This master's thesis explores the principal retention mechanism in reversed-phase liquid chromatography from two perspectives: an experimental comparison of various void volume determination techniques, and a theoretical statistical-mechanics study aimed at simplifying the highly complex separation mechanism.
In the comparative analysis of different void volume determination techniques, we investigated three commonly used void volume markers (uracil, N,N-dimethylformamide, and phloroglucinol) along with deuterated acetonitrile, using liquid chromatography-mass spectrometry. Our findings suggest that the selected void volume markers exhibit parabolic retention patterns, which depend on the composition of the mobile phase. Additionally, we discovered that deuterated acetonitrile itself demonstrates retention-like behaviour in reversed-phase liquid chromatography. To achieve the minor disturbance method in a water:acetonitrile mobile phase, we used deuterated acetonitrile as the deuterated mobile phase component. Our investigation concluded that the void volume determined using this method agrees with the thermodynamically defined column void volume. A comparison of void volumes, obtained using void volume markers, revealed that the former underestimate the column void volume. We used deuterated acetonitrile retention data to calculate excess adsorption isotherms of acetonitrile. Furthermore, we concluded that utilizing deuterated water in a manner similar to deuterated acetonitrile does not yield favourable results with the minor disturbance method, due to extensive isotopic exchange, leading to an increase of the limit of detection, along with instrumental limitations of measuring low nominal masses.
The theoretical study is divided into two parts: (i) by using the PROZA theory, based on Wertheim's integral equation theory for associating liquids, we constructed a model chromatographic stationary phase using a polymeric matrix of hard spheres, (ii) we used the replica Ornstein-Zernike theory to study the interactions between a binary fluid and the polymeric matrix. These interactions were modelled using a hard sphere and a hard sphere attractive Yukawa pair potential. Comparative analysis of results for an unpolymerized monomer solution and the polymer matrix suggests successful modelling of polymerization. Additionally, we successfully calculated adsorption isotherms at three fluid compositions for both model stationary phases.
The main findings of the determination of column void volumes study present a thermodynamically concise method for determining the column void volume, based on the minor disturbance method. Results from the theoretical study suggest potential research avenues for investigating the limitations and applications of the developed theoretical model.
|
