came out as the main peak.
What HPLC can't tell you
- It doesn't prove the main peak is the right peptide. That needs mass spectrometry.
- It doesn't see things that don't absorb UV, such as water, salts or counter-ions.
- Impurities that come out at the same time as the main peak can hide inside it.
Mass spectrometry: confirming identity
Mass spectrometry (MS) ionises molecules and measures their mass-to-charge ratio. The observed molecular mass is compared with the mass calculated from the expected sequence. A match within the instrument's tolerance confirms identity. Common configurations are LC-MS (HPLC coupled directly to MS) and MALDI-TOF. Tandem MS (MS/MS) can go further and fragment the peptide to confirm the sequence itself.
Purity is not the same as content
This is the most commonly misunderstood point. A lyophilised peptide powder isn't 100% peptide, even when HPLC purity is 99%. The powder also contains:
- Counter-ions, such as TFA or acetate, bound to charged residues
- Residual water, since peptides absorb moisture from the air
The share that is actually peptide is called the net peptide content. It's often 60–90% and is measured by methods such as amino acid analysis or nitrogen determination. Labs sometimes also report quantity: the actual mass of peptide in a sample versus the amount on the label.
Other tests you may see
| Test | What it checks |
|---|---|
| Endotoxin (LAL) | Bacterial endotoxin contamination |
| Sterility | Microbial contamination |
| Heavy metals | Trace metal contamination, e.g. by ICP-MS |
| Residual TFA / solvents | Left-over chemicals from synthesis and purification |
| Water content (Karl Fischer) | Moisture in the powder |
How to read a certificate of analysis (COA)
A credible COA should let you answer each of these questions:
- Who tested it? Is the lab named and independent of the manufacturer?
- What exactly was tested? Look for a batch or lot number that ties the report to a specific production run.
- When? Look for a test date. Results describe that batch on that date only.
- Identity: are the expected and observed masses both shown?
- Purity: is the method stated, and is the chromatogram included rather than just a number?
- Can it be verified? Some independent labs, including Janoshik Analytical, let you check a report against their own records online. A report that can't be traced back to the lab is much weaker evidence.
Red flags: no chromatogram, no batch number, a lab name with no traceable contact details, identical results across different batches, or a report issued by the seller's own lab.
Related articles
- How laboratories validate analytical methods
- Antimicrobial peptides: an ancient defence against modern superbugs
- How clinical trials are designed
Sources and further reading
- Mant CT et al. HPLC analysis and purification of peptides. Methods Mol Biol 2007;386:3–55. doi:10.1007/978-1-59745-430-8_1 · PMID: 18604941
- Steen H, Mann M. The ABC’s (and XYZ’s) of peptide sequencing. Nat Rev Mol Cell Biol 2004;5:699–711. doi:10.1038/nrm1468 · PMID: 15340378
- Fenn JB et al. Electrospray ionization for mass spectrometry of large biomolecules. Science 1989;246:64–71. doi:10.1126/science.2675315 · PMID: 2675315
- International Council for Harmonisation. Quality guidelines, including Q2(R2) Validation of Analytical Procedures. www.ich.org/page/quality-guidelines