Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.
J Sep Sci. 2013 Nov;36(21-22):3471-7. doi: 10.1002/jssc.201300725. Epub 2013 Oct 9.
A challenge for capillary LC (cLC) is fraction collection and the manipulation of fractions from microscale columns. An emerging approach is the use of segmented flow or droplet technology to perform such tasks. In this work, a fraction collection and postcolumn reaction system based on segmented flow was developed for the gradient cLC of proteins. In the system, column effluent and immiscible oil are pumped into separate arms of a tee resulting in regular fractions of effluent segmented by oil. Fractions were generated at 1 Hz corresponding to 5 nL volumes. The fraction collection rate was high enough to generate over 30 fractions per peak and preserve chromatographic resolution achieved for a five-protein test mixture. The resulting fractions could be stored and subsequently derivatized for fluorescence detection by pumping them into a second tee where naphthalene dicarboxyaldehyde, a fluorogenic reagent, was pumped into a second arm and added to each fraction. Proteins were derivatized within the droplets enabling postcolumn fluorescence detection of the proteins. The experiments demonstrate that fraction collection from cLC by segmented flow can be extended to proteins. Further, they illustrate a potential workflow for protein analysis based on postcolumn derivatization for fluorescence detection.
毛细管 LC（cLC）面临的一个挑战是馏分收集和从微柱上处理馏分。一种新兴的方法是使用分段流或液滴技术来执行此类任务。在这项工作中，开发了一种基于分段流的馏分收集和柱后反应系统，用于蛋白质的梯度 cLC。在该系统中，柱流出物和不混溶的油被泵入三通的两个臂中，从而使流出物的常规馏分被油分段。馏分以 1 Hz 的频率产生，对应于 5 nL 的体积。馏分收集率足够高，可以为每个峰生成超过 30 个馏分，并保持对五种蛋白质测试混合物实现的色谱分辨率。所得馏分可以储存，并通过将其泵入第二三通来随后衍生化以进行荧光检测，其中萘二羧酸醛，一种荧光试剂，被泵入第二臂并添加到每个馏分中。蛋白质在液滴中衍生化，从而能够对蛋白质进行柱后荧光检测。实验表明，通过分段流从 cLC 进行馏分收集可以扩展到蛋白质。此外，它们说明了基于柱后衍生化进行荧光检测的蛋白质分析的潜在工作流程。
