Espina-Benitez Maria Betzabeth, Randon Jérôme, Demesmay Claire, Dugas Vincent
Université de Lyon, CNRS, Université Claude Bernard Lyon 1, ENS de Lyon, Institut des Sciences Analytiques, UMR 5280, 5 rue de la Doua, F-69100 VILLEURBANNE, France.
Université de Lyon, CNRS, Université Claude Bernard Lyon 1, ENS de Lyon, Institut des Sciences Analytiques, UMR 5280, 5 rue de la Doua, F-69100 VILLEURBANNE, France.
J Chromatogr A. 2017 Apr 21;1494:65-76. doi: 10.1016/j.chroma.2017.03.014. Epub 2017 Mar 9.
An integrated, miniaturized and fully automated system was developed for the analysis (preconcentration/purification, separation and detection) of cis-diol containing molecules in complex matrices. This innovative in-line coupling system was achieved via the in-situ and localized synthesis of a short segment of silica-based monolith at the inlet of a 75-μm inner diameter fused silica capillary. The monolithic segment was locally functionalized with an acrylamide derivative of phenylboronic acid by free radical photopolymerization within 10min of irradiation time. Efficiency of the photopolymerization reaction was followed by frontal affinity chromatography of 1,2-dihydroxybenzene (catechol) as cis-diol model solute. An active-site amount of 0.43nmolcm (9.8nmolμL) of phenylboronic acid moieties was obtained, with a K value of about 290μM close to reported value for the phenyl boronate-catechol complex. The optimal conditions of use of the miniaturized boronate affinity monolithic column (μBAMC) were determined and adapted to the in-line coupling with capillary electrophoresis. Catechol was specifically preconcentrated in a pH 8.5 phosphate buffer/MeOH (80/20, v/v) mixture. A volume up to 20 times the monolith volume can be percolated with a quantitative recovery yield. Three catecholamines were purified, preconcentrated and in-line separated. Elution from the μBAMC was performed with a small plug of acidic solution, allowing field amplified sample stacking of solutes within the plug before their in-line electrophoretic separation at pH 8.75. This unique in-line coupling was successfully used for the fully automated analysis of catecholamines neurotransmitters in urine samples, highlighting the purification efficiency of the μBAMC and the potential of such a fully integrated approach. In addition to the low sample volume required (less than 2μL), the limits of detection (LOD) accomplished with this coupling were estimated at 9.0, 9.5 and 4.8ngmL for dopamine, adrenaline and noradrenaline respectively, which improves the LOD of theses solutes compared to other CE methods.
开发了一种集成化、小型化且全自动的系统，用于分析复杂基质中含顺式二醇的分子（预富集/纯化、分离和检测）。这种创新的在线耦合系统是通过在75μm内径的熔融石英毛细管入口处原位局部合成一小段基于二氧化硅的整体柱实现的。通过自由基光聚合，在10分钟的辐照时间内，整体柱段用苯基硼酸的丙烯酰胺衍生物进行局部功能化。以1,2 - 二羟基苯（儿茶酚）作为顺式二醇模型溶质，通过前沿亲和色谱法跟踪光聚合反应的效率。获得了0.43nmol/cm（9.8nmol/μL）的苯基硼酸部分的活性位点量，其K值约为290μM，接近报道的苯硼酸 - 儿茶酚络合物的值。确定了小型化硼酸亲和整体柱（μBAMC）的最佳使用条件，并使其适用于与毛细管电泳的在线耦合。儿茶酚在pH 8.5的磷酸盐缓冲液/甲醇（80/20，v/v）混合物中被特异性预富集。高达整体柱体积20倍的体积可以以定量回收率渗滤。三种儿茶酚胺被纯化、预富集并在线分离。用一小段酸性溶液从μBAMC进行洗脱，使得溶质在塞子内进行场放大样品堆积，然后在pH 8.75下进行在线电泳分离。这种独特的在线耦合成功用于尿液样品中儿茶酚胺神经递质的全自动分析，突出了μBAMC的纯化效率以及这种完全集成方法的潜力。除了所需的低样品体积（小于2μL）外，这种耦合实现的检测限（LOD）分别估计为多巴胺9.0、肾上腺素9.5和去甲肾上腺素4.8ng/mL，与其他CE方法相比，提高了这些溶质的LOD。
