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This thesis aims at a deeper understanding of Supercritical Fluid Chromatography (SFC). Although preparative SFC has started to replace Liquid Chromatography (LC) in the pharmaceutical industry - because of its advantages in speed and its less environmental impact - fundamental understanding is still lacking. Therefore there is no rigid framework to characterize adsorption or to understand the impact of changes in operational conditions. In Paper I we demonstrated, after careful system verification, that most methods applied to determine adsorption isotherms in LC could not be applied directly in SFC. This was mainly due to operational differences and to the fact that the fluid is compressible which means that everything considered constant in LC varies in SFC. In Paper II we showed that the most accurate methods for adsorption isotherm determination in LC, the so called plateau methods, do not work properly for SFC. Instead, methods based on overloaded profiles should be preferred. In Paper III a Design of Experiments approach was successfully used to quantitatively describe the retention behavior of several solutes and the productivity of a two component separation system. This approach can be used to optimize SFC separations or to provide information about the separation system. In Paper IV severe peak distortion effects, suspected to arise from injection solvent and mobile phase fluid mismatches, were carefully investigated using experiments and simulations. By this approach it was possible to examine the underlying reasons for the distortions, which is vital for method development. Finally, in Paper V, the acquired knowledge from Paper I-IV was used to perform reliable scale-up in an industrial setting for the first time. This was done by carefully matching the conditions inside the analytical and preparative column with each other. The results could therefore provide the industry with key knowledge for further implementation of SFC. This thesis aims at a deeper understanding of Supercritical Fluid Chromatography (SFC). Although preparative SFC has started to replace Liquid Chromatography (LC) in the pharmaceutical industry - because of its advantages in speed and its less environmental impact - fundamental understanding is still lacking. Therefore there is no rigid framework to characterize adsorption or to understand the impact of changes in operational conditions. In Paper I-II it was demonstrated why most methods applied to determine adsorption isotherms in LC could not be applied directly for SFC. Methods based on extracting data from overloaded profiles should be preferred. In Paper III a Design of Experiments approach was successfully used to quantitatively describe the behavior of several solutes in a separation system. This approach can be used to optimize SFC separations or to provide information about the separation system. In Paper IV severe peak distortion effects often observed in SFC were carefully investigated and explained using experiments and simulations. Finally, in Paper V, the prerequisites for performing reliable and predictable scale-up of SFC were investigated by small and large scale experiments. Paper 4 ("Evaluation of scale-up from analytical to preparative...") ingick som manuskript med samma titel i avhandlingen. Nu publicerad.