Analytical methods
How do you read the chromatogram and mass spectrum on a COA?
On a certificate, the HPLC chromatogram shows the separated components as peaks over time, and purity is the main peak's share of the total peak area. The mass spectrum shows ions by mass-to-charge ratio, and identity is confirmed when the mass worked out from those ions matches the peptide's expected molecular weight.
By Mason
What does an HPLC chromatogram show?
The horizontal axis is retention time, in minutes: how long each component took to pass through the column. The vertical axis is the detector's response, usually UV absorbance, often read near 214 or 220 nm where the peptide bond absorbs. Each component that separates from the rest draws its own peak.
How is purity read from the peaks?
The laboratory's software integrates each peak, measuring the area under it. Purity is the main peak's area as a percentage of the total area of the integrated peaks. Many certificates print a peak table beside the trace:
| Column | What it means |
|---|---|
| Peak | The peak's number, in order of retention time |
| Retention time | When the peak came off the column, in minutes |
| Area | The integrated area under the peak |
| Area % | The peak's share of the total area; for the main peak, this is the purity |
What do the small peaks mean?
Smaller peaks before and after the main one are usually related impurities from synthesis, such as chains missing a residue or still carrying a protecting group. A clean trace has one tall peak and small, well-separated neighbours. A main peak with a shoulder, or a broad hump, points to material the method did not fully separate.
What does a mass spectrum show?
The horizontal axis is m/z, the mass-to-charge ratio of each ion; the vertical axis is relative intensity. A peptide usually picks up one or more protons in the instrument, so the same molecule appears at several m/z values, one for each charge state: [M+H]+, [M+2H]2+, [M+3H]3+ and so on.
How do you check identity from the spectrum?
To recover the molecule's mass from an ion, multiply its m/z by its charge and subtract the mass of the added protons, about 1.007 each. An ion at m/z 710.8 carrying two charges works out to 2 × 710.8 − 2 × 1.007, about 1,419.6. Compare that with the expected molecular weight in the compound record; a match within the laboratory's stated tolerance confirms identity.
Check whether the certificate quotes a monoisotopic mass or an average molecular weight. For a mid-sized peptide they differ by about one unit, so the comparison has to use the same kind on both sides.
What if a certificate shows no trace or spectrum?
Some certificates print only the results table, with the purity percentage and the identity result. That is common, and the report is still checkable at the laboratory through its accession number. Where a trace or spectrum is printed, it is the evidence behind those two figures.
Questions
What is retention time on a chromatogram?
How long a component takes to pass through the HPLC column, in minutes. Under one method the same compound comes off at the same time, which is how its peak is recognised.
How is HPLC purity calculated?
As the main peak's area divided by the total area of all the integrated peaks, times 100.
What does m/z mean on a mass spectrum?
Mass-to-charge ratio. An ion carrying two charges appears at about half its mass, so the laboratory multiplies by the charge to recover the molecule's mass.
Why does one peptide show several peaks on a mass spectrum?
It carries different numbers of protons, and each charge state appears at its own m/z. Worked back to mass, they should all give the same molecular weight.
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