Analytical methods · 7 min read
Reading an HPLC Chromatogram, Step by Step
How to read the HPLC chromatogram on a peptide certificate: axes, retention time, main peak, impurity peaks, integration, and how purity is calculated.
A purity percentage is a summary. The chromatogram is the evidence behind it. Being able to read one takes about five minutes and lets a researcher judge a lot independently of the number printed at the top of the certificate.
1. The axes
The horizontal axis is retention time in minutes, the time each component takes to pass through the column. The vertical axis is detector response, usually UV absorbance in milli-absorbance units (mAU) at 214 or 220 nm.
2. The method conditions
A proper chromatogram lists the column type (commonly C18), the mobile phases (water and acetonitrile with an acid modifier), the gradient, flow rate, and wavelength. Without them the trace cannot be reproduced or compared.
3. The main peak
The tallest peak should be sharp and symmetrical. A shoulder on either side can indicate a closely related impurity that did not fully separate. Significant tailing can indicate column or method problems.
4. Impurity peaks
Small peaks before and after the main peak are typically synthesis by-products such as deletion sequences or oxidized forms; see how synthesis creates impurities. Peaks at the very start of the run, the solvent front, are generally excluded from integration.
5. Integration and the purity calculation
Software integrates the area under each peak. Purity equals the main peak area divided by the total integrated area, multiplied by 100. The peak table should list retention time, area, and area percent for every integrated peak, so the calculation can be checked by hand.
6. Warning signs
- A trace with no axis labels, scale, or method details
- A perfectly flat baseline with a single spike, which can indicate an edited image
- A purity figure that does not match the peak table
- The same chromatogram reused across different lots
Pair the chromatogram with an identity result; see HPLC vs. mass spectrometry. Example reference pages: GHK-Cu and Epitalon.
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