High-Performance Liquid Chromatography (HPLC) is the standard technique for measuring the purity of a peptide sample. It separates the components of a mixture based on how they interact with a stationary phase inside a chromatography column, and reports the result as a percentage area under the main peak.
How the separation works
A small volume of the dissolved sample is injected into a stream of solvent (the mobile phase) that flows through a packed column (the stationary phase). Different molecules travel through the column at different speeds depending on their affinity for the stationary phase. A detector at the far end records each component as it exits, producing a chromatogram of peaks over time.
Reverse-phase HPLC for peptides
Peptides are typically analysed by reverse-phase HPLC (RP-HPLC) using a C18 column with a water/acetonitrile gradient acidified with 0.1% trifluoroacetic acid. UV detection at 214 nm captures the absorbance of the peptide backbone, so every peptide-containing peak is visible regardless of aromatic residues.
Reading the chromatogram
The main peak represents the target peptide. Smaller peaks eluting before or after it are related impurities — typically truncated sequences, deletion products, or oxidised variants. Purity is calculated as the area of the main peak divided by the sum of all integrated peaks, expressed as a percentage.
What HPLC does not tell you
HPLC measures how much of the injected material elutes as a single peak. It does not confirm the identity of that peak. Identity is established separately by mass spectrometry. A COA that reports HPLC purity without a matching mass spectrometry result is incomplete.