Anatomy of an HPLC Chromatogram
An HPLC chromatogram is a graph plotting detector response (y-axis) against retention time (x-axis). For peptide purity analysis, the chromatogram reveals the composition of a sample — each peak represents a distinct chemical species, with the area under each peak proportional to its concentration.
Key Features to Examine
- Main peak: The dominant peak representing the target peptide. Should be sharp, symmetrical, and well-resolved from neighboring peaks.
- Retention time: The time at which the target peptide elutes. This is characteristic of the peptide's hydrophobicity and should be consistent across batches.
- Minor peaks: Smaller peaks representing impurities — truncated sequences, deletion peptides, or oxidation products.
- Baseline: Should be flat and stable. A noisy or drifting baseline may indicate column problems or gradient issues.
Calculating Purity
Purity is calculated by dividing the area of the target peptide peak by the total area of all detected peaks, multiplied by 100. For example, if the main peak area is 980 units and total peak area is 1000 units, purity is 98.0%. Research-grade peptides from suppliers like ROEHN typically achieve ≥98% HPLC purity, as documented on our COAs for products like BPC-157 and Semaglutide.
Common Impurity Peaks
Understanding what impurity peaks represent helps assess whether they impact your specific research application. Common impurities include deletion peptides (missing one amino acid), truncated sequences, oxidized forms, and residual protecting groups. Mass spectrometry data on the COA can identify specific impurity species. Always review the full COA for products from ROEHN's catalog.
Research Disclaimer
This article is for educational and informational purposes only. All compounds discussed are intended strictly for in-vitro and preclinical research use. They are not intended for human consumption. Always consult published scientific literature and institutional review protocols before initiating any research program.