What HPLC Measures
High-Performance Liquid Chromatography (HPLC) separates compounds in a mixture by their interaction with a stationary phase (a packed column) and a mobile phase (a solvent gradient). For peptide analysis, reverse-phase HPLC is the standard — the column is non-polar, the mobile phase starts polar and becomes increasingly non-polar over the run. Compounds elute (exit the column) at different times based on their hydrophobicity.
The Chromatogram Display
A chromatogram is a 2D plot. The horizontal axis is retention time in minutes — how long after injection a compound exited the column. The vertical axis is detector response, usually UV absorbance at 214 nm or 220 nm for peptide bonds. Each compound produces a peak. The target peptide ideally produces one large, symmetric peak.
Reading the Peaks
For a clean batch, expect a single dominant peak with retention time matching the peptide’s reference standard. Any additional peaks represent impurities — typically deletion sequences (peptide missing one or more amino acids), truncation products, or oxidation byproducts. A well-purified batch shows the main peak accounting for ≥99% of the total area under the curve.
Purity Calculation
HPLC purity is calculated as: (Area of main peak ÷ Sum of all peak areas) × 100%. This is an area normalization method — it assumes all detected compounds have similar UV absorptivity at the chosen wavelength, which is approximately true for peptide bonds at 214 nm.
What to Watch For
Beyond the purity percentage, examine the main peak’s shape. A sharp, symmetric peak indicates a homogeneous compound. Broad peaks or peaks with shoulders suggest the presence of closely-related impurities that didn’t fully resolve. A clean chromatogram shows a flat baseline before and after the main peak, with no rising drift.
Pairing HPLC With LC-MS
HPLC tells you purity. To confirm identity, you need LC-MS (Liquid Chromatography-Mass Spectrometry). LC-MS measures the molecular mass of the eluting peak and compares it to the theoretical mass calculated from the peptide sequence. A purity result without identity confirmation is incomplete — every batch in our catalog gets both.
See Real Chromatograms
Browse the COA Library to see actual chromatograms from current inventory batches — every product page links to its batch-specific COA. For more on storage, reconstitution, and shelf-life, see our Storage Protocol. Questions about COA interpretation? Contact our research team.