Understanding HPLC Testing
9 September 2026
What HPLC is
High-performance liquid chromatography (HPLC) is an analytical technique for separating, identifying and quantifying the components of a mixture. For peptides it is the standard method of assessing purity, and the figure on a certificate of analysis is usually an HPLC result.
How it works
A liquid sample is pushed under high pressure through a column packed with a solid adsorbent. Different molecules interact with that packing to different degrees, so they leave the column, or elute, at different times.
The parts of the system
- Mobile phase – the solvent that carries the sample through the column
- Stationary phase – the column packing material (typically C18 for peptides)
- Detector – usually UV absorbance at 214 nm or 280 nm
- Data system – records the chromatogram for analysis
Reading a chromatogram
The output is a chromatogram: a trace with one peak for each component that eluted.
- Main peak – the target peptide
- Peak area – proportional to the quantity of that component
- Purity calculation – (target peak area / total peak area) x 100
A purity of 99% or above means at least 99% of what was detected in the sample is the intended compound.
Why it matters in research
- Reproducibility – consistent material gives consistent results across experiments
- Contamination detection – synthesis by-products and degradation show up as extra peaks
- Quality verification – confirms the compound matches its specification
Complementary methods
| Method | Purpose |
|---|---|
| Mass Spectrometry (MS) | Confirms molecular weight / identity |
| HPLC | Quantifies purity |
| Endotoxin testing | Ensures absence of bacterial contamination |
Draft wording prepared 2026-09-09, awaiting approval by Aevryn Labs Pty Ltd.