High-Performance Liquid Chromatography (HPLC)

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HPLC is the gold-standard analytical method for determining peptide purity. It separates the components of a sample so the main peak’s area can be reported as a purity percentage.

In reverse-phase HPLC, the most common mode for peptide analysis, a pressurized mobile phase carries the dissolved sample through a column packed with a hydrophobic C18 stationary phase. Individual components separate according to how strongly they interact with that phase, emerging at distinct retention times that a UV detector records as peaks on a chromatogram.

Purity is calculated by integrating the area of the main peak relative to the total area of all peaks, so a single sharp dominant peak indicates a high-purity sample while additional peaks signal impurities or degradation. This chromatogram is the primary quantitative result reported on a lot-specific certificate of analysis.