Nonlinear behavior in preparative liquid chromatography: A method‐development case study for hydroxytyrosol purification

Abstract Preparative liquid chromatography has become an important purification method owing to its advantages of high separation efficiency, good reproducibility, and low solvent consumption. Because overloading in preparative liquid chromatography must be performed to increase the throughput in a cycle, nonlinear chromatographic behavior is observed. Therefore, it is crucial to carefully study nonlinear chromatography for the purification of a given product, which facilitates the efficient optimization of the purification parameters. In this work, a method for the development of a purification method using preparative liquid chromatography based on nonlinear chromatography is proposed. Hydroxytyrosol was selected as the subject for method demonstration. Using methanol and ethanol as organic modifiers, the optimum flow rate was determined on three commercial columns entitled C 8 TDE, C 18 ME, and C 18 TDE, respectively. The curves were fitted with the van Deemter equation, with thorough analysis of the A , B , and C terms. Adsorption isotherms were subsequently studied to explore the distribution of solutes between the stationary and mobile phases at equilibrium. C 18 TDE, 5 vol% ethanol–water, and 0.2 mL/min were selected as the optimal separation material, elution solvent, and flow rate, respectively. Purification of hydroxytyrosol was tentatively confirmed on a C 18 TDE column with 1.6% sample loading, 90.98% recovery, and 98.01% purity.