Column equilibration effects in gradient elution in reversed-phase liquid chromatography

The fundamental equations and conditions for linear and stepwise gradient elution in reversed-phase liquid chromatography are applied to a mixture of amino acids in their underivatized form in aqueous mobile phases modified by 2-propanol, acetonitrile or methanol for examining column equilibration effects. It was found in all cases systematic deviations between experimental and calculated retention times, which are prominent in 2-propanol, reduced in acetonitrile and practically negligible in methanol. These deviations appear within a chromatogram just after the first change in the composition of the mobile phase reaches the detector and last ca. 5 min, where the magnitude of errors reduces exponentially with time. Based on these observations we propose a simple way to correct the calculated from the gradient elution theory retention times of sample solutes. The origin of the discrepancies between theory and experiment as well as their impact on the resolution is also discussed.