High-performance liquid chromatography

High-performance liquid chromatography, usually abbreviated to HPLC, separates a mixture by passing it in solution through a packed column. Components interact with the stationary phase to different degrees and therefore emerge at different times. A detector at the outlet records a signal against time, producing a chromatogram.

Reversed-phase separation

Peptide work overwhelmingly uses reversed-phase chromatography, in which the stationary phase is non-polar and the mobile phase is aqueous. Separation is driven largely by hydrophobicity, so peptides differing in sequence but similar in hydrophobic character may be poorly resolved. This is a known limitation rather than a fault in any particular run.

Reading a chromatogram

• Retention time locates a peak but does not identify it. Retention shifts with column, temperature, gradient and instrument.
• Peak area, not peak height, is used for quantification.
• Peak shape carries information: tailing or fronting can indicate overload, secondary interactions or a column problem, and can also conceal a poorly resolved neighbour.
• Baseline behaviour matters. A drifting or noisy baseline changes how integration boundaries are drawn, which changes the reported percentage.
• The gradient and detection wavelength must be stated for the trace to be interpretable at all.