How to Read an HPLC Chromatogram on a Peptide COA
The purity percentage is one number derived from the trace. The trace shows how it was arrived at, and whether it should be believed.
The chromatogram is the part of a certificate of analysis that most people skip and that carries most of the information. A purity percentage is a single number derived from that image; the image itself shows how the number was arrived at, and whether it should be believed.
What the axes are
The horizontal axis is time — minutes since injection. The vertical axis is detector response, usually UV absorbance at 214 nm (the peptide bond) or 280 nm (aromatic residues). A trace with no axis labels is not evidence of anything.
Retention time
Reverse-phase HPLC separates by hydrophobicity. The column is non-polar; the mobile phase starts mostly aqueous and becomes increasingly organic — typically acetonitrile — over the run. Hydrophilic compounds elute early, hydrophobic ones late.
Retention time is reproducible on a given method but not transferable between methods. This is why comparing a retention time on one supplier’s certificate with another’s tells you nothing unless the gradient, column and flow rate match. The number is meaningful within a laboratory, as a batch-to-batch consistency check.
Reading the main peak
What a good main peak looks like:
- Symmetrical. A peak that rises and falls at similar rates. Asymmetry has diagnostic value — see below.
- Narrow relative to the run. Broad peaks suggest poor column condition, sample overload, or a heterogeneous population.
- Returning to a flat baseline on both sides. If it does not, integration has to guess where the peak ends, and the purity figure inherits that guess.
Tailing — a peak with a long trailing edge — usually indicates secondary interactions with the stationary phase, often involving basic residues. Fronting — a leading edge that rises slowly — usually indicates column overload. Shouldering, a bump on the flank of the main peak, is the one to watch: it is typically a co-eluting impurity that the integration has folded into the main peak, inflating the reported purity.
Reading the impurity peaks
Where an impurity sits matters as much as how large it is.
| Position | Likely identity |
|---|---|
| Very close to the main peak | Deletion sequence or a single-residue variant — chemically similar, hardest to remove |
| Slightly earlier | More hydrophilic: often a truncated or hydrolysed fragment, or an oxidised form |
| Much later | More hydrophobic: incompletely deprotected chains, dimers |
| At the very start (void volume) | Salts, unretained material — usually not counted |
A chromatogram with one dominant peak and two or three small, clearly separated neighbours is a normal, honest picture of a purified peptide. A chromatogram with a single peak and nothing else at all, on a long sequence, is worth a question.
How the percentage is calculated
Purity by HPLC is area percent: the area under the main peak divided by the total area under all integrated peaks. Two consequences follow, and both matter:
- It is relative, not absolute. It says nothing about counter-ions, water, or residual solvent — none of which absorb UV. This is why peptide content is a separate assay.
- It depends on the integration. Where the baseline is drawn and where peak boundaries are set changes the number. This is why an integration table with retention times and areas is worth more than the image alone, and far more than the percentage on its own.
What to ask for
A chromatogram image, an integration table, the method conditions (column, gradient, flow, detection wavelength), and the batch number the run belongs to. A laboratory that ran the analysis has all four.
Our certificate guide covers the rest of the document, and content versus purity covers what the percentage leaves out.
All products and information referenced are for in-vitro research and laboratory use only. Nothing here is medical advice, and no therapeutic claim is made or implied.