Purity of research peptides: what the percentages mean

Three different numbers

• Chromatographic purity (area percent): share of the main peak in the HPLC chromatogram. This is the figure stated in the COA.
• Net peptide content: share of pure peptide in the total weight of the powder. For synthetic peptides it is usually between 70 and 90 %, because counter-ions and bound water are weighed as well.
• Fill weight per vial: the amount stated on the label (e.g. 5 mg) refers to the total fill weight unless “net peptide” is explicitly stated.

What area percent measures

Area percent is a relative figure, not an absolute one. The detector records how strongly each eluting component absorbs at the chosen wavelength, and at 210–220 nm it is mainly the peptide bond that responds. Components that absorb weakly are therefore under-represented, and anything not eluting within the run does not enter the calculation at all. The same material can give slightly different values on a different column or gradient, which is why method details belong on the certificate.

Typical impurities

Solid-phase synthesis regularly produces truncated sequences (deletion sequences), sequences with incomplete removal of protecting groups and oxidised variants – for example at methionine or tryptophan. They appear in the chromatogram as small secondary peaks close to the main peak. Further common by-products are dimers formed through disulfide bridges, chains capped by acetylation, and deamidation products at asparagine or glutamine, which differ from the target molecule by a single mass unit and can be hard to separate.

Counter-ions

After purification a peptide is present as a salt, frequently as trifluoroacetate (TFA) or acetate. The counter-ion is not an impurity in the chromatographic sense, but it affects the weight and therefore the net peptide content. The salt form is relevant for many laboratory applications and should be known. Residual TFA can also interfere with certain cell-based assays, which is why some working groups ask for the salt form on the certificate.

Residual water

Lyophilised peptides are hygroscopic. A vial that has been opened several times absorbs water – this does not change the purity, but it does change the actual amount of peptide per milligram. Therefore: bring vials to room temperature before opening, open briefly, close again. Water content is measured separately, usually by Karl Fischer titration, and is not part of an HPLC result. Handling of the freeze-dried material is covered in the storage guide.

What a purity figure does not cover

Chromatographic purity says nothing about heavy metals, endotoxins or microbiological load. These are separate determinations with their own methods and their own units, and a high percentage on an HPLC report neither includes nor replaces them. The same applies to the identity question, which is answered by mass spectrometry and not by the size of a peak.

Comparing figures

• Check whether both values were produced with the same method and wavelength.
• Check whether the figure refers to chromatographic purity or to net peptide content.
• Check what was measured: a percentage always belongs to the material that was tested, not to a product name.
• Check the date; an older report describes older material.

What GPeptides states

The product pages state the chromatographic purity of the most recently tested material (HPLC). The tested product, test date and method are given in the certificate of analysis in the lab section, where the reports released so far are published; material without a published report carries the note pending. How such a document is read in detail is explained in the guide Reading a COA.

The two methods behind these figures, and what each of them can and cannot show, are described in the guide HPLC and mass spectrometry.