Determination of hydroxyl groups in biorefinery resources via quantitative 31P NMR spectroscopy

Abstract

The analysis of chemical structural characteristics of biorefinery product streams (such as lignin and tannin) has advanced substantially over the past decade, with traditional wet-chemical techniques being replaced or supplemented by NMR methodologies. Quantitative 31P NMR spectroscopy is a promising technique for the analysis of hydroxyl groups because of its unique characterization capability and broad potential applicability across the biorefinery research community. This protocol describes procedures for (i) the preparation/solubilization of lignin and tannin, (ii) the phosphitylation of their hydroxyl groups, (iii) NMR acquisition details, and (iv) the ensuing data analyses and means to precisely calculate the content of the different types of hydroxyl groups. Compared with traditional wet-chemical techniques, the technique of quantitative 31P NMR spectroscopy offers unique advantages in measuring hydroxyl groups in a single spectrum with high signal resolution. The method provides complete quantitative information about the hydroxyl groups with small amounts of sample (~30 mg) within a relatively short experimental time (~30–120 min).

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Fig. 1: A representative structural model of hardwood lignin, as predicted from NMR-based lignin analysis.
Fig. 2: Structural features of proanthocyanidin and its subunits.
Fig. 3
Fig. 4
Fig. 5
Fig. 6: Contents of different hydroxyl groups in organosolv poplar, switchgrass, and pine lignin determined by 31P NMR analysis.
Fig. 7: Representative 31P NMR spectra of condensed tannins using cholesterol as the IS.
Fig. 8: Representative 31P NMR of a complex tannin (epigallocatechin-3-O-gallate) using cholesterol as the IS: signals of esterified gallates and B rings are excellently resolved.

Data availability

All data generated during this study are included in this published article. The NMR integration data are available upon request. The software used for NMR data analysis is freely available (see ‘Software’).

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Acknowledgements

This research used resources of the Oak Ridge National Laboratory, which is managed by UT-Battelle, LLC, under contract no. DE-AC05-00OR22725 with the US Department of Energy (DOE). This study was supported and performed as part of the Center for Bioenergy Innovation (CBI). The CBI is a DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. The research effort that established the quantitative 31P NMR in wood chemistry protocol was supported by FP Innovations (formerly Pulp and Paper Research Institute of Canada) and the Natural Sciences and Engineering Research Council of Canada.

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A.J.R. proposed and designed the manuscript. X.M. wrote the manuscript with major input from D.S.A., C.C., H.B., Y.P., and A.J.R. X.M. and N.H. performed the organosolv pretreatment. X.M. and Y.P. performed the NMR experiments and data processing on lignins. C.C. carried out the NMR experiments and data processing on tannins. For specific questions regarding the conception and the foundations of the protocol, including selection of an appropriate solvent, relaxation time, and synthesis of phosphorus reagent, contact D.S.A. Questions about the characterization of tannin should be referred to C.C. For selection of an appropriate IS and the application of 31P NMR for the characterization of native/transgenic plants and untreated or pretreated biomass, please contact A.J.R. All authors approved the final version of the manuscript.

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Correspondence to Claudia Crestini or Arthur J. Ragauskas or Dimitris S. Argyropoulos.

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Peer review information: Nature Protocols thanks Luc Avérous and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Gioia, C. et al. J. Am. Chem. Soc. 140, 4054–4061 (2018): https://pubs.acs.org/doi/abs/10.1021/jacs.7b13620

Li, M. et al. Commun. Biol. 2, 22 (2019): https://www.nature.com/articles/s42003-018-0265-6

Granata, A. & Argyropoulos, D. S. J. Agric. Food Chem. 43, 1538–1544 (1995): https://pubs.acs.org/doi/10.1021/jf00054a023

Crestini, C., Lange, H. & Bianchetti, G. J. Nat. Prod. 79, 2287–2295 (2016): https://pubs.acs.org/doi/abs/10.1021/acs.jnatprod.6b00380

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Meng, X., Crestini, C., Ben, H. et al. Determination of hydroxyl groups in biorefinery resources via quantitative 31P NMR spectroscopy. Nat Protoc 14, 2627–2647 (2019). https://doi.org/10.1038/s41596-019-0191-1

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