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Development of ultra high-resolution capillary liquid phase chromatographic system

超高分辨率毛细管液相色谱系统的研制

基本信息

批准号：
16550071
负责人：
TAKEUCHI Toyohide
金额：
$ 1.86万
依托单位：
Gifu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16550071/
关键词：
liquid phase separation system capillary column monolithic column ion chromatography enrichment system bisphenol A on-line tryptic digestion protein フローセル粒子充填型カラムグラジエント溶離理論段数

项目摘要

1. Monolithic silica capillary columns dynamically modified with quaternary ammonium ions were evaluated for the determination of bromide in seawater samples. A quaternary ammonium ion such as cetyltrimethylammonium was dynamically introduced onto monolithic silica surfaces. The first layer of the modifier was introduced by electrostatic interaction, whereas the second layer was introduced by hydrophobic interaction. The latter layer worked as the anion-exchange sites. The modified monolithic silica capillary columns could be used for rapid separation of inorganic anions. Separation of authentic mixture of five anions was achieved within a few minutes. The addition of small amount of the modifier in the eluent improved the repeatability of the retention time. Seawater samples could be directly injected onto the prepared capillary columns, and bromide could be determined to be 63 mg/L.2. The application of a boronate precolumn in the trace determination of bisphenol A(4,4'-isopropyliden … More ediphenol or BPA) in aqueous solution was investigated by microcolumn liquid chromatography (LC). BPA was enriched by a precolumn packed with TSKgel boronate and it was then injected (back-flushed) into an ODS separation column. Comparing to a conventional syringe injection, a better peak shape was obtained via the boronate precolumn injection, and the calculated theoretical plate number improvement was approximately 2 folds. The relative standard deviations for the retention time, peak area and peak height were 0.12, 3.0 and 1.8%, respectively. The system is linear (R2 > 0.99) within the working sample concentration and sample-volume ranges, and it presents a detection limit of 0.09μgl^<-1> (S/N=3) when a 0.2 ml sample was enriched. Recovery values of the proposed method were between 107 and 123%, and this method was applied satisfactorily to the determination of BPA in commercially available bottled drink samples. The boronate precolumn was proven to produce better sensitivity than that of an ODS or C30 precolumn as long as the enrichment of BPA in aqueous samples is concerned.3. A novel enzyme-immobilized glass beads based micro column was developed for the on-line, simple and swift protein digestion, followed by peptide separation via reversed-phase HPLC. This inexpensive and easy-made enzyme-immobilized micro column was prepared by using aminopropyl-controlled pore glass that was first reacted with glutaraldehyde followed by reaction with trypsin under the presence of phosphate buffer. Tryptic digestion of bovine serum albumin (BSA) was carried out by simply passing the pre-treated protein solution through the laboratory made trypsin-immobilized column, then followed by separation of tryptic fragments via reversed-phase HPLC. The separation of peptides was found to be equivalent to those done by using a sample which was underwent a conventional enzymatic protein digestion performed in solution. Less

1.建立了季铵离子动态修饰硅胶整体毛细管柱测定海水中溴的方法。将季铵离子如十六烷基三甲基铵动态引入到整体式二氧化硅表面。第一层改性剂是通过静电作用引入的，第二层是通过疏水作用引入的。后一层起到阴离子交换的作用。改进后的整体硅胶毛细管柱可用于无机阴离子的快速分离。在几分钟内就实现了正宗的五种阴离子混合物的分离。在洗脱液中加入少量的改性剂，提高了保留时间的重复性。海水样品可直接进样到所制备的毛细管柱上，溴化物的测定浓度可达63 mg/L。硼酸盐预柱在痕量双酚A(4，4‘-异丙基亚丙基…)测定中的应用用微柱液相色谱(LC)分析了水溶液中较多的双酚A。双酚A用填充有TSKGel硼酸盐的柱前富集，然后进样(反冲洗)到ODS分离柱。与传统注射器进样相比，采用硼酸盐柱前进样可获得更好的峰形，理论板数约提高2倍。保留时间、峰面积和峰高的相对标准偏差分别为0.12、3.0和1.8%。该体系在工作样品浓度和进样量范围内呈线性关系(R2&gt；0.99)，当样品浓缩0.2ml时，检出限为0.09μgl^-1&gt；(S/N=3)。方法的回收率在107%~123%之间，用于市售瓶装饮料中双酚A的测定，结果令人满意。就水样中双酚A的富集度而言，硼酸盐预柱比ODS柱或C30柱具有更好的灵敏度。开发了一种新型的酶固定化玻璃微柱，用于蛋白质的在线、简单、快速的消化，然后用反相高效液相色谱分离多肽。在磷酸盐缓冲溶液中，氨丙基可控微孔玻璃首先与戊二醛反应，然后与胰酶反应，制备了这种廉价且易于制作的酶固定化微柱。牛血清白蛋白(BSA)的胰酶消化是通过实验室自制的胰酶固定化柱，然后用反相高效液相色谱分离胰酶片段来实现的。多肽的分离被发现等同于使用在溶液中进行常规酶蛋白消化的样品所做的分离。较少