| It is well known that there is abundant medicinal plants in our country, which play an important role in disease prevention. In this study, we make use of narural active components in Traditional Chinese Medicine as chromatographic ligands to design, prepare and apply new-style chromatographic separation materials. Direct exploitation of the natural ligands can not only reduce production cost, but also to avoid the complicate synthesis and purification process, and it also has an importantance in the development and utilization of Chinese herbal medicine deeply. In this thesis, a new emodin-bonded silica gel stationary phase (EDSP) with the ligand from Rheum officinale Baill for HPLC was prepared for the first time. Its structure were characterized by Fourier transform infrared spectrometry, elemental analysis and thermal analysis. The chromatographic performance of EDSP were evaluated. Related separation mechanism was systematically studied. The new stationary phase was used for the separations of complicate samples. The major contents are as follows.1. The recent advance in the proceeding from the matrix, preparation, chromatographic retention mechanism and their application of novel bonded silica gel stationary phases for HPLC at home and abroad, were reviewed. The study of bonded silica gel stationary phases for HPLC was reviewed from two aspects, including the improvement of traditional bonded-silica gel stationary phases and the design of new bonded phases. Above all will contribute to the research basis and start point of this thesis.2. Emodin was extracted and purfied from Rheum officinale Baill by organic solvent extraction method. And emodin was chemically immobilized to the surface of silica gel as stationary phase by the solid-liquid phase reaction with y-glycidoxy-propyltrimethoxy-silane (KH-560) coupling reagent for high performance liquid chromatography(HPLC). The new emodin-bonded stationary phase (EDSP) was brown-red monodispersal particles. According to. the preparative scheme, the reaction conditions were also partially optimized via investigation under different reaction ratios. The structure of new emodin-bonded stationary phase (EDSP) was characterized by Fourier transform infrared spectrometry, elemental analysis and thermal analysis. The results showed that emodin was successfully immobilized onto the surface of spherical silica gel with a 0.18 mmolg-1 of bonded amount and highly physical and chemical stability. In addition, the bonded amounts of the packings in different batches were similar to each other, which indicated that the above preparation procedure was practical and reliable.3. The slurry of EDSP was packed into a stainless steel column by using common LC pressure pump. The column efficiency was 8827 plates/m by using biphenyl as probe in methanol-water (50/50, v/v) mobile phase at room temperature. The chromatographic stability of EDSP in strong organic solvent and acidic mobile phase were investigated, respectively. That an authorized column validation method proposed by Kimata's group was used to systematically evaluate the basic chromatographic properties of EDSP, including hydrophobicity, charge transfer ability, hydrogen-bonding, steric selectivity and ion exchange interactions. The chromatographic performance of new EDSP was studied in detail by using different structural solutes as probes and ODS as reference. These chromatographic data can be used as the theoretical basis for its applications in future. The tested solutes included neutral aromatics, acid phenols and basic aromatic amines etc. The results indicated that EDSP performed as a versatile reversed-phase material with a relatively weak hydrophobicity, especially, various sites located in the emodin ligand, such as hydrophobic, hydrogen bonding,π-πor n-πcharge transfer and dipole-dipole interactions. EDSP has excellent abilities for the fast separations of neutral, acid and basic compounds. It is related with the existence of largeπsystem and planar rigid structure of EDSP. Hence, EDSP was endowed advantage over only hydrophobic ODS in rapid separations of polar compounds.4. In order to further evaluate chromatographic performance in the separation of polar compounds, the behavior of nine pyrimidines, four purines, three nucleosides, four pterins and five pesticides on EDSP were studied by HPLC. Meanwhile, the comparative study in the retention behavior of the above solutes on both EDSP and ODS were also done under the same conditions. The influence of mobile phase variables, such as methanol content and pH value on the retention behavior of the polar compounds were investigated. The results showed that baseline separation on EDSP were easily achieved by using common and cheap methanol-water mobile phases. The elution orders of 5-nitrouracil, isoxanthopterin and propanil were very different on EDSP and ODS under the same condition, which showed that EDSP and ODS had different retention mechanism. Obviously, the EDSP exhibited advantage over only hydrophobic ODS in seperations of the above polar compounds. The results can be explained as follows:theπ-πinteraction, hydrogen bonding, n-πinteraction existed between phenolic hydroxyl or carbonyl groups of emodin anthraquinone ligands containingπ-conjugated system and the solutes.5. To investigate the chromatographic performance in the separation of complicate compounds, EDSP was also employed for the separation of complex samples, which were extracted from common herbs, including polygonum cuspidatum S. et Z., Radices puerarire, Scutellaria barbatat and lobelia. Some major components were identified by LC-ESI-MS in the absence of standard substances. Good separations of the above samples were achieved without pre-treatment, which was because the carbonyl and hydroxyl on emodin anthraquinone ligands could provide various sites. In addition, new separation material with natural emodin ligand will exhibit a bright future for industrial chromatography separation applications because it has both pharmaceutical and chromatographic functions.
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