Multimode gradient elution in reversed-phase liquid chromatography: application to retention prediction and separation optimization of a set of amino acids in gradient runs involving simultaneous variations of mobile-phase composition, flow rate, and temperature.

The theory of multimode gradient elution in liquid chromatography involving combined gradients of the mobile-phase composition with flow rate and column temperature is presented, and a very simple stepwise method that allows for the calculation of the elution time of a sample solute under all gradient conditions is proposed. The theory is successfully applied to the separation of 12 o-phthalaldehyde derivatives of amino acids in eluting systems modified by acetonitrile. Average errors below 2.9% have been found in the retention prediction using the above method, which is supported by adequate models and algorithms capable of describing the chromatographic behavior of solutes upon changes in the separation factors, such as the modifier content, flow rate, and temperature.