Revisiting the definition and measurement of total accessible and hold-up volumes in liquid chromatography: Current understanding and advances

Abstract

The accurate determination of the retention factor, and particularly, the determination of the mobile phase volume that is needed for its computation, remains a fundamental, yet highly-debated topic in liquid chromatography. Two main approaches currently co-exist in literature: i) one based on the total accessible volume (= the total column volume accessible for solvent molecules after bonded phase coating) and ii) one based on the mobile phase volume that is accessible to unretained analytes (the hold-up volume). This review provides a clear graphical definition for both volumes and subsequently critically examines their use in current literature and reviews their methods of determination, emphasizing the need for careful experimental practice and standardized, universally accepted definitions. By comparing traditional experimental methods with findings from molecular dynamics simulations and Monte Carlo method studies, this review furthermore aims to clarify ambiguities and recommend best practices for future research. In addition, a comprehensive table linking the different names, symbols and definitions that have been used throughout literature to denote the different mobile phase volumes is provided, to help researchers navigate literature more easily.