What is Chromatogram?
Also called: HPLC trace, LC trace, Chromatographic trace
The plot produced by a chromatography run: detector response against time, with each separated component appearing as a peak whose area reflects its amount.
By the APL Research Team · Updated
A chromatogram is the record of a chromatographic separation: time along the x-axis, detector signal up the y-axis, and a peak wherever a separated component passes the detector. On a peptide certificate of analysis it is the evidence behind the purity percentage, and it can be checked in ways the percentage on its own cannot.
Anatomy of a peptide HPLC trace
- Axes. Time in minutes; absorbance in milli-absorbance units (mAU), usually recorded at 214–220 nm.
- Baseline. The signal between peaks. In gradient runs it often drifts as the proportion of organic solvent rises.
- Solvent front. A disturbance in the first minute or two from material the column does not retain, such as salts; it is normally excluded from integration.
- Main peak. The target peptide, at its characteristic retention time for that method.
- Impurity peaks. Smaller peaks nearby. Closely related impurities, such as sequences missing one residue, often elute just before or just after the main peak because their hydrophobicity is so similar.
- Peak table. Retention time, area, height and area percentage for every integrated peak.
Worked example: from peak table to purity
The table below is illustrative, not data from any batch.
| Peak | Retention time (min) | Area (mAU·s) | Area % |
|---|---|---|---|
| 1 | 11.92 | 12.2 | 0.50 |
| 2 (main) | 12.84 | 2,416.0 | 99.10 |
| 3 | 13.41 | 9.8 | 0.40 |
| Total | 2,438.0 | 100.00 |
Purity = 2,416.0 ÷ 2,438.0 × 100 = 99.10%. Three things can move that figure without the sample changing at all:
- Integration choices. If peak 3 were left unintegrated, for instance because it sat on the tail of the main peak or below a reporting threshold, purity would read 2,416.0 ÷ 2,428.2 = 99.50%.
- Resolution. If peak 1 co-eluted with the main peak, its area would be counted as product and the impurity would vanish from the report.
- Run length. Strongly retained impurities or aggregates that elute after the run ends are never recorded.
What to check on a COA chromatogram
| Check | Reason |
|---|---|
| Axes, units and detection wavelength are labelled | An unlabelled trace cannot be interpreted |
| Gradient and run time are stated | Shows whether late-eluting impurities had time to appear |
| A peak table accompanies the trace | Lets the purity figure be recalculated |
| The main peak is sharp and symmetrical | A shoulder or split peak can indicate a co-eluting species |
| Baseline noise differs from batch to batch | Identical noise on different lots suggests a copied image |
| The lot number on the trace matches the vial | Ties the data to the material in hand |
What a chromatogram cannot show
A UV trace records only what absorbs at the chosen wavelength under one method. It does not identify peaks, which is the job of mass spectrometry, often coupled directly to the column as LC-MS so that each peak gets its own mass. It also does not show water or counter-ions, so the 99.10% above describes the peptide-related material only; peptide purity explains what that number does and does not cover.
The HPLC entry describes how the separation is run, and the HPLC testing guide and COA guide cover how traces are presented in batch documentation.