How reversed-phase HPLC works
Most peptide purity analysis uses reversed-phase HPLC (RP-HPLC). A dissolved sample is pushed through a column packed with hydrophobic particles using a solvent that gradually changes from water-rich to organic-rich (commonly acetonitrile with a small amount of an ion-pairing acid). Components of the sample leave the column at different times depending on how strongly they interact with the packing, and a detector, usually UV absorbance near 214–220 nm for the peptide bond, records each as a peak.
Reading the chromatogram
- Retention time: when the peak elutes. Reproducible retention is a consistency check between lots.
- Peak area: proportional to the amount of absorbing material in that peak.
- Purity %: main-peak area divided by total integrated peak area, times 100.
- Baseline and small peaks: truncation sequences, deletion products, oxidized forms and residual protecting groups typically appear as small neighboring peaks.
What purity does not tell you
| Question | Answered by HPLC purity? |
|---|---|
| Is the main peak the correct compound? | No — needs mass spectrometry. |
| How much of the vial mass is peptide? | No — needs net peptide content or quantitative assay. |
| Is it free of endotoxin or microbes? | No — separate assays. |
| Is non-UV-absorbing contamination present? | Not reliably — salts and some solvents are invisible at 214 nm. |
Method details matter
Different gradients, columns and wavelengths can resolve different impurities. A report that states the method (column type, gradient, flow rate, wavelength) can be reproduced; a bare percentage cannot. Integration choices, such as where the software draws the baseline, also affect the reported figure, which is why the chromatogram should accompany the number.
Purity thresholds
Research applications set their own requirements. Many in vitro assays are run with material at or above 95%, while sensitive receptor-binding or analytical standards work often calls for 98% or higher. We target ≥99% by HPLC on released lots. See how to read a COA for the rest of the report.