The molecular mechanism of retention in reversed-phase liquid chromatography

The term “reversed-phase” chromatography seems at first inappropriate for what is by far the most popular mode of modern liquid chromatography. Estimates of the popularity of the technique range from 57% of all analytical chromatography1 to as high as 80-90% .* The term itself can be traced to Howard and Martin in 1950.3 In attempting the separation of long-chain fatty acids they realized that the “normal” mode of chromatography, using a polar stationary phase and nonpolar mobile phase, would not work, as the hydrophobic compounds had too little retention to effect a separation. They were able to treat Kieselguhr with dimethyldichlorosilane vapor and then coat this hydrophobic support with a nonpolar liquid stationary phase. Both the polarity of the phases and the respective elution order of solutes were reversed from traditional chromatographic systems, and they christened the technique “reversed-phase” partition chromatography. The popularity of reversed-phase liquid chromatography (RPLC), as practiced today, can be attributed to the development of chemically stable, microparticulate bonded phases that provide rapid mass transfer and a high degree of reproducibility. Attempts to utilize liquid-liquid chromatography, with a liquid stationary phase physically coated on an inert support, were rapidly abandoned with the introduction of commercially available bonded phases. Interesting perspectives on the early development of bonded phases for modern liquid chromatography can be found in a book devoted to the history of liquid chromatography.* H. A. Laitinen, in an editorial in Analytical Chemistry, described the seven ages of an analytical method from the birth of an idea to the ultimate replacement of the method by newer technique^.^ The fourth phase he described as “...detailed studies of principle and mechanisms are pursued with the aid of improved instrumentation. This represents the stage at which the method matures as an accepted procedure in competition and cooperation with other approaches. This stage represents the crest of analytical research as distinguished from instrumentation research.” This most clearly describes the present status of reversedphase liquid chromatography. Many methods of investigation are being brought to bear on the problem of understanding the molecular mechanism of retention of RPLC. These range from spectroscopic studies, including UV-visible, IR, NMR, fluorescence, and others, to thermal methods, to neutron scattering, to chromatographic methods themselves. Experimental studies alone are not enough. The cooperation of theorists and experimentalists is leading to dramatic advances in the understanding of the retention process. The goal of this review is to critically assess the current understanding of retention of small molecules in reversed-phase chromatography from both a theoretical and experimental perspective. We first discuss the synthetic methodologies for the preparation of reversed-phase stationary phases and deal next with the partitioning processes and stationary-phase structural details.