A detailed understanding of the structure of technical lignins is critically important in their valorization in high value-added applications. It should be pointed out once more that the structure of technical lignins is fundamentally different from the well-studied native lignins in plant material. However, technical lignins are the potential starting materials future biorefineries and chemical industries are dealing with. 

Nuclear magnetic resonance (NMR) spectroscopy is the analytical key method to characterize the structure of different lignins and their structural changes upon chemical modifications upon processing, such as depolymerization, condensation, oxidation, and many others.

A) One- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy

When the sample characterization requires in-depth structural analysis, NMR provides the most comprehensive information, depending on the respective experiment. Depending on the customers’ needs and sample features ALICE offers all relevant NMR experiments: 1H, 13C, COSY, TOCSY, ROESY, HSQC, HMBC, HSQC-TOCSY for liquid NMR and also CP/MAS NMR for the solid state NMR counterparts.

High quality measurements and results require some initial tests (purity of the samples, solubility in NMR solvents, viscosity of the solution etc.) prior to the actual NMR analysis. 

Sample amount required: 250 mg.

B) Quantitative 13C NMR

Most lignins possess pronounced structural heterogeneity and complexity, especially those that were strongly modified in technical processes. High-resolution 13C NMR spectroscopy can be utilized to obtain qualitative and quantitative information of various functional groups, linkages and moieties present. Though this measurement is rather time-consuming – a single analysis may take up to one day of instrument time – no other analytical method is able to provide such a comprehensive and quantitative description of structural features and functional group contents within just one experiment. The method requires several hundred milligrams of purified sample to be soluble in the corresponding NMR solvent, mainly DMSO-d6. For insoluble lignin samples, peracetylation of lignin is recommended beforehand. 

Quantitative 13C NMR provides data on condensed structures as well as on functional groups including also quaternary carbon atoms (substituted aromatic carbons, carbonyl, carboxyl), which are not covered by most 2D experiments (Capanema et al., 2004, Balakshin et al., 2015). Quantitative 13C NMR can serve as a measure of the severity of biomass processing conditions, to characterize lignin purity or to provide information about the reactivity in modification reactions for potential application.

The analysis is performed on Bruker Avance II 400 NMR spectrometer at 100.61 MHz (13C frequency).

Sample amount required: 250 mg.


Balakshin, M. Y., & Capanema, E. A. (2015).
Comprehensive structural analysis of biorefinery lignins with a quantitative 13C NMR approach.
Rsc Advances, 5(106), 87187-87199. doi: 10.1039/c5ra16649g

Capanema, E. A., Balakshin, M. Y., & Kadla, J. F. (2004).
A comprehensive approach for quantitative lignin characterization by NMR spectroscopy.
Journal of Agricultural and Food Chemistry, 52(7), 1850-1860. doi: doi 10.1021/Jf035282b

C) 2D NMR experiments for structure analysis of lignins

Qualitative heteronuclear single quantum coherence (HSQC) NMR techniques, requiring relatively short experimental time with high sensitivity and resolution has become one of the most important tools to study lignocellulosics and lignins in particular (Liitia et al., 2003, Mansfield et al., 2012). Moreover, for lignin characterization, HSQC over the past years has greatly benefitted from both the advancements in NMR technologies as such, and from the improved assignment of NMR resonances based on numerous model experiments and comparative lignin studies (Constant et al. 2016, Ralph et al. 2010).

The method provides comprehensive data on structure, i.e. functional groups, chains, the most important lignin interunit bonding motifs, such as β-O-4, β-5, β–β and others, as well as information on its purity, e.g. presence of carbohydrates or extractive substances.

The analysis is conducted on Bruker Avance II 400 spectrometer.

Sample amount required: 50-100 mg.


Constant, S., Wienk, H. L. J., Frissen, A. E., de Peinder, P., Boelens, R., van Es, D. S., Grisel, R. J. H., Weckhuysen, B. M., Huijgen, W. J. J., Gosselink, R. J. A., Bruijnincx, P. C. A. (2016).
New insights into the structure and composition of technical lignins: a comparative characterisation study.
Green Chemistry, 18(9), 2651-2665. doi: 10.1039/c5gc03043a

Liitia, T. M., Maunu, S. L., Hortling, B., Toikka, M., & Kilpelainen, I. (2003).
Analysis of technical lignins by two- and three-dimensional NMR spectroscopy.
Journal of Agricultural and Food Chemistry, 51(8), 2136-2143. doi: 10.1021/jf0204349

Mansfield, S. D., Kim, H., Lu, F. C., & Ralph, J. (2012).
Whole plant cell wall characterization using solution-state 2D NMR.
Nature Protocols, 7(9), 1579-1589. doi: 10.1038/nprot.2012.064

Ralph, J., Landucci, L. L. (2010).
NMR of Lignins. in Lignin and Lignans;
Advances in Chemistry, eds. C. Heitner, D. R. Dimmel and J. A. Schmidt, CRC Press (Taylor & Francis Group), Boca Raton, FL, 137-234. doi.org/10.1201/EBK1574444865