Key takeaways
- HPLC purity is the main peak’s share of all detected peptide-related signal.
- It does not count water, salts, endotoxins or heavy metals.
- Purity, identity and content answer three different questions.
- The chromatogram tells you more than the headline percentage.
“99% purity” is the most quoted number in the research peptide market, and one of the most misunderstood. Here is how that number is produced, what it tells you, and what it doesn’t.
How HPLC works
High-performance liquid chromatography (HPLC) separates the components of a sample by pumping it through a packed column under high pressure. For peptides, the standard method is reversed-phase HPLC: molecules separate by how strongly they interact with the column’s hydrophobic surface, so they exit at different times. A UV detector, usually at 214–220 nm where the peptide bond absorbs, records each component as a peak on the chromatogram.
What the purity number includes
HPLC purity captures peptide-related impurities, the by-products of solid-phase synthesis and of degradation:
- Deletion and truncated sequences
- Incompletely deprotected sequences
- Oxidized forms (methionine, tryptophan and cysteine are most prone)
- Deamidation products (asparagine and glutamine)
- Aggregates and diastereomers that resolve on the column
What it leaves out
- Water: lyophilized peptides retain some moisture.
- Counter-ions: most synthetic peptides are acetate or TFA salts, which add mass without appearing as impurity peaks.
- Endotoxins and heavy metals: these need separate assays.
- Whether the main peak is the right molecule: that’s identity testing.
This is why the net peptide content of a vial, the share of its mass that is actually peptide, is normally lower than its HPLC purity.
Purity vs. identity vs. content
| Question | Test | Answers |
|---|---|---|
| Is it the right molecule? | Mass spectrometry (MS / LC-MS) | Identity |
| How clean is it? | HPLC with UV detection | Purity |
| How much is in the vial? | Quantitative HPLC vs. reference standard | Content |
A sample can pass one and fail another, which is why a COA showing only a purity percentage is incomplete. Our guide to reading a peptide COA covers each section.
Reading the chromatogram yourself
- One sharp, symmetric main peakThe ideal: most of the signal in a single, well-defined peak.
- Watch for shouldersA bump on the side of the main peak can hide a closely related impurity.
- Count the small peaksMany small peaks across the run suggest synthesis by-products or degradation.
- Check the baselineA drifting or noisy baseline makes small impurities harder to quantify.
Our standard
After Hours Labs lots are independently tested for HPLC purity and mass-spec identity, and every COA is matched to its batch.
All After Hours Labs products are sold strictly for qualified in-vitro laboratory research. They are not for human or veterinary use, and nothing in this article is medical advice or a recommendation for use in people or animals.
