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.

<p>This thesis concerns the development and validation of methods for characterization of multi-component preparative LC systems. Measurements of competitive adsorption isotherms are performed to gain detailed information about the interactions inside the chromatography column. This information increases our understanding of the separation process and makes it possible to perform computer simulations and numerical optimizations to find optimal operating conditions.</p><p>The methods under focus are called “the tracer-pulse method”, “the inverse method”, and “the inverse method on plateaus”. They are extensions of existing methods, with new experimental and numerical procedures to enable rapid and accurate multi-component adsorption isotherm determination. In the validation it was shown that they can produce results agreeing with traditional methods and that the acquired adsorption isotherm parameters can be used in simulations to accurately predict the outcome of preparative LC separations.</p><p>The methods were used to characterize several complex LC systems and two phenomena were discovered and theoretically treated: 1) The presence of invisible deformed peaks in single-component systems. 2) Peak deformations encountered with modern chiral stationary phases, caused by strongly adsorbed eluent additives. The latter type of deformation was highly tuneable and it was possible to adjust the enantiomer peak shapes so that the peaks tailed in opposite directions with the sharp sides in between, yielding baseline resolution at remarkably high sample loads.</p><p>In a final applied study both the LC-based perturbation peak method and a biosensor method based on surface plasmon resonance (SPR) were used for the first time for detailed characterization of chiral drug-protein interactions. The fundamental properties of the two very different methods were compared and it was found that the LC method is more suitable for multi-component analysis and that the SPR method is more suitable for stronger interactions.</p>

This thesis concerns the development and validation of methods for characterization of multi-component preparative LC systems. Measurements of competitive adsorption isotherms are performed to gain detailed information about the interactions inside the chromatography column. This information increases our understanding of the separation process and makes it possible to perform computer simulations and numerical optimizations to find optimal operating conditions. The methods under focus are called “the tracer-pulse method”, “the inverse method”, and “the inverse method on plateaus”. They are extensions of existing methods, with new experimental and numerical procedures to enable rapid and accurate multi-component adsorption isotherm determination. In the validation it was shown that they can produce results agreeing with traditional methods and that the acquired adsorption isotherm parameters can be used in simulations to accurately predict the outcome of preparative LC separations. The methods were used to characterize several complex LC systems and two phenomena were discovered and theoretically treated: 1) The presence of invisible deformed peaks in single-component systems. 2) Peak deformations encountered with modern chiral stationary phases, caused by strongly adsorbed eluent additives. The latter type of deformation was highly tuneable and it was possible to adjust the enantiomer peak shapes so that the peaks tailed in opposite directions with the sharp sides in between, yielding baseline resolution at remarkably high sample loads. In a final applied study both the LC-based perturbation peak method and a biosensor method based on surface plasmon resonance (SPR) were used for the first time for detailed characterization of chiral drug-protein interactions. The fundamental properties of the two very different methods were compared and it was found that the LC method is more suitable for multi-component analysis and that the SPR method is more suitable for stronger interactions.