LC-MS peptide identity: mass confirmation versus purity

Two different questions

A chromatographic purity result can be excellent for a compound that is not the one you ordered. Purity describes homogeneity; identity describes what the homogeneous thing is. This is the single most common misreading of a peptide certificate.

How identity is established

In LC-MS, the HPLC eluate passes directly into a mass spectrometer, usually with electrospray ionisation. Peptides acquire multiple protons, producing a series of multiply charged ions; the instrument records mass-to-charge ratios which are deconvoluted into a neutral molecular mass.

That observed mass is compared against the mass calculated from the compound's molecular formula. Agreement within the instrument's stated tolerance supports the identity claim. Because the mass spectrometer is coupled to the separation, the analyst also learns which chromatographic peak corresponds to which mass — so related substances can be characterised rather than merely counted.

What mass agreement cannot resolve

• Isomers and sequence-scrambled species of identical composition share a molecular mass and are not distinguished by mass alone.
• A correct mass does not indicate quantity; content is measured separately by quantitative HPLC.
• Poorly ionising or suppressed species may be under-represented in the spectrum.
• Nothing about sterility, endotoxin or activity is established by mass measurement.

Calculated mass at Pure Helix

Every entry in the Pure Helix Molecular Research Index publishes the compound's molecular formula and its average molecular mass. Where a mass is not carried on the source record, it is computed arithmetically from the formula using IUPAC standard atomic weights and is labelled as calculated — never presented as a measured value.

Research use

Pure Helix supplies materials for laboratory research use only. Nothing on this page is medical advice, and none of the material described is for human or veterinary use.