Purity, HPLC and Mass Spectrometry Explained

High-performance liquid chromatography separates the components of a sample as it is pushed through a packed column. Most peptide methods use reverse-phase columns, where components separate by how strongly they interact with the column surface, and a UV detector (commonly at 214 to 220 nm, where peptide bonds absorb) records each component as a peak.

Purity is reported as the area of the main peak divided by the total area of all peaks. A result of 99% means the target peak accounts for 99% of the detected signal under that method.

• It only counts what the detector sees. Water, salts and counter-ions do not show up as UV peaks, so a 99% pure sample can still be well under 100% peptide by weight. That is what net peptide content measures.
• It depends on the method. Two labs using different columns, gradients or wavelengths can report different numbers for the same material.
• It does not confirm identity. A sample can be very pure and still be the wrong molecule.

Synthetic peptides are built one amino acid at a time, so the typical impurities are close relatives of the target: deletion sequences (a missing residue), truncations (a chain that stopped early), incompletely removed protecting groups, and oxidation or deamidation products that form during storage. These usually appear as small peaks near the main peak.

Mass spectrometry measures the mass of the molecules in a sample. Matching the observed mass to the theoretical mass of the intended sequence confirms the main component is the right molecule. HPLC and MS answer different questions, which is why a complete certificate reports both.

Results carry more weight when they come from a laboratory with no commercial stake in the outcome. ISO/IEC 17025 is the international standard for the competence of testing laboratories: an accredited lab has demonstrated validated methods, calibrated equipment and documented quality control to an external assessor.