High-performance liquid chromatography

High-performance liquid chromatography is liquid chromatography typically using a finely divided stationary packing and a pump supplying the pressure needed to drive the chromatographic mobile phase through it. A sample mixture is injected, its components interact differently with the chromatographic stationary phase, and a detector records their elution. IUPAC recognizes both high-performance and high-pressure terminology for this form of liquid chromatography.^[ref]

The pressure supplies flow through the column; differential interactions produce the separation. Raising pressure alone does not specify which component will elute first. The solvent composition, stationary material and analyte chemistry all matter. The useful result is a set of peaks separated sufficiently for the intended identification or measurement.

An original example illustrates how to assess a recorded separation. Suppose two peaks have chromatographic retention times $t_{R1}=5.0$ minutes and $t_{R2}=6.0$ minutes, with baseline widths $w_1=w_2=0.40$ minutes. Using widths at the base, their chromatographic resolution isIf the widths become $0.80$ minutes each while the peak times stay fixed, $R_s$ falls to $1.25$. Peak timing alone therefore does not establish separation quality. Widths measured at half-height require the corresponding formula rather than substitution into this baseline-width definition.^[ref]

Matching a chromatographic retention time to a standard under the same conditions is useful evidence, but different substances can coelute. Quantifying a peak also requires a calibrated detector response; its area is not automatically an amount independent of molecular identity and instrument settings.