When analysing a synthetic peptide, two questions come first: is the material the expected molecule? and how much of it is that molecule? Mass spectrometry answers the first; high-performance liquid chromatography (HPLC) answers the second.
HPLC: separate to measure
In reversed-phase HPLC, the sample passes through a column packed with hydrophobic particles. Each component interacts differently with the column and elutes at a characteristic time (retention time). A UV detector — usually at 214–220 nm, where the peptide bond absorbs — records the peaks.
Purity is calculated as the main peak area divided by the total peak area. That is why the chromatogram matters: it shows whether impurities sit close to the main peak, such as truncated or deletion sequences.
Mass spectrometry: confirm identity
MS ionises molecules and measures their mass-to-charge ratio (m/z). Peptides often appear with multiple charges ([M+2H]²⁺, [M+3H]³⁺…), and software reconstructs the original mass. If the observed mass matches the theoretical mass within instrument tolerance, identity is confirmed.
Why use both
A wrong molecule can show up as a single clean HPLC peak; a right molecule can be mixed with impurities that MS alone does not quantify. HPLC + MS — sometimes coupled as LC-MS — is the minimum standard expected for research materials.
Other relevant tests
- Peptide content (amino acid or nitrogen analysis): how much of the powder's mass is peptide, net of water and salts.
- Endotoxins (LAL): important for cell or animal experiments.
- Moisture and counter-ions: affect the calculation of the actual peptide amount.