Chromatography is a widely used separation technique including many different modes, for example supercritical fluid chromatography (SFC) which uses a supercritical fluid as mobile phase. A supercritical fluid is achieved when a substance is subjected to a temperature and pressure above the critical point and the boundary between the liquid phase and gas phase is erased. The interest for SFC has increased in recent years, mainly for separation of chiral molecules in the pharmaceutical industry. What makes SFC interesting is that it is a quick, cost-efficient and green method. This is in part due to less organic solvent used in the mobile phase in SFC compared with liquid chromatography and that the carbon dioxide that represents the major part of the mobile phase is a by-product from other processes. In SFC modifiers, often small alcohols, are added to carbon dioxide based mobile phase in order to increase the solubility of polar compounds. In this study the adsorption of methanol to two different stationary phases; Kromasil-Diol and chiral Lux Cellulose-4 were studied. Adsorption is a phenomenon where surface interactions crate a higher density of molecules at the surface than in the bulk. The aim of this work has been to study the adsorption of modifier (methanol) to the stationary phase both to determine the extent of adsorption and the kinetics for system equilibration. These findings were then put into perspective of normal use of SFC for separation of molecules. There are a number of techniques for measuring adsorption; in this study the tracer pulse method is used. This is a pulse method where a concentration plateau is created and an isotope labelled molecule is injected. This was performed in the mobile phase composition from pure carbon dioxide to pure methanol. In addition to the tracer pulse experiments the isotope effect, the eluent flow, equilibration times for the column and retention times for a set of analytes were measured. For the Diol column no large isotope effect was observed, the method was also proved to be highly reproducible since several runs gave consistent results. Calculations based on the experimental data showed that a 6.3 Å thick layer was built up at a methanol fraction of 13% (v/v), corresponding to a monolayer. Changes of the methanol fraction below the saturation level has has greater effect on the retention factor for the analytes than at higher methanol fractions, when the monolayer was saturated. The conclusion of this is that SFC is more stable in the area where the layer has been built up. A preliminary study has been made for the chiral Lux Cellulose-4 column which was not as conclusive as for the Kromasil-Diol column. This type of column needs further studies to confirm the deviating observations and to investigate the cause for these.

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.