微波辅助合成用于开管毛细管电色谱柱的制备及应用性能的研究

【作者】 鄂红军；

【导师】 杨屹；

【作者基本信息】 北京化工大学 ， 应用化学， 2009， 博士

【摘要】 开管毛细管电色谱作为毛细管电色谱的重要分支,结合了毛细管区带电泳的高柱效和高效液相色谱高选择性的优点,成为当今分析化学领域的研究热点。大量的末端官能团、紧密且精确控制的分子结构是树状大分子所具有的独特性质,PAMAM是通过支化基元逐步重复的反应得到的具有树枝状高度支化结构的大分子,将其引入到毛细管电色谱柱中可以大大增加柱的表面积、容量和选择性,提升柱的性能。微波加热作为一种不同于传统加热模式,具有内外同热、热应力小、效率高、加热速度快的特点。本文将开管毛细管电色谱柱的制备技术,PAMAM树枝状大分子和微波辅助合成方法相结合,开展以下研究工作。采用微波辅助合成方法快速地制备了PAMAM树枝状大分子为固定相的开管毛细管电色谱柱。在微波加热的条件下,通过丙烯酸甲酯与KH 550的端氨基发生Micheal加成反应,得到半代的PAMAM,之后端酯基与乙二胺发生酰胺化反应,得到一代的枝形大分子PAMAM,重复以上,制得二代、三代树枝状大分子修饰的毛细管开管电色谱柱。首次尝试用FITC荧光标记的方法对制得的PAMAM修饰的开管毛细管电色谱柱进行表征,结果表明微波辅助合成方法用于制备PAMAM树枝状大分子修饰毛细管柱是可行的。与常规方法相比,微波辅助合成方法大大地缩短了PAMAM/OTCEC柱的制备周期。获得的3G PAMAM/OTCEC柱对丙氨酸和辅氨酸达到了基线分离。用丙酮考察获得PAMAM/OTCEC柱,迁移时间的RSD＜3.O%,说明制备的毛细管柱具有良好的重现性。用丙氨酸和辅氨酸对3 GPAMAM/OTCEC柱的稳定性进行测试,10天后峰面积RSD＜6.0%,说明制得的开管毛细管色谱柱的具有良好的稳定性。利用微波辅助合成方法制备β-环糊精衍生物（Br-β-CD）为固定相的开管毛细管电色谱柱。通过FITC荧光标记的方法优化KH 550修饰毛细管柱的微波反应时间,在微波功率140 W,微波温度40℃的条件下,微波反应10 min,KH 550修饰毛细管柱的荧光强度最大。继续通入Br-β-CD,微波反应30 min,制得Br-β-CD/OTCEC柱。获得的毛细管柱对N-乙酰-DL-蛋氨酸、l-苯基-1,2-乙二醇达到基线分离。用丙酮和1-苯基-1,2-乙二醇考察制得毛细管柱的性能,实验结果表明,制得的Br-β-CD/OTCEC柱具有良好的稳定性和重现性。毛细管酶微反应器是从固定化酶术发展而来的,将酶固定在毛细管内,可以进一步减少酶的消耗量。本文以氨基酰化酶为模型,成功地制备3G PAMAM树枝分子修饰的毛细管柱酶微反应器。通过高分支PAMAM树枝状大分子的引入,可以大大增加表面固定酶的容量,并且减小固定化的酶分子在刚性基体表面出现构像变化的可能性,保持酶的活性。利用毛细管区带电泳（CZE）优化制备的酶微反应器的酶解条件,实验结果表明,酶解温度37℃,17.5μg mL^-1N-乙酰-DL-蛋氨酸磷酸缓冲溶液(pH7.5,0.02 moL L^-1),底物的流速4μL min^-1,制备的毛细管酶微反应器具有良好的酶解效果。在优化条件下,考察10天内（每天酶解5次）酶解产物峰面积的变化。实验结果表明,10天后酶解产物的峰面积的RSD<10%,说明制备的毛细管柱酶微反应器具有良好的稳定性。β-环糊精柱撑水滑石（Mg₂Al/SO₃-β-CD/LD）是一种新型有机/无机杂化的纳米材料。本文以Mg₂Al/SO₃-β-CD/LDH为固定相,制备水滑石内壁修饰的毛细管电色谱开管柱,获得的毛细管柱在pH8.0Tris-HCl缓冲溶液中对1-苯基-1,2-乙二醇达到基线分离（Rs=3.03）。更多还原

【Abstract】 Open tubular capillary electrochromatography(OTCEC) is one of the most important approaches in capillary electrochromatography(CEC). Microwave irradiation method has become increasingly popular for modern chemical synthesis strategies sine.it’s first application towards organic chemistry.Compared with conventional synthesis process, microwave irradiation method has the advantages such as,heating system with uniform temperature,selective heating and no hysteresis effect. Polyamidoamine(PAMAM) dendrimers stationary phase for OTCEC was prepared from silanized fused-silica capillaries by 3-Aminopropylmethyldimethoxysilane(KH 550) with repeating tertiary amine/amide branching units using microwave-assisted method. Fluorescein isothiocyanate(FITC) is one of the most commonly used fluorescent dyes which act as an amine reactive label in bioassay.FITC was used as a label for the characterization of PAMAM dendrimer modified capillary column.The results verified that the preparation of PAMAM dendrimer modified column with microwave irradiation is feasible.Comparing with the conventional synthesis,microwave irradiation significantly improved the reaction speed and shortened the preparation time.The separation of Alanine and Proline was demonstrated with prepared PAMAM/OTCEC column.The results showed that the separation was gradually improved with the increasing of PAMAM generation,and the baseline separation can be obtained by G3 PAMAM dendrimer modified capillary column.The reproducibility of the PAMAM/OTCEC column was evaluated by measuring the RSD of the migration time.The results showed that the reproducibility of the column was satisfactory(RSD＜3.0%).The rate of column efficiency decline was used to examine the stability of G3 PAMAM/OTCEC column. Alanine and Proline was used to determine the stability for consecutive 10 days,the results showed that the columns have a good stability.At present,cyclodextrins(CDs) and their derivatives are the most widely applied chiral selector due to its low price,low UV absorbance, and unique structure.In this study,FITC was used as label for the characterization of KH 550 modified capillary column prepared by microwave assisted method.The results verified that the preparation of KH 550 modified column with microwave irradiation is feasible. Bromoacetate-substituted-β-cyclodextrins(Br-β-CD) have been successfully bonded to KH 550 modified column as chiral stationary phase in OTCEC.Comparing with the conventional synthesis,microwave assisted process significantly improved the reaction speed and shortened the preparation time from 16h to 40min.Baseline chiral separation of N-acetyl-DL-methionine and 1-phenyl-1,2-ethanediol was obtained in Br-β-CD modified column.The reproducibility of the Br-β-CD/OTCEC column was evaluated by measuring the RSD of the migration times of chiral compound 1-phenyl-1,2-ethanediol.The migration time data indicated that the reproducibility of the column was satisfactory(RSD＜2 %).Separation efficiencies of acetone in the Br-β-CD/OTCEC column decreased 6.77%after 10 days,good stabilization was obtained.An enzyme is a biological catalyst,which has high efficiency under mild conditions and is highly selective.In the concept of utilizing the natural catalysts to manufacture various products,such as foods or pharmaceuticals,immobilizing enzyme technique was born.The ability to easily separate immobilized enzymes from the reaction mixture offers an additional advantage for multiple use of the enzyme,resulting in reduced cost and time to remove the enzyme from the measurement samples.The preparation of the immobilized microenzyme reactors inside capillaries or channels of the microfluidic devices represents another step in the development of the miniaturized systems for rapid analyses of very small sample sizes.The studied enzymes can be covalently attached on the surface of the capillaries or channels.The advantage of the covalent bonding is that the leakage of the immobilized enzyme is minimized. PAMAM dendrimers stationary phase for OTCEC was prepared successfully using microwave-assisted method.Through the high branches of the dendrimers,the capacity of the surface introduction can greatly increase the immobilized enzyme and reduced the enzyme molecules immobilized in the rigid matrix surface,thus reducing the possibility of changes in conformation to maintain the enzyme activity.In this work,the enzyme micro-reactor was prepared in PAMAM dendrimers modified capillary using glutaraldehyde as a cross-linking agent and aminoacylase as immobilized enzyme.The product of enzyme capillary micro-reactor was separated by capillary zone electrophoresis. Peak area of the product represented the enzyme activity. N-acetyl-DL-methioine was passed through a 15cm enzyme capillary micro-reactor.The results showed that the optimal conditions were, substrate concentration 17.5μg mL-1 having a flow rate of 4μL min-1 at 37℃using phosphate buffer of pH 7.5.Under the optimized conditions, the substrate N-acetyl-DL-methionine showed enzymatic degradation for 10 days consecutive(analyzed 5 times a day).The enzyme capillary micro-reactor showed good stability.Layered double hydroxides(LDHs) are layered compounds with anion-exchange ability.The intercalation of sulfatedβ-cyclodextrin into LDHs by the ion-exchange method was used.In this study,the sulfatedβ-cyclodextrin intercalated in layered double hydroxides (Mg2Al/SO3-β-CD/LDH) was chosen as stationary phase for open tubular capillary electrochromatography and was applied to separate the receme of 1-phenyl-1,2-ethanediol under the optimized conditions.更多还原