Institute of Physical and Theoretical Chemistry

Multidimensional separation techniques

A major interest of our group is related to the development of multidimensional electromigrative separation techniques. We aim at developing a modular analytical platform, which is based on electromigrative separation modes on the one hand and various detection techniques on the other hand. Due to this modular approach, the instrumental setup can be adapted to the respective analytical task. The capabilities and limitations of these new instrumental developments are investigated and validated in detail in the working group to judge and enhance their applicability for different analytical settings. This also includes basic research regarding the respective separation mechanisms which are involved as well as the effects during the transition of separation modes of the two dimensions.

We use a hybrid instrumental setup, which couples classical CE capillaries via a microfluidic chip interface. Design and optimization of the chip interface are currently in our focus. Our research aims at coupling the multidimensional separation platform with mass spectrometric detection in order to account for the complex analytical questions in the fields of environmental analytics, bioanalytics, diagnostics and pharmaceutical applications. For the realization of our approach, new solutions for the control of the electrical potentials within the microchips, especially at the interfaces as well as the development and implementation of a suitable high voltage source, are required. Furthermore, this approach includes the intensive development of on-chip detection systems which enable us to accurately monitor analyte separation in the interface which is required for the exact switching and sample transfer between the separation dimensions. In our current research, both fluorescence and conductivity detection are used for on-chip detection.

The first successful multidimensional separation was realized by the hyphenation of isotachophoresis with capillary zone electrophoresis for the separation of amino acids and peptides as model system. In this experimental setup, isotachophoresis in the first dimension is used for on-line analyte preconcentration and matrix removal. CE-MS in the second dimension enables high quality analyte separation with sensitive MS detection, providing both excellent detection limits as well as very high separation efficiency.

In a second application we investigate the coupling between isoelectric focusing (IEF) as first and CE-MS as second dimension. Biotechnologically produced recombinant proteins are separated and preconcentrated based on their respective pI values in the first dimension. In capillary IEF, direct MS detection after IEF-isoform separation is not possible due to the ampholytes which generate the pH gradient and hinder MS detection due to severe ion suppression. The second separation dimension allows separating proteins from the ampholytes, followed by sensitive MS detection and identification.

Cooperations:

  • Peter Zipfl, Optoelektronik, Hochschule Aalen, Aalen
  • Daniel Lutz, CalvaSens, Aalen
  • Pablo Kler, Centro de Investigación de Métodos Computacionales (CIMEC), Universidad Nacional del Litoral (UNL), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Santa Fe, Argentina
  • Marián Masár, Milan Hutta, Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slowakei

Publications:

  1. On-chip intermediate potential measurements for the control of electromigration in multi-channel networks in case of time-dependent potential changes

    D. Sydes, P.A. Kler, P. Zipfl, D. Lutz, H. Bouwes, C. Huhn, Sens. Actuat. B: Chem. 2017, 240, 330-337

  2. On-chip intermediate LED-IF-based detection for the control of electromigration in multichannel networks

    D. Sydes, P. A. Kler, H. Meyer, P. Zipfl, D. Lutz, C. Huhn, Anal. Bioanal. Chem. 2016, 408, 8713-8725

  3. Zero-dead-volume interfaces for two-dimensional electrophoretic separations
    D. Sydes, P. A. Kler M. Hermans, C. Huhn, Electrophoresis 2016, 37, 3020-3024

  4. Column–coupling strategies for multidimensional electrophoretic separation techniques
    P. A. Kler, D. Sydes, C. Huhn, Anal. Bioanal. Chem. 2014, DOI 10.1007/s00216-014-8099-7
  5. Non-aqueous electrolytes for isotachophoresis of weak bases and its application to the comprehensive preconcentration of the 20 proteinogenic amino acids in column-coupling ITP/CE-MS
    P. A. Kler, C. Huhn, Anal. Bioanal. Chem. 2014, 28, 7163-7174
  6. Column coupling isotachophoresis/capillary electrophoresis with mass spectrometric detection: characterization and optimization of microfluidic interfaces
    P. A. Kler, T. N. Posch, M. Pattky, R. M. Tiggelaar, C. Huhn, J. Chromatogr. A 2013, 1297, 204-212