Procházková Barbora, Glovinová Eliska, Pospíchal Jan
Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry in Brno, Brno, Czech Republic.
Electrophoresis. 2007 Jul;28(13):2168-73. doi: 10.1002/elps.200600762.
The use of carrier ampholyte-free IEF (CAF-IEF) with ITP mobilization and conductivity detection in ITP mode for preconcentration and analysis of amino acids is demonstrated. The analytical procedure consists of three subsequent steps. In the first step, amino acids are continuously dosed from an infinite volume reservoir by electromigration to the column, where a sharp, stationary neutralization reaction boundary (NRB) is created in between acidic and basic primary electrolyte. Here, amino acids are selectively focused (trapped), if their pI falls to the pH difference on both sides of the NRB (pH gap). Amino acids create sharp rectangular zones, arranged according to their pI values. In the second step, focused zones are mobilized. After accumulation of the detectable amount of amino acids, dosing electrolyte in the infinite volume reservoir is changed for the mobilizing electrolyte. The migration mode is changed from CAF-IEF to ITP and substances start to migrate toward the analytical capillary. In the third step, analytes are transferred into the analytical column equipped with a conductivity detector and are detected in the new leading electrolyte in an ITP migration mode. The presented CAF-IEF-ITP-ITP with time-dependent accumulation of the large-volume sample enables to achieve in a reasonable time a 100 times lower c-LOD (here in orders of nmol/L), than can be reached by conventional hyphenated ITP-ITP.
本文展示了使用无载体两性电解质等电聚焦（CAF-IEF）结合等速电泳（ITP）迁移和电导率检测，在ITP模式下对氨基酸进行预浓缩和分析。分析过程包括三个后续步骤。第一步，氨基酸通过电迁移从无限体积储液器连续进样到柱中，在酸性和碱性一级电解质之间形成一个尖锐的、稳定的中和反应边界（NRB）。在此，如果氨基酸的pI落在NRB两侧的pH差值（pH间隙）范围内，它们就会被选择性聚焦（捕获）。氨基酸形成尖锐的矩形区带，根据其pI值排列。第二步，聚焦区带被迁移。在积累了可检测量的氨基酸后，将无限体积储液器中的进样电解质换成迁移电解质。迁移模式从CAF-IEF变为ITP，物质开始向分析毛细管迁移。第三步，分析物被转移到配备有导电率检测器的分析柱中，并在ITP迁移模式下在新的先导电解质中进行检测。所提出的具有大体积样品随时间积累功能的CAF-IEF-ITP-ITP，能够在合理的时间内实现比传统联用ITP-ITP低100倍的c-LOD（此处为nmol/L量级）。
