Purity analysis

Purity is normally reported as the proportion of chromatographic peak area attributable to the target compound. The figure is meaningful only alongside the method that produced it, because the method determines what was separated and what was detected.

Structural limits of the figure

• Species that do not respond to the detector are not counted. Water, counter-ions and many salts are effectively invisible to ultraviolet detection at typical peptide wavelengths.
• Species that co-elute with the target are counted as target. Resolution is a property of the method, not of the material.
• Detector response varies between compounds, so area percentage approximates composition rather than measuring it.

For peptides specifically, the impurities that matter most are close structural relatives of the target: deletion sequences missing a residue, insertion sequences carrying one twice, incompletely deprotected chains, oxidised methionine and deamidated asparagine. Their similarity to the target is precisely what makes them difficult to resolve.

Orthogonal methods

Confidence increases when purity is assessed by methods that separate on different physical principles — for example reversed-phase chromatography alongside ion-exchange or capillary electrophoresis. An impurity hidden under the target peak in one system is unlikely to be hidden in the other. A single chromatogram, however clean, cannot demonstrate this.