Development of Methods for Phase System Characterization in Liquid Chromatography

Abstract: The aim of this thesis is first and foremost to improve the fundamental knowledge of nonlinear and preparative separation theory by focusing on some of the remaining “white spots” on the theoretical chromatographic map. Secondly, the acquired knowledge is used to develop, validate and execute new methods for phase characterization in liquid chromatography. The methodology used in this thesis is a combination of experiments, fundamental nonlinear theory and systematic computer simulations.A fundamental knowledge of the molecular interactions between the compounds to be separated and the separation media requires the determination of adsorption isotherms over a broad concentration range to give a complete picture of all interactions in the separation system - weak as well as strong. In addition, such adsorption data is essential for optimization in preparative chromatography.For the first time, it has been experimentally shown that the injected molecules are not present in the detected peak when a small excess of molecules are injected into a chromatographic system equilibrated with a constant stream of identical molecules. Several experimental procedures for this method were developed such as (i) the optimal injection strategy and (ii) different labeling methods for visualizing the injected molecules. Remarkable phenomena in the single-component case, such as invisible peak deformation and deformed (invisible) frontal chromatograms, are reported, investigated, and explained. This phenomenon has asides from its future practical implementation, also a large didactic value.The accuracy of the ECP method is experimentally improved, and used to characterize the separation of protolytic compounds at different pH on modern commercially available silica and hybrid silica column packing materials. That investigation enables us to answer why basic compounds give a much more compact preparative peak profile at pH 11 than they yields at lower pH.