High-performance liquid chromatography (revision 4)

High-performance liquid chromatography (HPLC) separates the components of a mixture by passing a solution through a column packed with fine particles. Components that interact more strongly with the packing move more slowly and emerge later; the detector records what emerges against time, producing a chromatogram.^[1]

For peptides the dominant mode is reverse phase, in which the stationary phase is hydrophobic and the mobile phase is a water–acetonitrile mixture whose organic content is increased during the run. Detection is usually by ultraviolet absorbance at 214 nm, where the amide bond absorbs, so response is broadly proportional to the number of peptide bonds rather than to any particular side chain.^[2]

A sample is injected into a stream of mobile phase and carried onto the column. Each component partitions continuously between the mobile phase, in which it moves, and the stationary phase, in which it does not. The fraction of time spent in each determines how long it takes to traverse the column — its retention time.^[1]

In reverse-phase separation the stationary phase is a hydrocarbon chain, commonly C18, bonded to silica particles. Hydrophobic molecules are retained more strongly. Increasing the proportion of organic solvent in the mobile phase weakens that retention, so a gradient of increasing organic content elutes components in approximate order of hydrophobicity.

Resolution between two peaks depends on their retention difference, on peak width, and on the efficiency of the column. Efficiency improves with smaller particles and with longer columns, at the cost of higher back-pressure — the constraint that drove the development of sub-2-micron particles and the instruments capable of running them.^[3]