What a chromatogram is and how to read one
The features of a genuine HPLC trace, how to read the main and minor peaks, and the tells of a fabricated or borrowed chromatogram.
The chromatogram is the single most informative thing on a research peptide's Certificate of Analysis, and the item most often missing from the COAs circulating in the UK market. If you can read one, you can tell in thirty seconds whether a purity figure was measured or typed. This note explains what the trace shows, the features to look for, and the tells of a poor or fabricated one. All Alphex products are for laboratory and in-vitro research only.
What you are looking at
A chromatogram is a plot of detector signal (absorbance at 220 nm, usually in milli-absorbance units, mAU) on the vertical axis against time in minutes on the horizontal axis. As the sample passes through the HPLC column, components separate and reach the detector at different times; each produces a peak. Its position (retention time) identifies the component within that method; its area is proportional to how much of it there is. Purity is calculated from the areas, as described in how HPLC purity is calculated.
Features of a genuine trace
- Axes and units. Time in minutes, signal in mAU, both labelled. A trace with no axes is a picture, not data.
- A solvent front. A disturbance in the first minute or two where unretained material and the injection solvent pass through. Its absence on a real run is unusual.
- A rising baseline. As the acetonitrile proportion increases through the gradient, absorbance drifts slightly. A perfectly flat baseline from start to finish is more typical of a drawn trace than a real one.
- The main peak. Sharp, roughly symmetrical, labelled with retention time and area percent. Fronting or tailing suggests overload or column issues; a doublet suggests two co-eluting species.
- Minor peaks. A real synthetic peptide at 99% purity has small resolved impurity peaks, usually close to the main peak, integrated and listed. A trace showing a single peak and literally nothing else at 99.4% is a reason to ask questions, particularly for a large peptide.
- An integration table. Retention time, area, and area percent for each peak, summing to 100%.
- Method conditions. Column, gradient, flow rate, wavelength, injection volume, sample concentration. Without them the trace cannot be reproduced.
- Sample identity. Batch number and sample name on the trace itself, matching the COA header and the vial label.
Reading the main peak
Retention time by itself does not identify the peptide (that is what mass spectrometry is for), but it should match the reference standard run under the same conditions, and it should be consistent from batch to batch. A main peak that has shifted by a minute between two batches on the same method is either a method change or a different compound. Peak width matters too: a broad main peak can hide co-eluting impurities that a slower gradient would resolve.
Reading the small peaks
Impurities elute close to the target because they are close in structure. Slightly earlier peaks are typically more polar (deletions of hydrophobic residues, deamidated or oxidised forms); slightly later peaks are more hydrophobic (incompletely deprotected forms). Their total should match 100 minus the stated purity. If the integration table lists 0.6% total impurities but the trace visibly shows more, the threshold or baseline is doing work it should not.
Tells of a fabricated or borrowed trace
- No axes, no units, no integration table, or a table that does not sum to 100%.
- The same image appearing on COAs for different batches or different compounds (retention time and peak shape identical).
- Sample name or batch number on the trace that does not match the COA header.
- Resolution or artefacts consistent with a screenshot of a screenshot.
- A perfectly flat baseline and a single Gaussian peak with no solvent front.
The wider checklist for a certificate is in how to read a research peptide COA; the red flags are in fake COA red flags.
What Alphex includes
Every Alphex COA carries the full chromatogram with axes, integration table, method conditions and batch number, Every certificate is published in the COA library, so you can read the chromatogram for a batch before you order it.
Alphex research peptides are supplied for laboratory and in-vitro research by qualified researchers only. They are not authorised for human or veterinary use.
Research use only. Alphex products are supplied exclusively for laboratory and in-vitro research by qualified researchers. They are not authorised for human or veterinary use, and nothing in this note is advice on any such use.
Published 8 July 2026. Alphex reviews research notes when the testing method, the catalogue or UK guidance changes.