Purity versus net peptide content: why 99% is not 99%

What HPLC purity measures

High-performance liquid chromatography separates the components of a sample. The purity figure is the proportion of the signal that comes from the target peptide, compared with truncated, deleted or otherwise modified peptide impurities.

It is blind to anything that does not absorb at the detection wavelength, which includes water and most salts.

What is actually in the vial

A lyophilised peptide is rarely 100% peptide by weight. The remainder is typically made up of:

• Counter-ions, most often trifluoroacetate (from purification) or acetate, which balance the charge on basic residues.
• Residual water, because freeze-dried peptides are hygroscopic.
• Small amounts of residual solvents or excipients.

Net peptide content

Net peptide content is the weight fraction that is genuinely peptide. It is determined by methods such as amino acid analysis or nitrogen content, and it commonly sits well below the HPLC purity. Peptides with many basic residues carry more counter-ions, so their net content tends to be lower.

Why it matters for concentrations

If you dissolve what you believe is 5 mg of peptide but only 80% of the powder is peptide, the true amount is 4 mg and every concentration you calculate is 20% off. For qualitative work this may not matter; for quantitative comparisons it does.

The stock solution calculator on this site includes a peptide content field so you can correct for it when the figure is known.