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Continuous purification of reaction products by micro free-flow electrophoresis enabled by large area deep-UV fluorescence imaging

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Abstract

Microreactors have gained increasing attention in their application toward continuous micro flow synthesis. An unsolved problem of continuous flow synthesis is the lack of techniques for continuous product purification. Herein, we present a micro free-flow electrophoresis device and accompanying setup that enables the continuous separation and purification of unlabeled organic synthesis products. The system is applied to the separation and purification of triarylmethanes. For imaging of the unlabeled analytes on-chip a novel setup for large area (3.6 cm2) deep ultra violet excitation fluorescence detection was developed. Suitable separation conditions based on low conductivity electrophoresis buffers were devised to purify the product. With the optimized conditions, starting materials and product of the synthesis were well separated (R > 1.2). The separation was found to be very stable with relative standard deviations of the peak positions smaller than 3.5% over 15 min. The stable conditions enabled collection of the separated compounds, and purity of the product fraction was confirmed using capillary electrophoresis and mass spectrometry. This result demonstrates the great potential of free-flow electrophoresis as a technique for product purification or continuous clean-up in flow synthesis.

Micro free-flow electrophoresis (μFFE) allows continuous separation and purification of small organic synthesis products. Enabled by a novel deep-UV imaging setup starting materials and product of a recently developed synthesis for triarylmethanes could be purified. Thereby demonstrating the potential of μFFE as continuous purification technique for micro flow synthesis.

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References

  1. Jensen KF. Microreaction engineering — is small better? Chem Eng Sci. 2001;56:293–303.

    Article  CAS  Google Scholar 

  2. Jähnisch K, Hessel V, Löwe H, Baerns M. Chemistry in Microstructured Reactors. Angew Chem Int Ed. 2004;43:406–46.

    Article  Google Scholar 

  3. McMullen JP, Jensen KF. Integrated microreactors for reaction automation: new approaches to reaction development. Annu Rev Anal Chem. 2010;3:19–42.

    Article  CAS  Google Scholar 

  4. Plutschack MB, Pieber B, Gilmore K, Seeberger PH. The Hitchhiker’s Guide to Flow Chemistry. Chem Rev. 2017; https://doi.org/10.1021/acs.chemrev.7b00183.

  5. Sahoo HR, Kralj JG, Jensen KF. Multistep Continuous-flow microchemical synthesis involving multiple reactions and separations. Angew Chem. 2007;119:5806–10.

    Article  Google Scholar 

  6. Hartman RL, Naber JR, Buchwald SL, Jensen KF. Multistep microchemical synthesis enabled by microfluidic distillation. Angew Chem Int Ed. 2010;49:899–903.

    Article  CAS  Google Scholar 

  7. Timmer BH, van Delft KM, Olthuis W, Bergveld P, van den Berg A. Micro-evaporation electrolyte concentrator. Sensors Actuators B Chem. 2003;91:342–6.

    Article  CAS  Google Scholar 

  8. Ley SV, Fitzpatrick DE, Ingham RJ, Myers RM. Organic synthesis: march of the machines. Angew Chem Int Ed. 2015;54:3449–64.

    Article  CAS  Google Scholar 

  9. Kralj JG, Sahoo HR, Jensen KF. Integrated continuous microfluidic liquid–liquid extraction. Lab Chip. 2007;7:256–63.

    Article  CAS  Google Scholar 

  10. Jezierski S, Tehsmer V, Nagl S, Belder D. Integrating continuous microflow reactions with subsequent micropreparative separations on a single microfluidic chip. Chem Commun. 2013;49:11644–6.

    Article  CAS  Google Scholar 

  11. Agostino FJ, Krylov SN. Advances in steady-state continuous-flow purification by small-scale free-flow electrophoresis. TrAC Trends Anal Chem. 2015;72:68–79.

    Article  CAS  Google Scholar 

  12. Turgeon RT, Bowser MT. Micro free-flow electrophoresis: theory and applications. Anal Bioanal Chem. 2009;394:187–98.

    Article  CAS  Google Scholar 

  13. Köhler S, Weilbeer C, Howitz S, Becker H, Beushausen V, Belder D. PDMS free-flow electrophoresis chips with integrated partitioning bars for bubble segregation. Lab Chip. 2011;11:309–14.

    Article  Google Scholar 

  14. Köhler S, Benz C, Becker H, Beckert E, Beushausen V, Belder D. Micro free-flow electrophoresis with injection molded chips. RSC Adv. 2012;2:520–5.

    Article  Google Scholar 

  15. Geiger M, Frost NW, Bowser MT. Comprehensive multidimensional separations of peptides using nano-liquid chromatography coupled with micro free-flow electrophoresis. Anal Chem. 2014;86:5136–42.

    Article  CAS  Google Scholar 

  16. Herzog C, Beckert E, Nagl S. Rapid isoelectric point determination in a miniaturized preparative separation using jet-dispensed optical pH sensors and micro free-flow electrophoresis. Anal Chem. 2014;86:9533–9.

    Article  CAS  Google Scholar 

  17. Geiger M, Harstad RK, Bowser MT. Effect of surface adsorption on temporal and spatial broadening in micro free-flow electrophoresis. Anal Chem. 2015;87:11682–90.

    Article  CAS  Google Scholar 

  18. Poehler E, Herzog C, Suendermann M, Pfeiffer SA, Nagl S. Development of microscopic time-domain dual lifetime referencing luminescence detection for pH monitoring in microfluidic free-flow isoelectric focusing. Eng Life Sci. 2015;15:276–85.

    Article  CAS  Google Scholar 

  19. Anciaux SK, Geiger M, Bowser MT. 3D printed micro free-flow electrophoresis device. Anal Chem. 2016;88:7675–82.

    Article  CAS  Google Scholar 

  20. Novo P, Jender M, Dell’Aica M, Zahedi RP, Janasek D. Free-flow electrophoresis separation of proteins and DNA using microfluidics and polycarbonate membranes. Procedia Eng. 2016;168:1382–5.

    Article  CAS  Google Scholar 

  21. Johnson AC, Bowser MT. High-speed, comprehensive, two dimensional separations of peptides and small molecule biological amines using capillary electrophoresis coupled with micro free-flow electrophoresis. Anal Chem. 2017;89:1665–73.

    Article  CAS  Google Scholar 

  22. Herzog C, Poehler E, Peretzki AJ, Borisov SM, Aigner D, Mayr T, Nagl S. Continuous on-chip fluorescence labelling, free-flow isoelectric focusing and marker-free isoelectric point determination of proteins and peptides. Lab Chip. 2016;16:1565–72.

  23. Kochmann S, Krylov S. Image processing and analysis system for development and use of free-flow electrophoresis chips. Lab Chip. 2017;17:256–66.

    Article  CAS  Google Scholar 

  24. Novo P, Janasek D. Current advances and challenges in microfluidic free-flow electrophoresis – a critical review. Anal Chim Acta. 2017; https://doi.org/10.1016/j.aca.2017.08.017.

  25. Chartogne A, Tjaden UR, Van der Greef J. A free-flow electrophoresis chip device for interfacing capillary isoelectric focusing on-line with electrospray mass spectrometry. Rapid Commun Mass Spectrom. 2000;14:1269–74.

    Article  CAS  Google Scholar 

  26. Benz C, Boomhoff M, Appun J, Schneider C, Belder D. Chip-based free-flow electrophoresis with integrated nanospray mass-spectrometry. Angew Chem Int Ed. 2015;54:2766–70.

    Article  CAS  Google Scholar 

  27. Park JK, Campos CDM, Neužil P, Abelmann L, Guijt RM, Manz A. Direct coupling of a free-flow isotachophoresis (FFITP) device with electrospray ionization mass spectrometry (ESI-MS). Lab Chip. 2015;15:3495–502.

    Article  CAS  Google Scholar 

  28. Kochmann S, Agostino FJ, LeBlanc JCY, Krylov SN. Hyphenation of production-scale free-flow electrophoresis to electrospray ionization mass spectrometry using a highly conductive background electrolyte. Anal Chem. 2016;88:8415–20.

    Article  CAS  Google Scholar 

  29. Köhler S, Nagl S, Fritzsche S, Belder D. Label-free real-time imaging in microchip free-flow electrophoresis applying high speed deep UV fluorescence scanning. Lab Chip. 2012;12:458–63.

    Article  Google Scholar 

  30. Poehler E, Herzog C, Lotter C, Pfeiffer SA, Aigner D, Mayr T, Nagl S. Label-free microfluidic free-flow isoelectric focusing, pH gradient sensing and near real-time isoelectric point determination of biomolecules and blood plasma fractions. Analyst. 2015;140:7496–502.

  31. Becker M, Budich C, Deckert V, Janasek D. Isotachophoretic free-flow electrophoretic focusing and SERS detection of myoglobin inside a miniaturized device. Analyst. 2008;134:38–40.

    Article  Google Scholar 

  32. Jezierski S, Klein AS, Benz C, Schaefer M, Nagl S, Belder D. Towards an integrated device that utilizes adherent cells in a micro-free-flow electrophoresis chip to achieve separation and biosensing. Anal Bioanal Chem. 2013;405:5381–6.

    Article  CAS  Google Scholar 

  33. Yin X-Y, Dong J-Y, Wang H-Y, Li S, Fan L-Y, Cao C-X. A simple chip free-flow electrophoresis for monosaccharide sensing via supermolecule interaction of boronic acid functionalized quencher and fluorescent dye. Electrophoresis. 2013;34:2185–92.

    Article  CAS  Google Scholar 

  34. Jezierski S, Gitlin L, Nagl S, Belder D. Multistep liquid-phase lithography for fast prototyping of microfluidic free-flow-electrophoresis chips. Anal Bioanal Chem. 2011;401:2651–6.

    Article  CAS  Google Scholar 

  35. Saha S, Alamsetti SK, Schneider C. Chiral Brønsted acid-catalyzed Friedel–Crafts alkylation of electron-rich arenes with in situ-generated ortho-quinone methides: highly enantioselective synthesis of diarylindolylmethanes and triarylmethanes. Chem Commun. 2015;51:1461–4.

    Article  CAS  Google Scholar 

  36. Schulze P, Ludwig M, Kohler F, Belder D. Deep UV laser-induced fluorescence detection of unlabeled drugs and proteins in microchip electrophoresis. Anal Chem. 2005;77:1325–9.

    Article  CAS  Google Scholar 

  37. Ohla S, Schulze P, Fritzsche S, Belder D. Chip electrophoresis of active banana ingredients with label-free detection utilizing deep UV native fluorescence and mass spectrometry. Anal Bioanal Chem. 2011;399:1853–7.

    Article  CAS  Google Scholar 

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Acknowledgements

The authors gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG) through grant FOR 2177.

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Authors and Affiliations

  1. Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, 04103, Leipzig, Germany

    Simon A. Pfeiffer, Benjamin M. Rudisch, Petra Glaeser, Stefan Nagl & Detlev Belder

  2. Institut für Organische Chemie, Universität Leipzig, Johannisallee 29, 04103, Leipzig, Germany

    Matthias Spanka & Christoph Schneider

  3. Center for Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany

    Felix Nitschke & Andrea A. Robitzki

  4. Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China

    Stefan Nagl

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  1. Simon A. Pfeiffer
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  2. Benjamin M. Rudisch
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  3. Petra Glaeser
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  4. Matthias Spanka
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  7. Christoph Schneider
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  8. Stefan Nagl
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  9. Detlev Belder
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Correspondence to Detlev Belder.

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Published in the topical collection celebrating ABCs 16th Anniversary.

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Pfeiffer, S.A., Rudisch, B.M., Glaeser, P. et al. Continuous purification of reaction products by micro free-flow electrophoresis enabled by large area deep-UV fluorescence imaging. Anal Bioanal Chem 410, 853–862 (2018). https://doi.org/10.1007/s00216-017-0697-8

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  • DOI: https://doi.org/10.1007/s00216-017-0697-8

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