A chromatogram is the record an analytical separation produces – a two-dimensional trace that shows what a detector saw as the components of a sample passed through it, one after another. It is the raw evidence behind figures like a purity percentage on a Certificate of Analysis. This glossary entry explains, in neutral terms, what the two axes of a chromatogram represent, how to read its peaks and baseline, and what the trace can and cannot establish on its own.
What a chromatogram is
A chromatogram is a plot of a detector’s signal over time, generated by a technique called chromatography. Chromatography separates a mixture into its individual components by making them travel through a system at different speeds; a detector at the far end registers each component as it arrives. The chromatogram is simply the graph of that detector’s output. It is the visual output common to many separation methods – High-Performance Liquid Chromatography (HPLC), gas chromatography (GC), and others – and it is the trace an analytical laboratory reproduces on a report so that a measured value can be audited rather than merely asserted.
In analytical documentation the chromatogram is the evidence layer beneath a summary number. A specification sheet may state a single purity figure, but the chromatogram is where that figure is actually visible: the plot shows every component the instrument detected, in the proportions it detected them.
The two axes
Every chromatogram shares the same basic layout, and reading it starts with knowing what each axis means.
- The horizontal axis is time – usually minutes elapsed since the sample entered the system. A component’s position along this axis is its retention time: how long it took to travel through the system. Under fixed conditions, a given compound elutes at a characteristic retention time, which is why the horizontal position of a peak is informative.
- The vertical axis is detector response – the strength of the signal the detector produced at each moment, in whatever units that detector reports (for a UV detector, absorbance, commonly measured at 214 nm for peptide bonds). The height of the trace at any point reflects how much material was passing the detector at that instant.
Anatomy of a chromatogram
Against those two axes, a small vocabulary describes everything on the plot:
| Baseline | The flat signal when only mobile phase, and no analyte, is passing the detector |
|---|---|
| Peak | An excursion above the baseline as a component elutes and passes the detector |
| Retention time | The time (horizontal position) at which a peak reaches its maximum |
| Peak area | The area enclosed between a peak and the baseline – proportional to the amount of that component |
| Peak height | The vertical distance from baseline to the peak apex |
| Resolution | How cleanly two neighbouring peaks are separated from each other |
The single most important distinction for interpreting a trace is area versus height. It is a peak’s area, not its height, that is proportional to the quantity of a component present. Two peaks can reach the same height while enclosing very different areas, because a broad peak represents more material than a narrow one of equal height. This is why quantitative figures – such as an area-percent purity value – are calculated from integrated peak areas rather than from how tall the peaks look.
How a chromatogram is produced
The trace is generated in a sequence of steps that is the same in principle across techniques. A measured sample is introduced into a stream (the mobile phase) that carries it through a column packed with a stationary material. Because different components interact with the stationary material to different degrees, they move through the column at different rates and emerge separated in time. As each separated band leaves the column it passes a detector, which converts the amount of material present into an electrical signal. Plotting that signal against elapsed time produces the chromatogram. The separation conditions – the column, the mobile-phase gradient, the flow rate, and the detection wavelength – are reported alongside the trace, because a retention time only has meaning relative to the conditions that produced it.
Area percent and what it represents
The figure most often derived from a chromatogram is area percent: the area of one peak expressed as a share of the total area of all peaks on the trace. For a purity assessment, the main peak’s area percent is reported as the purity value, and the smaller peaks – the related substances – account for the remainder. Area percent describes the relative composition the detector saw; it is a proportion within the sample, not an absolute mass. How much peptide is present by weight is a separate quantity, reported as net peptide content.
What a chromatogram does and does not establish
A chromatogram is powerful evidence for how a sample is composed – how many components are present and in what proportions. What it does not establish on its own is identity: the trace shows that one component dominates, but a peak at an expected retention time is consistent with, not proof of, a particular molecule. A different compound could elute at a similar time. Confirming that the main peak is the intended molecule is the job of mass spectrometry, which measures mass directly. A thorough analytical record therefore pairs the chromatogram (composition) with a mass-spectrometry result (identity). For a step-by-step reading of a purity chromatogram in particular, see HPLC purity explained; for the separation technique that most commonly produces these traces for peptides, see what HPLC is; and for how identity is confirmed separately, see what mass spectrometry is.
Key takeaways
- A chromatogram plots a detector’s signal (vertical axis) against elapsed time (horizontal axis) as separated components pass through.
- A peak’s horizontal position is its retention time; its area – not its height – is proportional to the amount of that component.
- Area percent is a proportion within the sample, calculated from integrated peak areas, and is the basis of a purity figure.
- The trace establishes composition, not identity; mass spectrometry confirms which molecule the main peak is.
- Retention times are only meaningful relative to the reported separation conditions (column, gradient, flow, detection).
A chromatogram is the reason a purity figure can be checked rather than taken on trust. PeptSelect publishes the certificate behind every lot in a public COA Vault, and describes the full testing pipeline on how we test.
Frequently asked questions
What is a chromatogram?
A chromatogram is a plot of a detector’s signal over time, produced by a chromatographic separation. As a sample’s components pass a detector one after another, each registers as a peak, so the trace shows how many components are present and in what proportions.
What do the two axes of a chromatogram represent?
The horizontal axis is time – usually minutes since the sample entered the system – and a peak’s position along it is its retention time. The vertical axis is detector response, the strength of the signal the detector produced, such as UV absorbance for a UV detector.
What is retention time?
Retention time is how long a component takes to travel through the chromatographic system before reaching the detector, read as the horizontal position of its peak. Under fixed separation conditions a given compound elutes at a characteristic retention time.
Why is peak area used instead of peak height?
A peak’s area is proportional to the amount of that component, whereas height is not: a broad peak and a narrow peak can reach the same height while representing very different quantities. Quantitative figures such as area-percent purity are therefore calculated from integrated peak areas.
What does area percent mean on a chromatogram?
Area percent is one peak’s area expressed as a share of the total area of all peaks on the trace. For a purity assessment the main peak’s area percent is reported as the purity value, and the smaller peaks – related substances – make up the remainder. It is a proportion within the sample, not an absolute mass.
Can a chromatogram confirm which molecule is present?
Not on its own. A chromatogram shows composition – how many components there are and their proportions – but a peak at an expected retention time is consistent with, rather than proof of, a particular molecule. Confirming identity is the role of mass spectrometry, which measures mass directly.
Is a chromatogram specific to HPLC?
No. A chromatogram is the general output of any chromatographic technique, including High-Performance Liquid Chromatography (HPLC) and gas chromatography (GC). HPLC is the method most commonly used to produce purity chromatograms for peptides.
