SEPARATION AND DETERMINATION OF NATURAL PRODUCTS BY HIGH RESOLUTION SEPARATION TECHNIQUES

Abstract

In this thesis, some new analytical methods were developed based on capillary electrophoresis, high performance liquid chromatography, and supercritical fluid extraction for the identification, separation and determination of natural products, such as stevia sweeteners and auxins. Capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) were used for the separation and determination of stevia sweeteners, a natural sweeteners extracted from the plant Stevia rebaudiana Bertoni (Compositae). A simple and efficient capillary electrophoretic method was developed. The results were compared with those obtained by HPLC. The individual steviol glycosides were obtained by 1-IPLC fraction collection, and peaks in the electropherograms of the sweeteners samples were identified by comparing with those of individual steviol glycosides. The method was applied to the determination of real samples. The extraction of steviol glycosides in Stevia rebaudiana, including stevioside, rebaudioside A, rebaudioside C and dulcoside A by subcritical fluid extraction (SubFE) was investigated. A simpler and more efficient subcritical fluid extraction method was developed, compared with conventional liquid extraction methods. The extraction conditions (extraction phase composition, extraction time, etc) were optimised. Recovery, coefficient of variation, quantification limits for SFE of stevia glycosides were determined by capillary electrophoresis. The separation of fifteen auxins, a group of plant hormones, by CE was also investigated. Satisfactory separation could be achieved by varying pH and modifiers added to the separation media. The overlapping resolution mapping (ORM) scheme was employed in the separation optimization. An interpretative optimization approach was utilised to predict the optimum buffer pH, and the concentrations of ?-, ?-, and ?-cyclodextrins. A four-dimensional ORM scheme was developed to optimize these four parameters simultaneously. By conducting sixteen preplanned experiments, global optimum conditions for the separation within an acceptable analysis time could be obtained. This method was applied to the determination of indole acetic acid (IAA), a ubiquitous auxin in plant samples, with cinnamic acid as the internal standard. The results obtained in this thesis show that capillary electrophoresis is a powerful analytical method capable of achieving rapid, simple and highly efficient separation of the components in natural products, and the technique can be combined readily with other separation techniques, such as high performance liquid chromatography and supercritical fluid extraction.