Anatomy of a Chromatogram
An HPLC chromatogram is a graph with time on the x-axis (measured in minutes) and detector response on the y-axis (measured in absorbance units, typically at 214nm or 220nm wavelength for peptides). Each compound in the sample appears as a peak at a characteristic retention time.
The Main Peak
The dominant peak on a peptide chromatogram represents the target compound. For a 99%+ purity peptide, this peak should tower above the baseline with minimal neighboring peaks. The retention time of the main peak should be consistent with the known hydrophobicity of the target peptide.
Key things to evaluate: Is the peak symmetric? A tailing or fronting peak can indicate column overloading or sample degradation. Is the peak sharp? A broad peak may suggest multiple co-eluting species that the method cannot resolve.
Impurity Peaks
Smaller peaks before and after the main peak represent impurities. Early-eluting peaks (shorter retention times) are typically more hydrophilic impurities — often truncated peptide sequences that are shorter than the target. Late-eluting peaks are more hydrophobic impurities — potentially oxidized forms or longer deletion sequences.
On a high-quality COA, impurity peaks should be barely visible relative to the main peak. If you can see substantial peaks other than the main compound, the purity may be lower than claimed.
Baseline
The baseline is the flat line when no compounds are eluting. A stable, flat baseline indicates a well-maintained HPLC system. Baseline drift, noise, or waviness can affect purity calculations and may indicate instrument issues rather than sample quality.
Purity Calculation
Purity is calculated as: (Area of main peak / Total area of all peaks) x 100. This is called area percent purity. For a COA stating 99.2% purity, the main peak represents 99.2% of the total integrated peak area, and all impurity peaks together represent 0.8%.
What to Look For on a COA
A trustworthy COA should include the actual chromatogram image, not just a purity number. It should list the HPLC method conditions (column type, mobile phase, gradient, detection wavelength), which allow the analysis to be reproduced. The retention time and purity percentage should be clearly stated alongside the chromatogram.
