Why one test isn't enough
No single method characterizes a peptide. HPLC separates components but doesn't identify them. Mass spectrometry identifies the molecule but doesn't say how much of it there is. Endotoxin testing looks for something else entirely. A complete CoA combines methods so each covers what the others miss.
HPLC: purity
Reverse-phase high-performance liquid chromatography (RP-HPLC) pushes a dissolved sample through a column packed with a hydrophobic stationary phase, typically C18. A gradient of water and acetonitrile, usually with 0.1 % trifluoroacetic acid, elutes components at different times according to their hydrophobicity.
A UV detector set near 214 nm, where peptide bonds absorb, records each component as a peak. Purity is the area of the main peak as a percentage of total peak area.
- What it shows: how cleanly the target separates from synthesis by-products such as truncated or deletion sequences.
- Its limits: impurities that co-elute with the main peak, or that don't absorb at the detection wavelength, are invisible. HPLC alone doesn't confirm identity.
Mass spectrometry: identity
Mass spectrometry ionizes the sample and measures mass-to-charge ratios. Electrospray ionization (ESI) and MALDI-TOF are the common techniques for peptides. The observed molecular mass is compared with the theoretical mass calculated from the sequence and its modifications.
- What it shows: that the main component has the expected mass, which confirms the sequence, modifications and disulfide state are consistent with the specification.
- Its limits: molecules with the same mass, such as D- and L- isomers, can't be told apart by mass alone, and MS isn't quantitative on its own.
Content: how much peptide is in the vial
Content compares the measured amount of peptide with the label claim. The common approach is an HPLC assay against a characterized reference standard. Amino acid analysis and nitrogen determination are alternatives.
Content matters because lyophilized powder also contains water and counter-ions. PepDistro specifies content at ± 10 % of the label claim.
Endotoxin: LAL testing
Endotoxins are lipopolysaccharides from the outer membrane of Gram-negative bacteria. Even trace amounts can interfere with cell-based and other sensitive assays, which is why research buyers check for them.
The Limulus amebocyte lysate (LAL) test detects endotoxin through a clotting cascade. In the kinetic chromogenic format, the reaction releases a colored product read over time against a standard curve. Results are reported in endotoxin units per milligram (EU/mg). Recombinant factor C (rFC) assays are an animal-free alternative.
At a glance
| Method | Measures | Reported as | Doesn't show |
|---|---|---|---|
| RP-HPLC | Separation of components | % main peak area | Identity; co-eluting impurities |
| Mass spectrometry | Molecular mass | Observed vs. theoretical mass | Quantity; same-mass isomers |
| HPLC assay | Peptide quantity | mg per vial vs. label | Identity on its own |
| LAL | Bacterial endotoxin | EU/mg | Other contaminants |
See the specifications PepDistro tests every lot against on Quality & testing.