| Capillary monolithic column as liquid chromatography stationary phase has attracted great attention from researchers due to its merits of easy preparation without frit, excellent permeability, high efficiency and low column pressure. Moreover, the result can be repeated and displayed easily, and achieve quickly sample separation, so it has been applied in capillary electrochromatography (CEC) and micro-high performance liquid chromatography (μHPLC). Boronate affinity chromatography (BAC) is an important tool for specific capture and separation of cis-diol containing compounds such as glycoproteins, RNA and carbohydrates. With the development of proteomics, metabonomics, glycomics and other research, boronic affinity chromatography, especially those based on nano-materials and monolithic materials, gets more attention.In this study, a Poly(VPBA-co-PETA) boronate capillary monolithic column was synthesized, characterized and utilized in the mode of both affinity and reversed phase chromatography. In order to optimize the properties, the orthogonal experiment was designed by the DOE software. We establishedμ-BAC/RPLC 2 dimensional separation platform with affinity/reversed separation mechanism. Then, the Poly(VPBA-co-PETA) capillary monolithic column was applied to enrich small molecules contained cis-dihydroxyl group in the traditional Chinese medicine.This thesis has five chapters.The first chapter described the background of Capillary monolithic column and Boronate affinity chromatography from methodology, theory and applications, respectively. The outlook of capillary electrochromatography and micro-high performance liquid chromatography separation were summarized, where the development of boronate affinity monolithic column was also outlined.Chapter 2 includes the preparation, characterization and applications of the poly(VPBA-co-PETA) monolithic capillary column. A boronate functionalized monolithic capillary column was synthesized, characterized and utilized in the mode of both affinity and reversed phase chromatography. The polymer was prepared using 4-vinylphenylboronic acid as the functional monomer, pentaerythritol triacrylate as crosslinker, azobisisbutyronitrile as trigger, ethanediol and diethylene glycol as porogenator. In order to optimize the properties, the orthogonal experiment was designed by the DOE software. The optimum preparation conditions were monomer (VPBA, 732 mg), cross-linking agent (PETA, 6 mg), trigger (AIBN, 1 mg) and pore-foaming agent (diethylene glycol/ethanediol = 2:8). It can be also seen that the monolithic bed exhibited a well-distributed open channel network and good permeability. The breakthrough capacity and the column efficiency are also improved.In Chapter 3, the column with the best proportion was characterized and applied in capillary high performance liquid chromatography (μHPLC) and pressurized capillary electrochromatography (pCEC). The results suggested the column persists affinity chromatography character because the boronate group located at the stationary phase could specifically absorb compounds containing cis-dihydroxy functionality at alkaline conditions; because the properties of PETA, the column also persists RP separation mechanism and separate different benzene series with 20% ACN in H2O.In Chapter 4, we established the BAC/RPLC 2 dimensional separation platform with affinity/reversed separation mechanism. The two types of chromatographic modes can be performed consecutively on the same column during one complete run, making it a 2-D chromatography. We also examined the influence of pH and the concentration of salt to the separation. After optimization, we separated 6 compounds in 28 min on the 2-D chromatography system.Chapter 5 focused on the applications of Poly(VPBA-co-PETA) in the separation and enrichment of traditional Chinese medicines, Herba Taraxaci and Thorn fruit of Euonymus extract. The extract was separated onμHPLC and the fractions at acidic conditions were collected and examined by RP chromatography. The contract of the chromatograms before and after enrichment on the boronate affinity column showed the column can be used to enrich small molecules contained cis-dihydroxyl group efficiently, such as caffeic acid and chlorogenic acid. |
