Analysis Of Carotenoids And Their Isomers In Tobacco

The point of this thesis was to explore a simple analytical method to separate and determine carotenoids and their isomers in tobacco, and apply this methodology to give comprehensive information about carotenoids degradation in tobacco leaves during flue-curing period. Through the investigation of a lot of literatures and experiments, a fast and effective method using reversed phase high-performance liquid chromatography (RP?HPLC) with the photodiode array detection (DAD) has been developed for the determination of composition and content of carotenoids and their isomers in tobacco leaves; explored the content and composition in representative fresh, flue-cured and aging tobacco leaves. Furthermore, the dynamic change of plastid pigments in tobacco leaves from different stalk positions during flue-curing period was analyzed. These studies provided reference information for industrial and agricultural production of tobacco and cigarette.Three main aspects were included in the thesis:1. A fast and effective method using RP-HPLC-DAD for quantitative analysis of carotenoids and their isomers in tobacco leaves was established. According to modern analysis theory and on the premise of reducing analytical time and amount of poisonous solvent, a series of optimization were done to find the most effective extraction and separation, such as extraction solvent, sample preparation, mobile phase composition, gradient elution, and other chromatographic conditions. Finally, the best determination conditions were chosen after a series optimization, and the method was validated by linearity, limit of detection (LODs), repeatability (RSD) and recovery. LODs were ranged from 18~42 ng/mL, RSD value were from 3.01%~4.29%, and the recovery varied from 87.7%~94.6%, all these results indicated that this method was to be sensitive, reliable, reproducible and accurate for the carotenoid analysis in tobacco leaves.2. The contents and varieties of plastid pigments in tobacco leaves from different stalk positions during flue-curing period were determined. Not only the degradation of plastid pigments with the change of temperature during different stages of flue-curing period was analyzed, but also the distribution pattern of plastid pigments in tobacco leaves from different stalk positions at each stage of flue-curing period was compared. Meanwhile, the relationship between the maintenance of moisture content and the declines of pigments contents in tobacco leaves during flue-curing period was also investigated. The results provide useful information for investigating flue-curing characteristics of high-quality tobacco leaves from different stalk positions and evaluating the relationships between tobacco quality and safety and tobacco compositions.3. Four carotenoids were separated from spinach using open column chromatography. Thin-layer chromatography, ultraviolet-visible spectrophotometry and chemical reaction were used to identify these four carotenoids, and which were finally identifiedβ-carotene, lutein, violaxanthin and neoxanthin. Through the calculation of carotenoids purity and concentration, all separated carotenoids were used as referenced standard for the analysis of carotenoids in tobacco leaves, and the results were reached quantitative analysis requirement.