Quantitative Characterization of a Hardwood Milled Wood Lignin by Nuclear Magnetic Resonance Spectroscopy EWELLYN A. CAPANEMA,* ,² MIKHAIL YU.BALAKSHIN, ² AND JOHN F. KADLA ‡ Department of Wood and Paper Science, North Carolina State University, Raleigh, North Carolina, 27695-8005, and Department of Wood Science, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 The structure of Eucalyptus grandis milled wood lignin (MWL) was investigated by 2D 1 H- 13 C HSQC, HMQC, and 1 H- 1 H TOCSY correlation NMR techniques and by quantitative 13 C NMR as well as by the permanganate oxidation degradation technique. The combination of 2D NMR and quantitative 13 C NMR spectroscopy of nonacetylated and acetylated lignin preparations allowed reliable identification and calculation of the amount of different lignin structures. About 85% of side-chain moieties were estimated on the structural level. This information was substantiated by data on the quantity of various functional groups and interunit linkages as a whole. A modified method for calculation of the h:g:s ratio has been suggested and compared with previously suggested approaches. E. grandis MWL has been determined to have an h:g:s ratio of 2:36:62. The amounts of various phenolic/etherified noncondensed/condensed guaiacyl and syringyl moieties were ap- proximately estimated. E. grandis MWL contained ∼0.60/Ar of -O-4 moieties along with small amounts of other structural units such as pino/syringyresinol (0.03/Ar), phenylcoumaran (0.03/Ar), and spirodienone (0.05/Ar). The degree of condensation was estimated at ∼21%; the main condensed structures are 4-O-5 moieties (∼0.09/Ar). The structure of E. grandis MWL was compared with those of other lignin preparations isolated from various hardwoods. KEYWORDS: Correlation 2D NMR; hardwood; lignin; milled wood lignin (MWL); quantitative 13 C NMR INTRODUCTION Lignin is a very complex irregular polymer formed by random dehydrogenative polymerization of lignin precursors of the cinnamyl type. Most softwood lignins consist predominantly of guaiacyl (g) units, whereas the structure of hardwood lignins is more complex due to the presence of both guaiacyl (g) and syringyl (s) units (Figure 1). There are a few comprehensive models on the structure of hardwood lignins (Table 1). Nimz (1) suggested the structure of beech lignin on the basis of thioacidolysis and nonquantitative 13 C NMR studies, whereas Adler (2) has drawn a scheme of birch lignin based predomi- nantly on the results of permanganate oxidation. However, despite extensive investigations during the past five decades, the structure of lignin, especially that of hardwood lignins, is not well understood. Therefore, the development of methodolo- gies for lignin investigation is of primary importance to enable further progress in the elucidation of lignin structure. NMR spectroscopy is one of the most powerful tools in lignin chemistry. Some of the first works on the quantification of birch milled wood lignin (MWL) with 13 C NMR were published more than 20 years ago (3, 4). Later, Chen and Robert (5, 6) and Kanitskaya et al. (7, 8) reported methods for the quantification of hardwood lignins by 13 C and 1 H NMR. In the 1990s, various hardwood lignins were analyzed by multidimensional correlation NMR techniques (9-13). Most recently, a combination of quantitative 13 C NMR of nonmodified lignin with 1 H NMR of the acetylated preparation has been suggested (14). In our previous study (15) we reported a comprehensive approach for the quantification of lignin substructures using a combination of correlation 2D NMR methods and quantitative 13 C NMR of nonacetylated and acetylated lignin preparations. Using this approach we obtained information on lignin structure comparable to that reported from other wet chemistry techniques, but requiring only rather short experimental times and small amounts of sample. The results obtained for a spruce MWL were in good agreement with the vast databases for this lignin preparation and showed some of the advantages of the NMR method. However, the calculation algorithm should be modified for the quantification of hardwood lignins. For further development of the NMR analysis of hardwood lignins we used a MWL isolated from Eucalyptus grandis plantation wood. The importance of plantation Eucalyptus species for the industry, particularly for the pulp and paper industry, is increasing, especially in southern Europe and South America. However, information on the structures of eucalypt * Corresponding author [telephone (919) 515-5797; fax (919) 515-6302; e-mail capanema@unity.ncsu.edu]. ² North Carolina State University. ‡ University of British Columbia. J. Agric. Food Chem. 2005, 53, 9639-9649 9639 10.1021/jf0515330 CCC: $30.25 © 2005 American Chemical Society Published on Web 11/17/2005