A purity figure is only as informative as the method behind it. Two suppliers can quote the same percentage while having measured different things. This overview explains how the common techniques work, and where their limits sit.

Reverse-phase HPLC and area percentage

Reverse-phase high-performance liquid chromatography separates components of a mixture by hydrophobicity. The sample travels through a column packed with a non-polar stationary phase, carried by a polar mobile phase. More hydrophobic molecules interact longer with the stationary phase and elute later; more polar molecules elute earlier. A detector records each eluting band as a peak.

The percentage quoted from such a run is usually an area percentage: the area of the main peak divided by the total area of all peaks, multiplied by one hundred. This is a relative measure. It describes the proportion of the detected signal, not the absolute mass of the target compound in the vial. A sample can be ninety-eight percent pure by area percentage and still contain material the column did not resolve, or material that does not absorb at the chosen wavelength.

Detection wavelengths and what they capture

Most peptide work uses ultraviolet detection at 214 nm. The peptide bond absorbs strongly there, so nearly every peptide-containing species in the sample produces a signal, which suits the purpose of seeing everything present. Detection at 280 nm instead responds to aromatic residues, principally tryptophan and tyrosine, so it is far less sensitive to peptides lacking those residues. A trace collected at one wavelength therefore reports a different picture from a trace collected at another.

Mass spectrometry confirms identity, not purity

Mass spectrometry is typically run by electrospray ionisation, which transfers the analyte into the gas phase as multiply charged ions. The instrument records a mass-to-charge spectrum, and software reconstructs the observed molecular mass. That value is compared against the theoretical mass calculated from the peptide sequence. Agreement within a stated tolerance supports the conclusion that the expected sequence is present. It does not establish how much of the sample is that sequence. Identity and purity are separate questions requiring separate measurements.

Water content, counter-ions and trace residuals

A purified peptide powder is rarely just peptide. Water is quantified by Karl Fischer titration, a specific chemical reaction that measures water content. Residual trifluoroacetic acid, carried over from the purification step, is measured separately, and in some batches it represents a significant fraction of the powder mass. Moisture and counter-ion content both reduce the proportion of the stated compound, so a purity figure quoted without them is incomplete. Amino acid analysis and capillary electrophoresis serve as complementary or alternative techniques, each with its own bias.

Limits of detection and of quantification

The limit of detection is the lowest amount an instrument can reliably distinguish from background. The limit of quantification is the lowest amount it can measure with acceptable precision. Impurities below the limit of detection are not reported because the method cannot see them. Impurities between the two limits may be seen but not quantified. Reported impurity levels are therefore shaped by the method chosen, not only by the contents of the vial.

A single HPLC trace at one wavelength cannot exclude co-eluting isomers. Two compounds of similar hydrophobicity can leave the column at the same time and appear as one peak, so a clean trace is evidence of separation achieved, not proof of structural homogeneity.

Questions worth putting to a supplier

• Which method and column were used, and at what wavelength was the trace collected?
• Is the quoted percentage an area percentage from one run, or an average of several?
• Was mass spectrometry performed, and what tolerance was applied to the theoretical mass?
• Were water content and residual counter-ion content measured, and are those values reported?
• Which limits of detection and quantification apply to the method used?

Reading a certificate of analysis critically means asking what was measured and by which method, rather than accepting a single number at face value.

Documentation that answers these questions lets a laboratory judge whether a material suits its work. Every compound supplied by SkyCorPeps is offered strictly for laboratory research.

Research use only. Not for human or veterinary consumption, diagnostic or therapeutic use.