Stastna Miroslava, Slais Karel
Institute of Analytical Chemistry of the Czech Academy of Sciences, v. v. i, Brno, Czech Republic.
Electrophoresis. 2015 Oct;36(20):2579-86. doi: 10.1002/elps.201500223. Epub 2015 Aug 21.
This study concentrates on development of instrumentation for focusing and separation of analytes in continuous flow. It is based on bidirectional ITP working in wide pH range with separation space of closed void channel of trapezoidal shape and continuous supply of sample. The novel instrumentation is working with electrolyte system formulated previously and on the contrary to devices currently available, it allows preparative separation and concentration of cationic, anionic, and amphoteric analytes simultaneously and in wide pH range. The formation of sharp edges at zone boundaries as well as low conductivity zones are avoided in suggested system and thus, local overheating is eliminated allowing for high current densities at initial stages of focusing. This results in high focusing speed and reduction of analysis time, which is particularly advantageous for separations performed in continuous flow systems. The closed void channel is designed to avoid basic obstacles related to liquid leakage, bubbles formation, contacts with electrodes, channel height and complicated assembling. The performance of designed instrumentation and focusing dynamics were tested by using colored low molecular mass pH indicators for local pH determination, focusing pattern, and completion. In addition, feasibility and separation efficiency were demonstrated by focusing of cytochrome C and myoglobin. The collection of fractions at instrument output allows for subsequent analysis and identification of sample components that are concentrated and conveniently in form of solution for further processing. Since the instrumentation operates with commercially available simple defined buffers and compounds without need of carrier ampholytes background, it is economically favorable.
本研究专注于开发用于连续流中分析物聚焦和分离的仪器。它基于双向等速电泳，在宽pH范围内工作，具有梯形封闭空通道的分离空间，并能连续供应样品。这种新型仪器采用先前配制的电解质系统，与目前可用的设备相反，它允许在宽pH范围内同时对阳离子、阴离子和两性分析物进行制备性分离和浓缩。在建议的系统中避免了在区域边界形成尖锐边缘以及低电导率区域，因此消除了局部过热，从而在聚焦初始阶段允许高电流密度。这导致了高聚焦速度并减少了分析时间，这对于在连续流系统中进行的分离尤为有利。封闭空通道的设计是为了避免与液体泄漏、气泡形成、与电极接触、通道高度和复杂组装相关的基本障碍。通过使用有色低分子量pH指示剂来测定局部pH、聚焦模式和完成情况，测试了所设计仪器的性能和聚焦动力学。此外，通过细胞色素C和肌红蛋白的聚焦证明了其可行性和分离效率。在仪器输出端收集馏分，便于后续分析和鉴定浓缩的样品成分，这些成分以溶液形式方便地用于进一步处理。由于该仪器使用市售的简单定义的缓冲液和化合物，无需载体两性电解质背景，因此在经济上是有利的。
