Pospíchal J, Glovinová E
Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry in Brno, Czech Republic.
J Chromatogr A. 2001 May 18;918(1):195-203. doi: 10.1016/s0021-9673(01)00753-1.
The applicability of carrier ampholyte-free isoelectric focusing (CAF-IEF) for analyses of ampholytes is demonstrated. The suggested method is based on the principle of both side regulated ionic matrix in CAF-IEF. A sharp step of pH is created in the column filled with a sample dissolved in a background electrolyte by influence of current and solvolytic fluxes. Here, ampholytes are focused upon. The magnitude of the step, its velocity and direction of its movement can be regulated electrically. In this manner, favorable separation properties of the system can be set up, even during the run. This brings several advantages over conventional methods. The principles of the separation can be easily changed, permitting selective pre-concentration (trapping) of minor components by processing large amounts of a sample to be preformed, effective isotachophoresis or IEF pre-separation and final electrophoretic analysis in one run. Advantages of these combinations are discussed together with the right choice of the working electrolyte. A 1000-fold increase in amount of substance in a column can be achieved for both isotachophoresis and capillary zone electrophoresis combined with CAF-IEF pre-concentration at reasonable working conditions. It enables a limit of detection at the nmol/l level with a concentration factor of about 10(7) to be reached.
证明了无载体两性电解质等电聚焦（CAF-IEF）在两性电解质分析中的适用性。所建议的方法基于CAF-IEF中双侧调节离子基质的原理。通过电流和溶剂解通量的影响，在填充有溶解于背景电解质中的样品的柱中产生pH的急剧变化。在此，两性电解质被聚焦。该变化的幅度、其速度及其移动方向可以通过电进行调节。以这种方式，即使在运行过程中也可以建立系统的良好分离特性。这相对于传统方法具有几个优点。分离原理可以很容易地改变，通过处理大量待预形成的样品，允许对微量成分进行选择性预浓缩（捕获），在一次运行中进行有效的等速电泳或IEF预分离以及最终的电泳分析。讨论了这些组合的优点以及工作电解质的正确选择。在合理的工作条件下，结合CAF-IEF预浓缩，等速电泳和毛细管区电泳在柱中的物质量可增加1000倍。它能够达到约10(7)的浓缩因子，检测限达到nmol/l水平。
