How to read a CoA and a chromatogram

What is a certificate of analysis (CoA)

A certificate of analysis, abbreviated CoA, is an analytical document linked to a specific batch of a substance, presenting the results of laboratory testing. A CoA for a peptide typically contains at least two types of analysis: an HPLC analysis for determining purity, and a mass spectrometry analysis (MS) for confirming the molecular weight. The document usually also states the batch number, the test date, and the analytical method used.

Reading the chromatogram: retention time and main peak

An HPLC chromatogram shows time, expressed in minutes, on the horizontal axis, and the signal intensity measured by the detector on the vertical axis. As a substance flows through the column, it reaches the detector after a specific retention time, which is characteristic of that substance under the analytical conditions used. This produces a peak on the chromatogram. The largest peak, occupying the greatest share of the area, is called the main peak and represents the main component of the sample.

Peak area percentage and secondary peaks

Alongside the main peak, smaller peaks may appear at other retention times. These secondary peaks represent related substances or impurities present at lower concentrations. Software calculates the area for each peak and expresses it as a percentage of the total area of all peaks combined. The area percentage of the main peak is the purity percentage stated on a CoA. The smaller and fewer the secondary peaks, the higher the reported purity percentage.

Mass spectrometry: confirming the molecular weight

In addition to the HPLC analysis, a complete CoA includes a mass spectrometry result. In this technique, molecules are ionised and separated according to their mass to charge ratio. The result is a measured molecular weight, which is compared to the theoretically calculated molecular weight of the intended peptide sequence. A match between the measured and expected mass confirms the identity of the substance. Without this MS confirmation, an HPLC result only provides insight into purity, not into whether the main component actually corresponds to the intended peptide sequence.

Assessing a CoA in practice

When assessing a CoA, it is important to check whether the batch number corresponds to the substance received, whether both an HPLC and an MS result are present, and whether the reported retention time and molecular weight are consistent with the intended substance. A chromatogram with a single sharp main peak and minimal secondary peaks, combined with an MS result matching the expected molecular weight, together form the basis of a well documented analysis.