Reversed-Phase HPLC on Monomeric Reversed Phases: Factors Determining Adsorbate Retention

In this brief review, we consider various characterizations of “monomeric” reversed phases for elucidating the interactions governing adsorbate retention in liquid chromatography. Conventional methods related to the assessment of retention capacity and hydrophobicity (specifically methylene selectivity) using single mobile phase compositions are discussed with a focus on dispersion interactions, along with their inherent strengths and limitations. An alternative approach involving separation maps through relative retention analysis is proposed. It is noted that, in real reversed-phase adsorbents, the density of the attached alkyl chains is typically one half of that of solid n-alkanes. In this case, adsorbate molecules to penetrate into the attached phase, and the process depends on the molecular shape. Consequently, conventional “monomeric” reversed phases exhibit specific selectivity towards substances with specific structures. The review also notes that current analytical methods often do not pay sufficient attention to the difference between the substance retention mechanisms, absorption and adsorption, because the predominant parameters of these mechanisms are quite different. Moreover, in the two most widely used very interesting and informative methods, linear solvation energy relationships (LSERs) and the hydrophobic-subtraction model, this characteristic has not received due attention. Taking into account that the method does not distinguish adsorbates retained by different mechanisms, absorptive versus adsorptive, to the obtained significant discrepancies between the calculated and experimental data do not seem extraordinary. The interpretation of the results of an LSER analysis is also complicated by uncertainties in the contributions of partial properties of adsorbates in both mobile and stationary phases to the total solvation energy, as only their difference is typically calculated. Nonetheless, a comparison of different columns in identical mobile phases can yield informative insights. A drawback of the second approach is the necessity of using multiple columns with substantial qualitative differences in the adsorbate retention among them. Furthermore, a possibility of the decomposition of all interactions into distinct types seems questionable, because the method does not involve any orthogonal (independent of the applied calculation method) properties.