How HPLC Works
A small amount of peptide is dissolved and injected into a column packed with a stationary phase. A mobile phase (a controlled mixture of solvents) carries the sample through the column. Different molecules travel at different speeds based on their interactions with the stationary phase, so they exit the column at different times.
Reading the Chromatogram
A detector — typically UV at a wavelength such as 214 nm for peptide bonds — measures absorbance as compounds elute. The output is a chromatogram: a plot of detector signal versus time, with peaks corresponding to individual compounds. The area under the principal peak, divided by the total area of all peaks, gives the purity percentage.
What 'HPLC Verified' Means in Practice
When a peptide is labeled ≥99% HPLC purity, the principal peak accounts for at least 99% of the total area. The remaining ≤1% is composed of impurities, related sequences, or solvent artifacts. A Certificate of Analysis records the assay conditions and result for the specific lot.
Why Independent Verification Matters
An in-house assay is a starting point. Independent third-party HPLC adds an additional check — a second laboratory, a second instrument, a second analyst — to confirm the supplier's purity claim. For reproducible research, this independence reduces the risk of upstream characterization errors propagating downstream.
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