Understanding HPLC: how purity is measured
What is behind the percentage on the certificate of analysis, how a chromatogram is generated and why the method matters as much as the result.
High performance liquid chromatography, or HPLC for short, is the standard method for determining the purity of a peptide. For peptides, the reverse-phase variant (RP-HPLC) is used almost exclusively.
The principle in three sentences
The sample is pumped in a liquid stream through a column packed with a non-polar material. Each substance adheres to it with different strength and therefore leaves the column at its own time, the retention time. A UV detector at the outlet measures when and how much substance arrives, typically at 214 to 220 nm, where the peptide bond absorbs.
Reading the chromatogram
The result is a curve over time. Each deflection (peak) represents a substance.
- Main peak: the target peptide
- Minor peaks: impurities, such as truncated sequences or oxidation products
- Area instead of height: the calculation is based on the area under each peak
The stated purity is the area share of the main peak relative to all peaks. 99.1 % therefore means: 99.1 % of the substance visible at this wavelength is the target peptide.
A purity figure is only as meaningful as the method used to measure it.
What the number does not say
HPLC purity is a relative value. It says nothing about the peptide content (the share of peptide in the total weight, which is below 100 % due to water and counter-ions such as acetate or TFA) and does not confirm identity. A wrong peptide can likewise appear as a clean single peak. That is why mass spectrometry is required for every batch in addition.
What to look for in a report
| Item | Why it matters |
|---|---|
| Column and gradient | Only this makes the result reproducible |
| Detection wavelength | Determines which impurities become visible |
| Chromatogram as an image | The percentage alone cannot be verified |
| Batch number | Links the report to exactly one vial |
| Testing laboratory and date | Makes the source traceable |
In our reports, these details are openly available for every batch, including an interactive chromatogram.