HPLC answers “how pure is it?”
High-performance liquid chromatography (HPLC) pushes the dissolved sample through a column that separates its components. A detector, typically reading ultraviolet light at about 214 nm where peptide bonds absorb, draws a chromatogram: one peak per component.
Purity is the main peak’s share of the total peak area, reported as area %. A result such as “99.7 area %” means the main peak makes up 99.7% of what the detector saw.
What HPLC does not tell you: what that main peak actually is. A wrong compound can still produce one clean, tall peak.
Mass spec answers “is it the right molecule?”
Mass spectrometry (MS), often run after a liquid chromatography step (LC-MS), measures the mass of the molecules in the sample. The lab compares the observed mass with the compound’s theoretical mass in daltons (Da). A close match, for example within ±1 Da, confirms identity.
What mass spec does not tell you on its own: how much of the material is that molecule.
Why a certificate needs both
| Question | HPLC | Mass spec |
|---|---|---|
| Is it the right compound? | No | Yes |
| How pure is it? | Yes | No |
| How much is in the vial? | Yes, with a reference standard (quantitative HPLC) | No |
A certificate with a high purity figure but no identity data could describe a very pure sample of the wrong thing. A certificate with identity but no purity says nothing about what else is in the vial.
What to look for
- Both an HPLC purity result and a mass-spec identity result.
- The specification each was judged against, not just “pass”.
- For blends, identity confirmed for each component.