Reversed Phase Liquid Chromatography

Abstract

Reversed phase liquid chromatography ( RPLC ) is a mode of high‐performance liquid chromatography ( HPLC ) that employs a nonpolar stationary phase (most frequently a hydrocarbon chain chemically bonded to porous silica particles) and a polar mobile phase constituted by water and at least a water‐miscible organic solvent, which performs as a modifier. RPLC is a mature technique, employed in science and technology by chemists, biochemists, and pharmacists for analysis and purification. Applications cover environmental control, food, clinical, pharmaceutical, and industrial analyses, drug and chemical manufacturing (at both quality control and preparative scales), biomedical studies, and measurement of physicochemical properties. The development of RPLC has been based primarily on empirical observations. The diversity of retention mechanisms has received considerable attention, sometimes with conflicting results. This reflects the difficulty in understanding the underlying principles. In this chapter, the theoretical background and technical traits of RPLC are outlined. The factors that contribute to the separation are described, together with the main approaches proposed to understand the observed behaviors. The advantages and limitations of using either isocratic or gradient elution are discussed. A picture of the retention behavior at the molecular level, including a description of the dewetting effect of highly aqueous mobile phases, is also given. The trends in the current evolution of the technique are commented upon. The overview contains over 100 references.

Protein Hydrolysate from Underutilized Legumes: Unleashing the Potential for Future Functional Foods

“…RPLC offers numerous advantages, including its compatibility with aqueous samples, gradient elution capability, the ability to separate ionic, nonpolar, and polar compounds within a single run, a wide range of commercially available stationary phases, and its extensive application in various fields ( García-Alvarez-Coque et al, 2016 ).…”

A Review of Analytical Methods for Codeine Determination

“…In addition, these systems have also been used to detect codeine and its active metabolites in biological samples [ 86 , 87 ]. C-18 columns are popular in RP-HPLC systems due to their relatively high organic/carbon contents, allowing for better interaction between the organic solutes/analytes with the stationary phase [ 88 ]. These columns are also more stable at very low or very high pHs compared with columns with shorter alkyl chains [ 89 , 90 ].…”

Estimation of Solute-Stationary Phase and Solute-Mobile Phase Interactions in the Presence of Ionic Liquids

“…On the other hand, it should be noted that the peaks are highly symmetrical, especially in the presence of [C 6 MIM] + . This suggests that all these additives efficiently hinder the access of the β-adrenoceptor antagonists to the residual free silanols on the stationary phase (even for [C 2 4 ], for which the retention of the probe compounds did not change at increasing concentrations of the additives-at least not significantly). On the other hand, it should be noted that the peaks are highly symmetrical, especially in the presence of [C6MIM] + .…”

“…Alkyl-bonded stationary phases (commonly octadecylsilane or C18) with silica as support are extensively used in reversed-phase liquid chromatography (RPLC) due to their capability to separate a wide variety of compounds [1,2]. Solutes are mainly retained according to their hydrophobicity.…”