Hybrid capillary-microchip setup for multidimensional electrophoretic separations

KLER, PABLO A.; SYDES, DANIEL; HUHN, CAROLIN

Essen (NRW)

Congreso; ANAKON 2013; 2013

GDCH

Due to the complexity of biological systems, the need for more accurate and efficient analytical tools is continuously growing. Despite the success of liquid chromatography or capillary electrophoresis (CE) coupled to mass spectrometry (MS), many analytical questions remain unsolved due to the large number of analytes in a wide range of concentrations, as is the case in proteomics and metabolomics. In order to face these challenges, we develop novel analytical platforms based electromigrative separation techniques to combine excellent separation performance, on-line pre-concentration and different detection techniques for the analysis of complex biological samples. We here describe a novel hybrid capillary-microchip module for 2D-electrophoretic separations coupling different separations methods including capillary isotachophoresis (ITP), capillary isoelectric focusing (IEF) and capillary electrophoresis. This instrumental setup combines the versatility of commercial CE equipment with the precision and accuracy of microfluidic chips: we use the classical equipment for channel filling, injection, and electric potential control, and the microphip as an interface for optimal sample transfer between columns. In order to control the system we have developed an advanced implementation of the high voltage regimes including a bi-polar multilevel source. Moreover, to monitor the sample transfer process we also implemented an integrated on-chip contactless capacitively coupled conductivity detector (C4D). Final detection is accomplished with UV, on-column C4D or mass spectrometric detection. This multimodular analytical platform enables us to sequentially perform electromigrative separation techniques for first on-line pre-concentration, isolation or matrix removal, and second, a highly efficient CE separation coupled to MS detection for identification and quantification of compounds of biological interest. Presented instrumentation and methods enable us the separation and identification of different peptides and proteins exploiting the improved analytical capabilities of the developed (ITP/IEF)-C4D-CE-(UV/MS) system. As an application example, we developed the separation and identification of a mixture of four different human angiotensins in order to demonstrate the analytical capabilities of a ITP-C4D-CE-MS system. We performed comparisons between our method and the direct CE-MS and ITP-MS methods. Considerable improvements on detection limit and peak resolution were reached.