Why mass spectrometry confirms identity where HPLC confirms purity, and how the observed mass on a COA should match the theoretical molecular weight of the sequence.
Purity versus identity
HPLC answers ‘how much of the sample is one thing’. Mass spectrometry answers ‘what is that thing’. Both are needed: a chromatographically pure vial of the wrong sequence is still the wrong sequence, and this is precisely the failure mode that unverified suppliers produce.
LC-MS couples the two, separating the sample chromatographically and then measuring the mass-to-charge ratio of each species as it elutes.
Matching observed mass to theoretical mass
Every peptide sequence has a calculable monoisotopic and average molecular weight. Electrospray ionisation usually produces multiply charged ions — [M+2H]²⁺, [M+3H]³⁺ and so on — so the raw spectrum shows a series of peaks that deconvolute to a single molecular weight. On a certificate, the observed mass should agree with the theoretical mass within the instrument’s stated tolerance, commonly a fraction of a dalton on a high-resolution instrument.
A mass that is 16 Da high often indicates oxidation. A mass low by the weight of one residue indicates a deletion sequence. A mass matching a different sequence entirely indicates you should stop and contact the supplier.
What to check on the certificate
- Theoretical molecular weight, stated alongside the sequence and molecular formula.
- Observed molecular weight from the mass spectrometry run for that specific batch.
- The ionisation mode and instrument type, where reported.
- That the batch code on the MS report matches the vial in front of you.
References
Supplied for laboratory research use only. Not for human or veterinary use.