Short answer: most peptide purity results come from HPLC (high-performance liquid chromatography). The sample is pushed through a column that separates its components, a UV detector records each one as a peak, and purity is the main peak’s area as a percentage of the total detected peak area.
Separation
Peptides are usually analyzed by reversed-phase HPLC (RP-HPLC) on a C18 column. Components leave the column at different times (retention times) depending on how strongly they interact with it.
Detection
A UV detector, commonly set around 214–220 nm where peptide bonds absorb, measures what leaves the column. The result is a chromatogram: a graph of signal against time, with one peak per detected component.
Calculation
Purity (area %) = area of the target peak ÷ total area of all detected peaks × 100. A result of 99.6% means 99.6% of the UV-detected material was the target peptide.
What HPLC purity does not tell you
- Identity: a clean peak shows one main component, not which compound it is. Identity needs mass spectrometry or another specific method.
- Quantity: purity is a ratio. It does not say how many milligrams are in the vial; net content testing does.
- Undetected material: water, salts and counter-ions do not appear as UV peaks, so they are not counted.
Why methods differ between labs
Column, gradient, wavelength and integration settings all affect the result. That is why a COA should state its method, and why results from different labs are not always directly comparable.
LC-MS
Many labs couple HPLC with mass spectrometry (LC-MS), which confirms the identity of the main peak in the same run.
See also what ≥99% purity means and our article How Researchers Test Peptides for Accurate Results.
Related guides
← All Research & Education guides · Certificates of Analysis
All Guardian Labs products are sold for laboratory research use only. They are not for human or veterinary use, and nothing on this page is medical advice.