Reversed-phase chromatography, principle and application for prediction of biological activity

Abstract

Reversed-phase chromatography is the term used to describe the chromatographic method in which the stationary phase is hydrophobic or non-polar (e.g. octadecyl group), while the starting mobile phase (e.g. water) must be more polar than the stationary phase. The distribution of the solute between the two phases depends on the binding properties of the stationary phase, the hydrophobicity of the solute and the composition of the mobile phase. Reversed phase chromatography is an adsorptive process, which relies on a partitioning mechanism to effect separation. Elution can be performed isocratic (the water-solvent composition does not change during the separation process) or by using a gradient (the water-solvent composition does changes during the separation process). Reversed phase chromatography has found both analytical and preparative applications in the area of biochemical separation and purification. Lipophilicity is an important physicochemical property of bioactive compounds, related to the ability of molecule to be transported through biological membranes. Reversed-phase thin layer chromatography is frequently used to estimate the lipophilicity of organic compounds. It is considered that the very similar principal intermolecular interactions determine the behavior of chemical compounds in both biological and chromatographic environments. From the obtained retention parameters (RF) the lipophilicity parameters (RM° and C°) of the investigated compounds can be determined. The chromatographically obtained lipophilicity parameters describes intra- and intermolecular interactions that actually exist under the experimental conditions, thus give more realistic description of examined system, than calculated values and can be used for further quantitative structure-activity relationship studies. As the example, the determination of the lipophilicity of some Schiff bases by RP TLC and their correlation with antibacterial activity was described.