Column Characterization and Selection Systems in Reversed-Phase High-Performance Liquid Chromatography

Abstract

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.

A Critical Review of Bottom-Up Proteomics: The Good, the Bad, and the Future of This Field

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Extraction and Determination of Polar Bioactive Compounds from Alfalfa (Medicago sativa L.) Using Supercritical Techniques

“…In case of naringenin and apigenin, were observed strongest retention, which is probably results of π-π interactions and hydrogen bonding with proton-donors between aromatic moieties from flavonoids and polar stationary phase [30]. This affects the band broadening during the chromatographic separation and also affects mass transfer, which includes thermodynamic processes responsible for system selectivity [31].…”

The challenge of separating and determining biologically active electrostatically stabilized silanates using the high‐performance liquid chromatography technique

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