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Solution-state nuclear magnetic resonance spectroscopy of crystalline cellulosic materials using a direct dissolution ionic liquid electrolyte

Nature Protocols volume 18, pages 2084–2123 (2023)Cite this article

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Abstract

Owing to its high sustainable production capacity, cellulose represents a valuable feedstock for the development of more sustainable alternatives to currently used fossil fuel-based materials. Chemical analysis of cellulose remains challenging, and analytical techniques have not advanced as fast as the development of the proposed materials science applications. Crystalline cellulosic materials are insoluble in most solvents, which restricts direct analytical techniques to lower-resolution solid-state spectroscopy, destructive indirect procedures or to ‘old-school’ derivatization protocols. While investigating their use for biomass valorization, tetralkylphosphonium ionic liquids (ILs) exhibited advantageous properties for direct solution-state nuclear magnetic resonance (NMR) analysis of crystalline cellulose. After screening and optimization, the IL tetra-n-butylphosphonium acetate [P4444][OAc], diluted with dimethyl sulfoxide-d6, was found to be the most promising partly deuterated solvent system for high-resolution solution-state NMR. The solvent system has been used for the measurement of both 1D and 2D experiments for a wide substrate scope, with excellent spectral quality and signal-to-noise, all with modest collection times. The procedure initially describes the scalable syntheses of an IL, in 24–72 h, of sufficient purity, yielding a stock electrolyte solution. The dissolution of cellulosic materials and preparation of NMR samples is presented, with pretreatment, concentration and dissolution time recommendations for different sample types. Also included is a set of recommended 1D and 2D NMR experiments with parameters optimized for an in-depth structural characterization of cellulosic materials. The time required for full characterization varies between a few hours and several days.

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Fig. 1: Preparation of the IL electrolyte [P4444][OAc]:DMSO-d6 (1:4 wt%) and dissolution of cellulose samples.
Fig. 2: Comparison of the obtainable resolution in cellulose NMR investigation of the presented quantitative solution-state NMR protocol and commonly performed solid-state CP MAS NMR protocol for MCC (Avicel PH-101).
Fig. 3: Preferred dissolution setup for the NMR sample preparation.
Fig. 4: Quantitative 1H spectrum ([P4444][OAc]:DMSO-d6 1:4 wt%, 65 °C, 600 MHz) of MCC (Avicel PH-101, 5 wt%).
Fig. 5: Suppressing (editing-out) resonances of low-molecular-weight compounds in the 1H spectra by diffusion editing.
Fig. 6: Multiplicity-edited HSQC spectrum ([P4444][OAc]:DMSO-d6 1:4 wt%, 65 °C) of MCC (Avicel PH-101, 5 wt%).
Fig. 7: Qualitative 13C spectrum ([P4444][OAc]:DMSO-d6 1:4 wt%, 65 °C, 150 MHz) of MCC (Avicel PH-101, 5 wt%).
Fig. 8: Quantitative 13C spectrum ([P4444][OAc]:DMSO-d6 1:4 wt%, 65 °C, 150 MHz) of MCC (Avicel PH-101, 5 wt%).
Fig. 9: HSQC–TOCSY spectrum (short range, 15 ms TOCSY mixing time) with a multiplicity-edited HSQC overlay ([P4444][OAc]:DMSO-d6 1:4 wt%, 65 °C) of MCC (Avicel PH-101, 5 wt%).
Fig. 10: HMBC spectrum ([P4444][OAc]:DMSO-d6 1:4 wt%, 65 °C) of MCC (Avicel PH-101, 5 wt%).
Fig. 11: Resolution of key signals in the technical cellulose backbone spectral regions.
Fig. 12: Application of the initial NMR experiments to distinguish between covalent modifications and impurities in cellulose chemistry.
Fig. 13: Utility of single and multiple bond 2D heteronuclear-correlated spectroscopy (HSQC and HMBC) in the assignment of a TEMPO-oxidized cellulose nanocrystal ([P4444][OAc]:DMSO-d6 1:4 wt%, 65 °C, 5 wt%, 600 MHz 1H frequency).
Fig. 14: 1D NMR spectra ([P4444][OAc]:DMSO-d6 1:4 wt%, 65 °C, 2.5 wt%, 600 MHz) for ‘Asplund’ wood (spruce fibers produced through low-energy thermomechanical refining).

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Data availability

The data related to the results shown in this protocol are included in this document and in the supporting primary research papers25,26.

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Acknowledgements

The authors want to acknowledge the fundamental contributions of A. Holding, V. Mäkelä and S. Heikkinen in the early stages of the development of this method. A.W.T.K. gratefully acknowledges funding by the Academy of Finland (project no. 311255, ‘WTF-Click-Nano’). K.H. gratefully acknowledges the postdoctoral grant received from the Academy of Finland (project no. 333905).

Author information

Authors and Affiliations

  1. Department of Bioproducts and Biosystems, Aalto University, Espoo, Finland

    Lukas Fliri, Katja Heise & Michael Hummel

  2. Materials Chemistry Division, Department of Chemistry, University of Helsinki, Helsinki, Finland

    Tetyana Koso, Aleksandar R. Todorov, Daniel Rico del Cerro, Sami Hietala, Ilkka Kilpeläinen & Alistair W. T. King

  3. Fibre Science and Communication Network (FSCN), Mid Sweden University, Sundsvall, Sweden

    Juha Fiskari

  4. VTT Technical Research Centre of Finland Ltd, Espoo, Finland

    Alistair W. T. King

Authors
  1. Lukas Fliri
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  2. Katja Heise
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Contributions

A.W.T.K and T.K., designed and developed the workflows presented in this protocol. L.F., K.H. and M.H. implemented the protocol in a more technology-orientated environment and addressed the occurring translational barriers. L.F. and A.R.T. contributed optimized metathesis schemes for the ionic liquid starting from commercial sources. S.H. provided solid-state NMR spectra and expertise. D.R.d.C. and J.F. provided samples, discussion and experimentation regarding the adaptation of the protocol to other substrates, as presented in the ‘Anticipated results’ section. L.F. and A.W.T.K. drafted, reviewed and edited the manuscript with significant input from K.H., T.K. and M.H. I.K. provided funding for the basic research (initial articles) and advice on presentation of the subject matter. All authors read and agreed on the final version of the manuscript.

Corresponding authors

Correspondence to Michael Hummel or Alistair W. T. King.

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The authors declare no competing financial interests.

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Nature Protocols thanks Jun Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

King, A. W. T. et al. Biomacromolecules 19, 2708–2720 (2018): https://doi.org/10.1021/acs.biomac.8b00295

Koso, T. et al. Cellulose 27, 7929–7953 (2020): https://doi.org/10.1007/s10570-020-03317-0

Extended data

Extended Data Fig. 1 Multiplicity-edited HSQC of acetylated MCC.

Multiplicity-edited HSQC of acetylated MCC showing significant peak superposition ([P4444][OAc]:DMSO-d6 1:4 wt%, 65 °C, 5 wt%; 600 MHz 1H frequency. For multiplicity edited HSQC green = CH, blue = CH2).

Extended Data Fig. 2 Effect of diffusing-editing on the 1H 1D data for surface acetylated MCC.

Comparison of the quantitative 1H spectrum (a) with the diffusion edited 1H spectrum (b) allows to quickly assess the introduction of functionalities of species exhibiting resonances in the heavily crowded IL spectral region ([P4444][OAc]:DMSO-d6 1:4 wt%, 65 °C, 5 wt%; 600 MHz 1H frequency).

Extended Data Fig. 3 Utility of the 2D HSQC-TOCSY experiment for further peak assignment of cellulose derivatives.

(a) HSQC-TOCSY in the full view allows to further assign the AGA moiety over interactions of the C1 signal with peaks in the crowded areas., (b) HSQC-TOCSY with zoom into the C2–C5 region shows that full characterisation of the spin system can be possible. However, owing to strong superpositions with the AGU, NRE and RE moieties the peak assignments can become tedious. Spectra shown with diffusion-edited 1H trace (top trace) and 13C trace (left trace). AGU = anhydroglucose unit; AGA = anhydroglucopyranosiduronic acid unit; NRE = non-reducing end; RE = reducing end. In the spectra HSQC correlations are shown in green (CH) and blue (CH2) and TOCSY correlations are shown in gray.

Extended Data Fig. 4 Diffusion-edited 1H spectra of food insects.

Diffusion-edited 1H spectra ([P4444][OAc]:DMSO-d6 1:4 wt%, 65 °C, 5 wt%, 600 MHz) for fruit flies, damselfly tail and whole food crickets, after Wiley milling and dissolution.

Supplementary information

Supplementary Information

Supplementary Figs. 1–12 and Note.

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Fliri, L., Heise, K., Koso, T. et al. Solution-state nuclear magnetic resonance spectroscopy of crystalline cellulosic materials using a direct dissolution ionic liquid electrolyte. Nat Protoc 18, 2084–2123 (2023). https://doi.org/10.1038/s41596-023-00832-9

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