Extraction and chromatography of bioactive compounds in complex samples using supercritical CO2 technology

Bioactive compounds found in plants have been of interest to man since ancient times. These compounds have the ability to modulate the metabolic processes in our bodies, which suggests that they may promote better health. Bioactive compounds vary in their chemical structure, polarity, stability and biological activity. This diversity makes the study of bioactive compounds challenging from the perspective of analytical chemistry. The extraction of bioactive compounds using conventional solid–liquid extraction (SLE) is slow due to mass transfer limitations. While increasing the temperature speeds up the mass transfer, it also leads to degradation and oxidation. Supercritical CO2 (ScCO2) extraction offers high mass transfer at low temperature, but it has selective solubility towards nonpolar compounds.This thesis describes the development of techniques and methods for the extraction and chromatographic analysis of bioactive compounds leading to improvements in mass transfer, solubility and resolution, using ScCO2 technology. Ultrahigh- pressure supercritical fluid extraction improved the solubility and extractability of oil from moringa seeds due to an increase in the density of the solvent. Extraction at 80 MPa increased the amount of oil extracted by about 30% in a short time, compared to extraction at 40 MPa. The selectivity was also affected, as higher content of polyunsaturated fatty acids and some phospholipid species were detected in the oil extracted at 80 MPa. CO2 expanded liquid extraction (CXLE) combined enhanced mass transfer and high solubility, which resulted in a high extraction rate. The addition of CO2 to a liquid organic solvent decreased the viscosity and changed the solubility parameters. CXLE showed a 10 times faster extraction rate of cis- verbenol from Boswellia sacra resin compared to supercritical fluid extraction (SFE) and SLE. A combination of sonication and CXLE improved the solubility and extractability of the oil from different berry seeds. Sonication increased the amount of oil extracted using CXLE 3-fold. The composition of the oil obtained using CXLE showed significant increases in the levels of phospholipids and glycolipids compared to the oil obtained by SLE.A method of supercritical fluid chromatography (SFC) was developed based on a Diol column, which showed the highest peak height, a small peak width and high resolution between and within lipid classes. Stationary phases with a β-amino alcohol ligand showed a very strong retention of the zwitterionic lipids with terminal primary amines such as phosphatidylethanolamines. The sensitivity of mass spectrometry (MS) was found to be dependent on the composition of the SFC mobile phase. Optimization of the ion source settings in MS is important to achieve a compromise between the detection sensitivity of early and late eluting peaks.The impact of bioactive compounds in lingonberries on metabolites in plasma was also investigated. The results showed that the intake of lingonberries could improve the liver function and decrease the effects of high-fat diet. The intake of lingonberries could also prevent the formation of metabolites associated with an unhealthy phenotype such as sphingomyelins by decreasing the level of serine