Two questions every sample has to answer
Any analysis of a synthetic peptide comes down to two questions. Is the main component the molecule it is supposed to be? And how much of the sample is that molecule rather than something else? Chromatography answers the second. Mass spectrometry answers the first. Neither can stand in for the other.
How HPLC measures purity
Peptides are usually analyzed by reversed-phase HPLC. The sample is loaded onto a column packed with a nonpolar material (commonly C18) and carried through it by a solvent gradient, typically water and acetonitrile with an acid modifier. Components that interact more strongly with the column come off later, so each one leaves at its own retention time.
A UV detector set to the wavelength where peptide bonds absorb records each component as a peak. Purity is the main peak's area as a share of the total area of all peaks, reported as a percentage. The remainder is usually related substances: sequences missing a residue, sequences with a protecting group left on, oxidized or deamidated forms.
What HPLC can't tell you
- Identity. A clean single peak shows that the sample is mostly one thing. It doesn't show which thing.
- Hidden impurities. A contaminant that leaves the column at the same time as the main peak, or doesn't absorb UV, won't appear as a separate peak.
- Non-peptide content. Water and salt counter-ions are invisible to the method, which is why purity is not the same as net content.
How mass spectrometry confirms identity
A mass spectrometer turns molecules into charged ions and measures their mass-to-charge ratio. With electrospray ionization, the method most often paired with HPLC, a peptide picks up several charges at once and appears as a series of peaks; software combines them to calculate the molecule's mass. MALDI-TOF is a common alternative that usually produces a single charge state.
The observed mass is compared with the theoretical mass calculated from the sequence. A match within the instrument's tolerance confirms that the sample is the expected molecule. A mismatch means a different molecule, a modified one, or a synthesis error.
What mass spectrometry can't tell you
- Purity or quantity on its own. Different molecules ionize with different efficiency, so peak height is a poor measure of how much of each is present.
- Same-mass variants. Molecules with identical mass, such as a sequence with one amino acid in its mirror-image form, can look the same without further work.
LC-MS: both in one run
Liquid chromatography–mass spectrometry connects the two instruments. The HPLC separates the sample, and the mass spectrometer weighs what comes off the column. That shows the mass of the main peak and of the impurities around it, which is often how a lab identifies what an impurity actually is.
Reading the two results together
| HPLC result | Mass result | What it means |
|---|---|---|
| High purity | Matches the target | The right molecule, with little else present |
| High purity | Doesn't match | A clean sample of the wrong molecule |
| Lower purity | Matches the target | The right molecule, alongside related impurities |
| Not run | Matches the target | Identity confirmed, purity unknown |
| High purity | Not run | A clean sample of an unconfirmed molecule |
What to look for on a report
- Both methods named, not only a purity percentage
- The chromatogram, or at least the main peak's retention time and area
- An identity result stated against the expected molecule
- The lot number the analysis applies to
Every certificate in the Lumina Labs COA Library lists the methods behind its figures. For an example of purity and identity reported together, see the Semax certificates. For a field-by-field guide to the rest of a report, read How to read a peptide COA.
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