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Novel approach for the synthesis of a neutral and covalently bound capillary coating for capillary electrophoresis-mass spectrometry made from highly polar and pH-persistent N-acryloylamido ethoxyethanol

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Abstract

Statically adsorbed or covalently coupled capillary coatings are of crucial importance in capillary electrophoresis-mass spectrometry for the separation of peptides and proteins. So far, published coating strategies and commercially available coated capillaries have a limited pH-stability so that the analysis at strongly acidic pH is limited, or harsh rinsing procedures for biological sample analysis cannot be applied. We here present a capillary coating based on Si-C linkages to N-acryloylamido ethoxyethanol (AAEE) with a new synthetic strategy including LiAlH4 surface reaction. We optimized the coating method with emphasis on stability and reproducibility applying harsh rinsing procedures (strong acid, strong base and organic solvent), using the electroosmotic mobility and separation efficiency of tryptic peptides as performance measure. Complete synthesis is performed in less than 2 days for up to 8 capillaries in parallel of more than 16 m total length. Intra- and inter-batch reproducibility were determined regarding electroosmotic mobility, separation efficiency and migration time precision in CE-MS separations of tryptically digested bovine serum albumin. Coating stability towards rinsing with strong acid (1 mol/L HCl), organic solvent (acetonitrile) and strong base (1 mol/L NaOH) was investigated. Outstanding performance was found for single capillaries. However, inter-capillary reproducibility is discussed critically. The new coating was successfully applied for reproducible CE-MS separation of large proteins in diluted serum, medium-sized peptides and small and highly charged polyamines in fish egg extracts using a very acidic background electrolyte containing 0.75 mol/L acetic acid and 0.25 mol/L formic acid (pH 2.2).

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Acknowledgements

We thank Uwe Irion, Max Planck Institute for Developmental Biology, Tübingen, for providing the fish egg samples and discussion. We want to thank the workgroup of Prof. Hermann A. Mayer (Eberhard Karls Universität Tübingen), especially Dipl. Chem. Thomasz Mistal, M. Sc. Dennis M. Meisel and Dr. Farhad Jafarli, for supplying us with dry solvents, support and discussion. Also, we would like to thank M. Sc. Sandra Köhn, M. Sc. Ronja Jordan, Sarah Köhn and B. Sc. Jacqueline Kieler who contributed during internships to early phases of this work.

Funding

This work was funded by the Karl & Anna Buck Stiftung and the Excellence Initiative, a jointly funded program of the German Federal and State governments, organized by the German Research Foundation (DFG).

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  1. Institute of Physical and Theoretical Chemistry, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 18, 72076, Tübingen, Germany

    Martin Meixner, Martin Pattky & Carolin Huhn

  2. Institute of Life Technologies, Route du Rawyl 64, 1950, Sion, Switzerland

    Martin Pattky

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  1. Martin Meixner
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Correspondence to Carolin Huhn.

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Meixner, M., Pattky, M. & Huhn, C. Novel approach for the synthesis of a neutral and covalently bound capillary coating for capillary electrophoresis-mass spectrometry made from highly polar and pH-persistent N-acryloylamido ethoxyethanol. Anal Bioanal Chem 412, 561–575 (2020). https://doi.org/10.1007/s00216-019-02286-z

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