Two techniques dominate peptide certificates of analysis: HPLC and LC-MS. They are often listed side by side as if they measure the same thing. They do not. HPLC tells you how pure a sample is; LC-MS tells you what the sample is. A complete COA needs both, and understanding why is the difference between reading a certificate and merely glancing at it.
What HPLC purity proves
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 depending on how strongly they interact with the stationary phase. As each component exits, a detector records a signal. The result is a chromatogram: a trace of detector response over time.
Purity is calculated from the area under each peak. The target peptide's peak area is divided by the total area of all peaks, and the result is reported as a percentage, commonly with a specification of ≥98%.
What HPLC proves is relative abundance: how much of the detectable material is one dominant species versus everything else. What it does not prove is the identity of that dominant species. The column and detector do not know whether the big peak is your peptide or a different molecule that happens to elute at a similar time.
How to read a chromatogram
Three features carry the information:
- Peaks — each vertical spike is a resolved component. One tall, sharp main peak with a few tiny impurity peaks is the expected shape for a high-purity sample.
- Retention time (RT) — the time in minutes at which a peak elutes. It is characteristic for a given compound and method, and is one of two things (the other being mass) used to argue identity.
- Area % — the integrated area of each peak relative to the total. This is the actual purity math; peak height alone can mislead because peaks differ in width.
A purity percentage with no chromatogram attached is an unverifiable claim. You cannot see whether the number came from one clean peak or from software integrating a messy baseline. The chromatogram is the evidence; the percentage is only its summary.
UV versus ELSD / CAD detection
Most peptide HPLC uses UV detection, typically at 214 nm, where the peptide bond absorbs strongly. UV is sensitive but response depends on a molecule's chromophores, so it can under- or over-represent species that absorb differently. ELSD (evaporative light scattering) and CAD (charged aerosol detection) respond more to mass than to optical properties, which can reveal non-UV-absorbing impurities such as counter-ions or excipients. A thorough lab may report more than one detection mode; the detection method should always be stated on the certificate.
What LC-MS identity proves
LC-MS couples the same liquid-chromatography separation to a mass spectrometer. After a component elutes, it is ionized and its mass-to-charge ratio (m/z) is measured. Because every peptide sequence has a specific, calculable mass, the measured mass is a direct test of identity.
Monoisotopic mass, ESI and m/z
A few concepts make the identity line readable:
- Monoisotopic mass — the mass calculated using the most abundant isotope of each element. It is the precise target value a lab compares against, distinct from the average molecular weight used elsewhere on the COA.
- ESI (electrospray ionization) — the standard soft-ionization method for peptides. It adds one or more protons, producing ions such as [M+H]⁺, [M+2H]²⁺, and so on.
- m/z — the axis the instrument actually measures. For a doubly charged ion the observed m/z is roughly half the molecular mass, which is why the certificate should state the charge state it is reporting.
When the found mass matches the expected mass for the sequence at the top of the certificate, identity is confirmed. When it does not, no purity figure can rescue the batch.
Why you need both: "98% of the wrong molecule"
The single most important idea on any peptide COA is that purity and identity are independent. Imagine a batch that runs as one clean, sharp peak at 99% area. HPLC alone would call that excellent. But if the mass spectrometer measures a mass that does not match the intended sequence, that clean 99% peak is simply 99% of the wrong compound. It is pure, and it is not what the label says.
HPLC answers "how much of one thing?" LC-MS answers "which thing?" Purity without identity, or identity without purity, is half a certificate. Reputable third-party laboratories such as Janoshik report both because either number alone is incomplete.
What a complete analytical section looks like
Pulling it together, a certificate you can actually verify shows:
- An HPLC purity percentage with a stated method and detection wavelength.
- The chromatogram image, so peaks, retention time and area can be inspected, not just trusted.
- An LC-MS identity result giving the found versus expected mass and the ion / charge state.
- Ideally the MS spectrum alongside the number, following the same evidence-not-assertion logic as the chromatogram.
These practices align with pharmacopeial expectations (for example USP method chapters) and with ISO/IEC 17025 competence requirements: a result should be reproducible, method-referenced, and backed by the raw data it was derived from.