Analysis

Purity by chromatography

Reversed-phase high-performance liquid chromatography pushes a dissolved sample through a column that holds back molecules according to how water-repellent they are. A detector measures ultraviolet absorption, usually around 214 to 220 nanometres, where the peptide bond itself absorbs. Purity is reported as the area of the main peak as a percentage of all peaks.

Identity by mass spectrometry

Mass spectrometry measures the mass of the molecule, typically after electrospray ionisation or with MALDI time-of-flight instruments. A measured mass that matches the mass calculated from the sequence confirms that the main component is the intended chain. It also reveals deletions, where one residue is missing.

Purity is not content

A powder that is 99 percent pure by chromatography is not 99 percent peptide by weight. Counter-ions from purification and residual water are invisible to the ultraviolet detector and can make up a noticeable share of the mass. Net peptide content is measured separately, classically by amino acid analysis.

Other measurements

• Water content, by Karl Fischer titration.
• Counter-ion content, such as acetate or trifluoroacetate, by ion chromatography.
• Bacterial endotoxins, by the Limulus amebocyte lysate test or recombinant alternatives.
• Sterility, by culture-based methods defined in pharmacopoeias.

Reading a certificate of analysis

A certificate of analysis summarises the results for one production batch: which methods were used, what was measured and when. It describes the sample that was tested. It is not evidence that a substance is safe, approved or suitable for any use, and it says nothing about a different batch.

General chemistry, written for orientation. It does not describe how to use any substance.