Every certificate of analysis for a research peptide carries a purity percentage, and that figure is almost always determined by HPLC. This guide explains what the technique does, how that percentage comes about and — most importantly — what it does not say.
HPLC separates the components of a sample and expresses the target peptide as a percentage of total peak area. So it is a ratio, not a weighing: residual water and counter-ions stay out of view, and HPLC does not confirm the identity of the molecule.
What is HPLC?
HPLC (High-Performance Liquid Chromatography) is a separation technique that pulls the components of a sample apart and makes each of them measurable. The sample is dissolved and driven under high pressure through a column packed with fine particles. Each component has its own affinity for that packing material and therefore takes more or less time to appear at the other end.
A detector sits at the end of the column. It registers what passes and when, and the result is a chromatogram: a trace with peaks, where each peak stands for a component that eluted at that moment.
How does HPLC separate a peptide sample?
For peptides, reversed-phase HPLC is almost always used: a non-polar column combined with an aqueous mobile phase to which an organic solvent is gradually added. The more hydrophobic a component, the longer it stays on the column and the later it elutes. That arrival time is called the retention time.
Because the composition of the mobile phase shifts during the run — a gradient — components that sit close together in hydrophobicity still come out as separate peaks. Which is exactly what you want with a peptide: related by-products from the synthesis resemble the target molecule closely.
Where does that purity percentage come from?
The purity percentage is an area percentage: the area of the target peptide's peak divided by the total area of all peaks. For peptides the detector typically measures UV absorption near 214 nm, the wavelength at which the peptide bond itself absorbs — useful, because then every peptide-like component is seen, not only those carrying an aromatic amino acid.
So 99.2% on a certificate means: 99.2% of the peak area belonged to the target molecule and 0.8% to something else. How to read such a report is covered in how to read a COA.
Purity is not the same as peptide content
This is where most of the confusion sits: 99% purity does not mean 99% of the vial's contents is peptide. Besides the molecule itself, a lyophilised peptide contains residual water and counter-ions — often trifluoroacetic acid (TFA) or acetate from peptide synthesis. Those add to the weight in the vial but not to the peak area in the chromatogram.
Net peptide content is therefore a separate determination, which a certificate may or may not include. For research where the exact quantity of substance matters, that is the figure to look at — not the purity percentage. Our own thresholds are on quality standards.
What HPLC does not tell you
HPLC quantifies but does not identify: a retention time is consistent with a substance without proving which substance it is. That needs mass spectrometry, which measures the molecular weight and so confirms molecular identity. Those two measurements together are the minimum for considering a batch characterised.
And beyond that: a purity measurement says nothing about sterility, endotoxins or suitability for any application. Who measures our batches, and why that is a third party, is on Janoshik Analytical.
This information is general and educational in nature. Peptide Lab supplies research peptides for laboratory and research purposes only. Not for human or animal consumption, not a medicine and not medical advice.
Frequently asked questions about HPLC
What does HPLC stand for?
HPLC stands for High-Performance Liquid Chromatography. It is a separation technique in which a sample is driven through a column under high pressure, so its components arrive at the far end at different moments and can be measured individually.
How is purity determined by HPLC?
From the area of the peaks in the chromatogram. The area of the target peptide's peak is expressed as a percentage of the total area of all peaks. So 99.1% on a certificate is an area percentage, not a weighing.
Is 99% purity the same as 99% peptide in the vial?
No. HPLC purity is the ratio between the target peptide and other peptide-related components the detector sees. Residual water and counter-ions from the synthesis are not counted in it. Net peptide content is a separate determination.
Can HPLC confirm which peptide it is?
Not on its own. A retention time is consistent with a substance but does not prove its identity. That needs mass spectrometry, which measures the molecular weight. HPLC says how much, mass spectrometry says what.
