Peptide purity and testing methods explained

Three techniques do most of the work in peptide quality control. Each answers a different question, and none of them alone is sufficient.

HPLC for purity

Reverse-phase HPLC separates the components of a sample by how strongly they interact with a hydrophobic column as a solvent gradient is run through it. The target peptide elutes as a main peak; truncated sequences, deletion products and other impurities elute as smaller peaks. Purity is reported as the main peak's share of the total peak area. HPLC is excellent at showing how much of the sample is not the main component, but it cannot on its own confirm what the main component is.

Mass spectrometry for identity

Mass spectrometry measures the molecular mass of the compound, typically by electrospray ionisation (ESI) or MALDI. Comparing the measured mass with the mass calculated from the intended sequence confirms that the main peak is the peptide it should be. It also reveals common synthesis errors — a missing residue shows as a specific mass deficit. Mass spectrometry confirms identity; it is not a purity measurement.

Amino acid analysis for net content

Amino acid analysis hydrolyses the peptide into its constituent amino acids and quantifies them, giving both a check on the composition and a measurement of how much peptide is actually present by weight. This is the method for net peptide content, which HPLC purity does not provide. Elemental analysis or a peptide-content assay may be used instead.

Why the combination matters

A sample can be 99% pure by HPLC and still be the wrong peptide; mass spectrometry catches that. A sample can be the right peptide at high purity and still contain far less material by weight than the label implies; net content catches that. A thorough Certificate of Analysis reports identity and purity at minimum, and net content where the compound's use depends on accurate dosing of an experiment.

For laboratory research use only — not for human or veterinary use. This article is general laboratory reference information and is not advice on the use of any product.