A Certificate of Analysis (COA) is the definitive proof of a compound's identity and purity. Yet, for many researchers new to peptide sourcing, reading a COA can feel like deciphering an ancient language.
Here is a straightforward guide to interpreting the two most critical components of a peptide COA: the HPLC chromatogram and the Mass Spectrometry (LC-MS) report.
1. High-Performance Liquid Chromatography (HPLC)
The HPLC report is your primary tool for determining purity.
During HPLC, the peptide sample is dissolved and pumped through a column under high pressure. Different molecules travel through the column at different speeds, separating the pure peptide from any impurities or synthesis byproducts.
What to look for:
- The Main Peak: You should see one dominant, sharp peak on the graph. This represents your target peptide.
- Area Percentage (Area %): Below the graph, a table will list the area under each peak. The Area % of the main peak is the purity of the peptide. Our standard demands that this number is ≥99.0%.
- Impurity Peaks: Small bumps before or after the main peak represent impurities. The table will detail exactly what percentage of the sample is comprised of these impurities.
2. Mass Spectrometry (LC-MS)
While HPLC tells you how pure the sample is, Mass Spectrometry tells you what the sample actually is (Identity).
Mass spec measures the mass-to-charge ratio of the ions in the sample. Every peptide sequence has a specific, calculable molecular weight based on its amino acid chain.
What to look for:
- The Target Mass: Check the theoretical molecular weight of the peptide you ordered.
- The Observed Mass: Look at the highest peaks on the mass spec graph. You will typically see peaks corresponding to the peptide carrying different numbers of protons (e.g., [M+H]+, [M+2H]2+).
- The Match: The observed mass must precisely match the theoretical mass. If it doesn't, the amino acid sequence is incorrect, and the batch is invalid.
Trust, but Verify
Never accept a COA that lacks either HPLC or LC-MS data. Furthermore, ensure the COA clearly lists the batch number and date of testing. Transparency is the cornerstone of rigorous science.