High speed gradient elution reversed phase liquid chromatography of bases in buffered eluents: Part II. Full equilibrium

In this work we determined when the state of thermodynamic (full) equilibrium, i.e. time-invariate solute retention, was achieved in gradient elution reversed-phase chromatography. We investigated the effects of flow rate, temperature, organic modifier, buffer type/concentration, stationary phase type, n-butanol as eluent additive, and pore size. We also measured how selectivity varied with reequilibration time. Stationary phase wetting and the ability of the stationary phase to resist changes in pH strongly affect the time needed to reach full equilibrium. For example, full equilibrium is realized with many endcapped stationary phases after flushing with only two column volumes of acetonitrile–water containing 1% (v/v) n-butanol and 0.1% (v/v) trifluoroacetic acid. Trends in retention time (<0.010 min) and selectivity become quite small after only five column volumes of reequilibration. We give practical guidelines that provide fast full equilibrium for basic compounds when chromatographed in buffered eluents.