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Preparation, Characterization And Application Of Novel Stationary Phases For High Performance Liquid Chromatography

Posted on:2017-09-24Degree:DoctorType:Dissertation
Country:ChinaCandidate:Q WangFull Text:PDF
GTID:1311330482994339Subject:Drug Analysis
Abstract/Summary:PDF Full Text Request
With the rapid development of proteomics, metabonomics, environmental monitoring, traditional Chinese medicine development, food safety, etc., more and more severe challenges appeared in analytical chemistry and other related fields. On the one hand, strong polar and hydrophilic molecules are rapidly becoming the important research subjects. However, sufficient separation of these compounds is difficult to complete on conventional reversed-phase liquid chromatography (RPLC) columns; on the other hand, the samples to be analyzed are becoming increasingly complicated, especially for the protein, peptides, Chinese medicine ingredients, etc. It is usually necessary to simultaneously separate the components with quite different properties. Therefore, the chromatographic mode possessing a single retention mechanism, is not sufficient to meet the separation and analysis demand.As a novel chromatographic separation approach, hydrophilic interaction liquid chromatography (HILIC) was proved to be a reliable approach for qualitative and quantitative analysis of strong polar and hydrophilic molecules. The stationary phase is the core of HILIC, whose performance influences the nature of the whole system and restricts the application and development of HILIC. In the face of the separation demand for complicated samples, increasing investigations are focused on the development of mixed-mode chromatography (MMC) stationary phases to solve the problem. MMC stationary phases could provide multiple retention mechanisms for the analyte. Compared with the stationary phase with one single mode, MMC stationary phases are characterized with good selectivity, high loading capacity and low cost.Based on the above analysis, with "Preparation of novel chromatographic stationary phases" as the subject, the preparation and characterization of a series of HILIC and MMC stationary phases were introduced in the present thesis. The chromatographic performance, retention mechanism and application were also investigated in detail. The contents are as follows:1. Zirconia (ZrO2) and magnesium oxide (MgO)-ZrO2 microparticles were prepared and acted as the HILIC stationary phases for retention mechanism exploration. The result indicated that the two ZrO2-based stationary phases exhibited RPLC and HILIC property in the low and high acetonitrile-content mobile phase conditions, respectively; the increasing concentration of buffer would decrease the retention of the analytes; the process of transfering the analytes from the mobile phase to the ZrO2-based stationary phases was endothermic and high column temperature would facilitate the retention. The main retention mechanism of the ZrO2-based stationary phases was adsorption interaction. Compared with the bare ZrO2 column, the chromatographic performance of the MgO-ZrO2 one was greatly improved in the column efficiency and separation resolution, demonstrating that the addition of MgO would improve the HILIC performance of ZrO2.2. N-methylene phosphonic acid chitosan modified MgO-ZrO2 HILIC stationary phase (P-CTS-MgO-ZrO2) was prepared via Lewis acid-base adsorption. Its dominating retention mechanisms included ion-exchange, hydrogen bonding and electrostatic interactions. Compared to the bare MgO-ZrO2, P-CTS-MgO-ZrO2 showed excellent separation performance for the basic analytes. In addition, the acids, which could not be eluted from the bare MgO-ZrO2 column, exhibited proper retention on the novel stationary phase. The result signified that the P-CTS molecule not only blocked the Lewis acid sites on the surface of MgO-ZrO2 stationary phase, but provide new retention mechanism for the analytes in HILIC mode. Finally, the exploration in the separation of monosaccharides, phospholipids and peptides demonstrated the great potential of P-CTS-MgO-ZrO2 in the analysis of saccharides and bio-samples.3. The ZrO2-coated silica (ZrO2/SiO2) core-shell substrate was prepared via layer-by-layer self-assembly method and sol-gel technology. Adenosine 5'-monophosphate (5'-AMP) was self-assembled onto ZrO2/SiO2 via Lewis acid-base interaction, generating 5'-AMP-ZrO2/SiO2. The novel stationary phase possessed favorable hydrophilicity and could provide electrostatic, hydrogen bonding and ion-exchange interactions for the retention of the analytes. The ZrO2/SiO2 core-shell substrate possessed the suitable porous structure of SiO2 and good chemical stability of ZrO2 coating, exhibiting great improvement in the column efficiency and stability, and showing great separation performance for both the acidic and basic compounds.4. (i) A RPLC/HILIC mixed-mode chromatographic packing material (C18-Diol-SiO2), containing hydrophobic C18 alkyl chain and hydrophilic diol group, was prepared with controllable hydrophobicity and hydrophilicty via adjusting the ratio of two silylating reagents (octadecyltrichlorosilane and 3-glycidoxypropyltrimethoxysilane) in the synthesis. Two orthogonal modes, RPLC and HILIC, existed on C18-Diol-SiO2. Hence, its separation selectivity was greatly improved. A series of analytes comprising acids, based and neutrals could be separated in a single run on the novel column. Owing to the existence of polar diol group, C18-Diol-SiO2 could be used in pure aqueous mobile phase and exhibited good stability. Compared to the bare C18 column, it showed improved separation performance for the basic analytes. (ii) Taking advantage of the RPLC/HILIC mixed-mode property of C18-Diol-SiO2, the two dimensional liquid chromatography on one column (2D-LC-1C) system was set up. The offline 2D-LC-1C was used for the analysis of the extract of Fritillaria hupehensis; the online 2D-LC-1C was used for the analysis of the extracts of Rhizoma chuanxiong Hort. and Lonicera japonica. The proposed system offered a resolution for simultaneously separating of hydrophobic and hydrophilic components in complex samples, revealing more chemical information of the components. The result demonstrated that C18-Diol-SiO2 was a potential stationary phase for 2D-LC-1C system based on RPLC/HILIC MMC.5. (i) A RPLC/weak cation exchange (WCX)/HILIC trimodal MMC stationary phase, which modified with hydrophobic C18 alkyl chain and hydrophilic ionizable-COOH group, was prepared via "alkene-thiol" click chemistry. The prepared C18-COOH-SiO2 stationary phase could provide RPLC, HILIC, hydrogen bonding and WCX interactions for the retention of the analytes. Under different elution modes, alkaloids, non-steroidal anti-inflammatory drugs, nucleotides, nucleobases, amino acids and sartans could be well separated. The online 2D-LC-1C system based on the RPLC and HILIC mode was employed to separate the extract of Scutellaria baicalensis. (ii) The C18-COOH-SiO2 material was employed in the enrichment of quaternary ammonium alkaloids from the crude extract of Coptis chinensis. Compared to the traditional method (at least 15 h), column chromatography based on C18-COOH-SiO2 was simple and convenient, time-saving (within 1 h), besides, the obtained extract was in high purity. C18-COOH-SiO2 showed satisfactory reusability, providing a new approach for extracting quaternary ammonium alkaloids from complicated matrices.6. The relations between the above investigations were summarized. Based on the limits of the present research, the prospect of the subsequent investigation was finally outlined.7. The development and application of stationary phases for mixed-mode chromatography in recent years was reviewed, and the future development directions were outlined.
Keywords/Search Tags:Hydrophilic interaction liquid chromatography, Stationary phase, Reversed-phase liquid chromatography, Mixed-mode chromatography, Chromatographic evaluation, Retention mechanism
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