Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most popular chromatographic mode, accounting for more than 90% of all separations. HPLC itself owes its immense popularity to it being relatively simple and inexpensive, with the equipment being reliable and easy to operate. Due to extensive automation, it can be run virtually unattended with multiple samples at various separation conditions, even by relatively low-skilled personnel. Currently, there are >600 RP-HPLC columns available to end users for purchase, some of which exhibit very large differences in selectivity and production quality. Often, two similar RP-HPLC columns are not equally suitable for the requisite separation, and to date, there is no universal RP-HPLC column covering a variety of analytes. This forces analytical laboratories to keep a multitude of diverse columns. Therefore, column selection is a crucial segment of RP-HPLC method development, especially since sample complexity is constantly increasing. Rationally choosing an appropriate column is complicated. In addition to the differences in the primary intermolecular interactions with analytes of the dispersive (London) type, individual columns can also exhibit a unique character owing to specific polar, hydrogen bond, and electron pair donor–acceptor interactions. They can also vary depending on the type of packing, amount and type of residual silanols, “end-capping”, bonding density of ligands, and pore size, among others. Consequently, the chromatographic performance of RP-HPLC systems is often considerably altered depending on the selected column. Although a wide spectrum of knowledge is available on this important subject, there is still a lack of a comprehensive review for an objective comparison and/or selection of chromatographic columns. We aim for this review to be a comprehensive, authoritative, critical, and easily readable monograph of the most relevant publications regarding column selection and characterization in RP-HPLC covering the past four decades. Future perspectives, which involve the integration of state-of-the-art molecular simulations (molecular dynamics or Monte Carlo) with minimal experiments, aimed at nearly “experiment-free” column selection methodology, are proposed.

The selectivity of chromatographic separation depends mostly on the stationary phase and mobile phase composition. Despite being a material with bonded simple organic molecule, a wide group of stationary phases contain immobilized compound that possesses biological activity. Stationary phases that contain amino acids and peptides as well as enzymes and proteins are alternative materials that may be used for liquid chromatographic separations and are reviewed in this work. In the case of peptide-bonded stationary phases, most of these types of materials were elaborated in the 1970s and 1980s; however, over the last few years a growing interest has been observed which is connected with hydrophilic interaction liquid chromatography. The most important application of amino acid and peptide-bonded stationary phases is connected with separation of amino acids, their derivatives, and peptides. The main advantage of such materials is the ability for chiral separations.

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In this article, we review the preparation methods of silica and silica hybrid materials, as well as their surface modification and application in liquid chromatography (LC). First we discuss the synthesis and properties of silica and silica hybrids. In the second part, we present the modification methods leading to their stationary phases. The current state of the art of the stationary phase's synthesis during silanization, hydrosilation, and coating is discussed. A detailed characterization of both native silica and its modifications is provided. The most common stationary phases for reversed‐phase, normal‐phase, and hydrophilic interaction LC are featured. Unconventional stationary phases, for example, with bonded cholesterol molecules, are also included. The potential applications of the described stationary phases are presented.

A series of phenyl-bonded stationary phases with incorporated polar functional groups was subjected to an adsorption investigation. Measurement of acetonitrile and methanol adsorption was obtained using the minor disturbance method. It was observed that adsorption of organic solvent strongly depends on the presence of polar functional groups in the bonded phases that influence the hydrophobicity and polarity of the stationary phase surface. Additionally, relative adsorption of acetonitrile and methanol confirms earlier observations, that the presence of amine and amide groups in the stationary phase changes the relative elution strength of organic solvents. The heterogeneous surface of the stationary phase makes it possible to observe the competitiveness of the water and organic solvent adsorption.