How to read a chromatogram

A chromatogram is the primary evidence behind a purity figure. Reading it takes five minutes to learn and changes what you can tell about a certificate.

By Canada Peptide Testing. Published September 1, 2026, updated September 13, 2026. 9 minute read.

Most people receiving a Certificate of Analysis look at the percentage and move on. The chromatogram above it is the reason the percentage exists, and it carries information the percentage cannot: how well the method separated, what else was there, and whether the number should be trusted at the precision it is quoted to.

When labels are too small to read in a certificate preview, open the full-size analytical attachment. Compare its sample reference and detector information with the report before interpreting the peak table.

The two axes

Time runs left to right, in minutes from injection. Detector response runs bottom to top, usually in milli-absorbance units. That is the whole coordinate system. Everything else is interpretation.

Both axes need labels for the trace to mean anything. A chromatogram with an unlabelled y-axis is a picture, not data, because the reader cannot tell whether a small peak is genuinely small or whether the scale has been chosen to make it look that way.

The four features worth finding

Void volume

The first sharp spike, usually inside the first minute or two. This is unretained material passing straight through: sample solvent, salts, counter-ion, anything with no affinity for the stationary phase. It is often the tallest feature on the trace and it is not an impurity. Methods normally exclude it from integration, and a method that quietly includes it is inflating its denominator.

Principal peak

The peak assigned to the target analyte, identified by retention time against a reference run under identical conditions and, in a complete analysis, supported by separate identity evidence for the compound. Its area divided by the total integrated area is the purity figure.

Related substances

The small peaks near the principal one. In a synthetic peptide these are usually truncation and deletion sequences, oxidation products, or partially deprotected material: chemistry closely related to the target, which is exactly why it elutes nearby. Their presence is normal. Their being resolved is the sign of a method doing its job.

Baseline

The response with nothing eluting. A flat, quiet baseline lets integration boundaries be set consistently. A drifting or noisy one makes small peaks unmeasurable, and is a reason to repeat a run rather than report from it.

Four things a clean trace does not prove

1. That nothing co-eluted. Two compounds leaving the column together integrate as one peak. A single sharp peak proves this method did not separate them, not that there was nothing to separate.
2. That everything present was detected. Ultraviolet detection only sees what absorbs at the detection wavelength. Salt, water, and residual solvent are in the vial and not on the trace.
3. That the peak is the right compound. Retention time is characteristic but not unique. Identity comes from comparing the sample against reference data for the named compound; the chromatogram supplies the separation that makes that evidence interpretable.
4. That there is as much as the label says. Area purity is normalised. It cannot detect an underfilled vial, because underfilling shrinks numerator and denominator together.

Reading the header, not just the picture

Everything above depends on conditions stated outside the plot. Before trusting a purity figure, find these on the certificate:

• Detection wavelength. The same sample gives different area percentages at 220 nm and 280 nm. Without it, the number is not interpretable.
• Column and gradient. These are the method identity. A different gradient on the same column is a different method and can legitimately give a different figure.
• Run length. A late-eluting hydrophobic impurity is invisible if the run ended before it came off. A short run and a clean trace are a suspicious pair.
• Integration treatment. Which regions were excluded, and on what basis.

Three warning signs

An unlabelled y-axis. If the scale is not stated, small peaks cannot be assessed and the reader is being asked to take the shape of the trace on trust.

A trace with exactly one feature and no baseline noise at all. Real detectors have noise. A perfectly flat baseline with one peak is more often a redrawn illustration than an instrument output.

A run that ends immediately after the principal peak. The region after the target is where hydrophobic impurities appear. A method that stops there has chosen not to look.

Why this laboratory prints it at full resolution

A certificate could carry the number alone and be shorter. It carries the trace because the number is a conclusion drawn from it, and a conclusion whose evidence is withheld is a claim rather than a result. The figures on our certificates are drawn at a scale where the small resolved peaks are visible, not at one where the principal peak fills the frame.

Common questions