Preparation, Characterization And Applications Of Quercetin-bonded Silica Gel Stationary Phase For HPLC

Chinese herbal medicine is a traditional medicine treasures. The use of Chinese herbal medicine to prevent and cure diseases has a long history in China, of which the most widely used medicinal plants. The pharmacological function of the natural active ingredients has attracted much attention, but few studies in other areas. The chemical structure of many natural compounds often contain a variety of active groups with various sites for solutes, which are precious natural chromatographic ligands of stationary phases. Large-scale development and utilization of natural ligands will help meet the needs of the industrial chromatographic separation materials, which is natural non-toxic, but also has antimicrobial properties. The prepared stationary phase was expected to use in the separation and purification of green chemical products with high added value. To select cheap and easy to get natural medicinal ingredients as ligands, without undergoing complicate syntheses, the use of Chinese herbal medicine resources has more important significance and value.In this thesis, a natural quercetin-bonded silica gel stationary phase (QUSP) for HPLC was prepared for the first time, which is a new type of separation materials with various interactions. The chemical structure of QUSP was characterized by solid state NMR, infrared spectroscopy, thermogravimetric analysis, elemental analysis and chemical analysis. Its basic chromatographic performance was systematically evaluated by using a series of solutes as probe. Following that the property of the new stationary phase was studied by different ionized polar acidic and basic compounds. The separation mechanism was also discussed. The new stationary phase was used to separate the complicate samples of Chinese herbal medicines, which was expected to provide the theoretical basis and experimental examples for industrial chromatography in the future. The main contents of this thesis is as follows:1. The recent research progress in the stationary phases for HPLC was comprehensively reviewed from different aspects, such as matrix, ligand, preparation, characterization, chromatographic performance, separation application at home and abroad. The application prospects of Chinese herbal ingredients for chromatographic separation was also discussed, which was used as the basis for this research work.2. A natural quercetin-bonded silica gel stationary phase (QUSP) for HPLC was first prepared via a KH-560coupling reagent by the solid-liquid surface continuous reaction, which is a brown solid. The chemical structure of QUSP was characterized by solid state NMR, infrared spectroscopy, thermalgravimetric analysis, elemental analysis and chemical analysis. The results show that the quercetin ligand have been successfully bonded to the surface of silica gel, and the ligand bonded was calculated to be0.139mmol g-1. Additionally, the stationary phase has good chemical and thermal stability. The prepared products in different batches have similar bond amount.3. QUSP was packed into a stainless steel column (4.6i.d.×150mm) under a constant pressure. The theoretical plate number of new column was16627/m by diphenyl as probe. The performance of QUSP were systematically evaluated according to a authority Kimata’s method, including its hydrophobic property, hydrogen bonding capacity, stereoselectivity and ion exchange capacity. The results show that the new stationary phase has an excellent reversed-phase chromatography with strong hydrogen bonding capacity and stereoselectivity. A more comprehensive study of the separation ability and mechanism to different polar compounds were also carried out by neutral, acidic and basic aromatic probe and comparison with ODS under the same conditions. The results showed that QUSP has a excellent reversed-phase chromatographic property with weak hydrophobicity. The hydrogen bonding, dipole-dipole and π-π interactions with solutes can effectively suppress the silanol effect for better separation of various basic compounds, to overcome the shortcome of a single hydrophobic interaction. The various interactions with solutes could improve the separation selectivity, and its advantages in rapid separation of polar compounds.4. The separation ability of a new stationary phase (QUSP) for polar compounds, especially nitrogen-containing basic solutes were further studied. In this experiment the silanophilic interaction was first investigated by using five common basic compounds as probes, including pyridine,3-methyl pyridine,1-naphthylamine, 4-methyl aniline and4,4*-diphenylenediamine. The negative "U" curve could not be observed, indicating quercetin ligands can suppress the silanol group effects and suitable for the separation of basic compounds. The separation mechanism was further studied through the investigation of chromatographic behavior of purines, pyrimidines, pyridines, anilines on QUSP, respectively. The results show that under the same conditions a single hydrophobic ODS has a limitable selectivity for the above basic compounds. However, QUSP exhibited the novel fast baseline separation of the polar nitrogen-containing compounds using simple methanol or acetonitrile-water mobile phases in isocratic elution mode, without the use of buffer with precise control of pH value. Obviously, various forces between quercetin ligand and solutes enhanced the separation selectivity.5. In addition to separation of basic compounds, more separations of easily ionized polar compounds have also been studied, such as phenols, benzoic acids, water-soluble vitamins and flavonoids. The chromatographic behavior of these polar compounds were studied. Their retention mechanism were proposed. The results show that QUSP has baseline separation ability of the above polar compounds in a relatively short time by using only simple methanol or acetonitrile-water mobile phase isocratic elution mode, without buffer for ionization suppression. Despite using gradient elution and optimize condition, only partial separations of the polar compounds on ODS were obtained. The retention order of solutes in the two columns showed obvious differences, suggesting new retention mechanism exists. QUSP with synergistic interaction has a good application prospects.6. To investigate the chromatographic performance in the separation of complicate compounds, QUSP was also employed for the separation of complicate samples, which were extracted from common herbs, including Tieguanyin tea, Honeysuckle and Polygonum cuspidatum. Some major components were identified by LC-ESI-MS in the absence of standard substances. Good separations of the above samples were achieved in the simple methanol or acetonitrile-water mobile phase in isocratic elution mode without pre-treatment. The high efficient separation paves the way for mass spectrometry for rapid and sensitive detection. Obviously, it was because the quercetin ligand could provide various sites.