tive syringyl/guaiacyl composition of hardwood lignins. By this technique, red oak fiber and middle lamel- Methoxyl

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1 Bd. 37 (1983) H. 6 Characterization of Hardwood Lignin: Investigation of Syringyl/Guaiacyl Composition by 13 C NMR 297 Holzforschung 37 (1983) Characterization of Hardwood Lignin: Investigation of Syringyl/Guaiacyl Composition by 13 C Nuclear Magnetic Resonance Spectroscopy By John R. Obst* and John U.S. Department of Agriculture, Forest Service, Forest Products Laboratory, Madison, WI 53705, U.S.A. Department of Foresty, University of Wisconsin-Madison, Madison, WI U.S.A. Keywords Characterization of Hardwood Lignin: Investigation of Syringyl/Guaiacyl Composition Lignin by 13 C Nuclear Magnetic Resonance Spectroscopy Syringyl/guaiacyl ratio Milled wood lignin Summary 13 C nuclear magnetic Carbon-13 nuclear magnetic resonance spectroscopy was investigated as a method to determine the relaresonance tive syringyl/guaiacyl composition of hardwood lignins. By this technique, red oak fiber and middle lamel- Methoxyl la milled wood lignins (MWLs) were similar while white birch MWLs from different morphological Ultraviolet spectroscopy regions gave apparent differences in their syringyl/guaiacyl ratios. However, methoxyl contents of MWLs Ray cells were similar, indicating the unsuitability of carbon magnetic resonance spectroscopy, as performed, as a Crill quantitative method. A correlation was noted between the measured carbon magnetic resonance syringyl/ Red oak guaiacyl ratio and the molecular weight distribution of the MWLs. Molecular weight distributions of the White birch MWLs suggested that birch middle lamella lignin was higher in molecular weight than fiber cell wall lignin. Quercus rubra L. Betula papyrifera Marsh. Schlüsselwörter Charakterisierung von Laubholz-Lignin: Untersuchung über die Syringyl/Guajacyl-Zuammensetzung (Sachgebiete) mittels 13 C NMR Spektroskopie Lignin Syringyl(Guajacyl Verhältnis Zusammenfassung MWL 13C NMR Spektroskopie wurde untersucht im Hinblick auf ihre Brauchbarkeit, die relative Syringyl/Gua 13C NMR jacyl-zusammensetzung in Laubholz-Ligninen zu bestimmen. Bei dieser Methode zeigte sich, daß bei Methoxylgehalt Roteiche Fasern- und Mittellamellen-MW Lignine ähnlich waren, während bei Birkenholz MW Lignine UV Spektroskopie aus verschiedenen morphologischen Zonen offenbar unterschiedliche Syringyl/Guajacyl-Verhältnisse Fasern aufwiesen. Dennoch stimmten die Methoxylgehalte weitgehend überein; dies weist auf die Unzulänglich- Strahlenzellen keit der C NMR Spektroskopie als quantitative Methode hin. Eine Korrelation zwischen gemessenem C Mittellamellenfraktion NMR Syringyl/Guajacyl-Verhältnis und der Molekulargewichtsverteilung der MWL wurde festgestellt. Roteiche Letztere deutet darauf hin, daß das Birkenholz-Mittellamellenlignin ein höheres Molekulargewicht be- Papierbirke sitzt als das Faser-Zellwandlignin. Introduction Although lignin structure influences the chemical and physical behavior of wood during pulping and the use of the resulting fibers, no widely accepted description of the lignin in hardwoods has been presented. Carbon-13 nuclear magnetic resonance, ultraviolet (UV) absorbance, methoxyl analysis, and molecular weight distribution of nine lignins isolated from white birch and red oak are presented in this study to further define the nature of hardwood lignin. Whereas softwood lignins are composed of mainly guaiacyl phenylpropanoid units (derived from coni * Research Chemist ** Chemist feryl alcohol and have one methoxyl substituent on the aromatic ring), hardwood lignins additionally contain significant amounts of syringyl phenylpropanoid units (derived from sinapyl alcohol and have two methoxyl substituents on the aromatic ring). The controversy regarding the occurrence of syringyl/guaiacyl copolymeric hardwood lignin as opposed to discrete syringyl and discrete guaiacyl lignins in hardwoods has been briefly reviewed (Obst 1982b). Studies continue to produce conflicting conclusions: Chemical and infrared and UV spectroscopic characterizations of several hardwood lignins indicated a syringyl/guaiacyl copolymeric structure (Obst 1982b) while 13 C magnetic resonance (cmr) spectroscopy of poplar lignins (Lapierre et al. 1982) and chemical analyses of birch

2 298 John R. Obst, and John Ralph Holzforschung milled wood lignins (MWLs) (Lee et al. 1981) led to the conclusion that hardwood lignins were heterogeneous in their syringyl and guaiacyl composition. Because cmr spectroscopy of MWLs could differentiate between hardwood and softwood lignins (Nimz and Lüdemann 1976; Nimz et 1981) it seemed likely that cmr could provide a useful tool to investigate the controversy regarding the monomeric composition of hardwood lignins. Cho et (1980) found no significant differences in the cmr spectra of MWLs from a birch pulp fiber fraction and a separate fraction enriched in middle lamella, nor did Lee et (1981) for birch MWLs isolated after various milling times even though chemical characterizations indicated significant differences in syringyl/guaiacyl ratios. However, Lapierre et al. (1982) concluded on the basis of cmr that poplar lignin was heterogeneous with respect to monomer composition. Techniques for fractionation of hardwood cell types and cell fragments (Hardell et al. 1981; Obst 1982b) now allow preparation of MWLs from various morphological regions of trees in quantities suitable for cmr analysis. Lignin cmr spectra (Lapierre et al. 1982; Lee et 1981; Cho et al. 1980) typically have been obtained under conditions which are unsuitable for quantitative analysis. Very long acquisition times (1 to 2 days) are required to meet the criteria for fully integrable spectra, notably elimination or leveling of nuclear Overhauser enhancement (NOE), complete relaxation of all carbons to their Boltzmann equilibrium distribution, and acceptable signal to noise ratios. Without a spectrometer dedicated to obtaining lignin spectra, these long times are prohibitive. However, it has been demonstrated theoretically (Doddrell, Glushko, and Allerhand 1972) and experimentally (Allerhand and Hailstone 1972) that NOES of all tertiary carbons in large polymers are equivalent, and it is reasonable to assume (Doddrell, Glushko, and Allerhand 1972) that the relaxation times of the tertiary carbons in both syringyl and guaiacyl units in the polymer are equivalent. Therefore it was anticipated that comparison of hardwood lignin spectra obtained in this study with identical acquisition conditions would allow estimation of the relative amounts of the monomeric lignin units. However, because the cmr method for determining the relative syringyl/guaiacyl composition was untested, an independent measure of the monomer composition was required. Methoxyl analysis and UV absorption of the lignins were used to provide such a measure. Experimental Cell Component Isolation Red oak (Quercus rubra, L.) and white birch (Betula papyrifera Marsh.) kraft pulps were prepared in 67% yield at 87 Kappa number and 75% yield at 94 Kappa number. The pulp was fiberized in a 12-inch Sprout-Waldron disc refiner at inch clearance. Ray cells were isolated as the <200-mesh fraction after 1 minute on a vibratory screen. The pulp was fiberized again at inch clearance and fines-free pulp was obtained as the > 100-mesh fraction after 10 minutes of vibratory screening. After beating the fines-free pulp at 10% consistency at 5,000 revolutions in a Papirindustriens Forskningsinstitutt (PFI) mill at 70 C, crill - a fraction enriched in middle lamella (Obst 1982b) - was isolated as the <200-mesh fraction using a Bauer-McNett classifier. The Klason lignin contents of the oak fiber and crill were determined to be 14.2 and 32.3%. The Klason lignin contents of the birch fiber, crill, and ray cells were 13.2, 38.9, and 39.4%, respectively. Lignin Isolation Milled wood lignins were prepared in varying yields (Table 1) with dioxane: water (9: 1 v/v) extraction of vibratory ball-milled pulp fractions. The dioxane extracted residue was exhaustively digested with polysaccharidases (Onozuka SS, Yaakult Biochemicals Co., Ltd., Nishinomiya, Japan) at 45 C and ph 4.6 to give a lignin fraction designated milled wood enzyme lignin ( MWEL ) (Fig. 1) to differentiate this lignin from true MWEL obtained after digestion of whole wood (Obst 1982a). Acetylation of the lignins was with acetic anhydride:pyridine (1:1) overnight. The portion of the acetylated MWEL soluble in acetone was used for cmr analysis. Klason lignins were prepared according to Effland (1977); sintered glass crucibles were used to avoid contamination of the acid-insoluble lignin. Fig. 1. Lignin isolation scheme. Analytical Procedures Proton-decoupled, NOE cmr spectra of the acetylated lignins (200 to 800mg) in acetone-d6 (2.5ml) in a 10-mm tube were recorded at a frequency of 50.1 MHz on a JEOL FX 200 FT spectrometer with a 10-mm broad band probe at 35 C. Spectra were obtained from 30,000 to 50,000 transients over a sweep width of 12 khz with 8K data points in the JEOL Double Precision mode which extended the dynamic range from 16 bit to 32 bit. A pulse angle of 70 (9.5 µs) was used with a pulse delay of 0.5 s for a total pulse repetition time of 0.84 s. Phasing was carefully maintained in the aryl region to ensure reproducible integral measurements.

3 Bd. 37 (1983) H. 6 Characterization of Hardwood Lignin: Investigation of Syringyl/Guaiacyl Composition by 13 C NMR 299 Methoxyl analyses were by a modified Zeisel method (Clark 1929). Methoxyl analyses of acetylated lignins gave spurious results. Therefore, the methoxyl values of the acetylated MWELs were obtained only on their corresponding Klason lignins. Ultraviolet spectra were recorded with a Beckman DK-2A with about 1 mg of MWL dissolved in 25 ml of 50% dioxane. Borohydride reduction was by addition of a few milligrams of sodium borohydride to the solution and allowing it to remain overnight at room temperature. Molecular weight distributions of MWLs were obtained with a cm column of Sephadex G-100, µ (Pharmacia), by elution with 0.1N sodium hydroxide. Similarly, molecular weight distributions of acetylated lignins were obtained with a cm column of Octyl-Sepharose CL-4B (Pharmacia) by elution with 1% acetic acid and 0.1% water in dimethylformamide. The eluents were monitored at 280nm. Results and Discussion Cmr of Oak and Birch Lignins The cmr spectrum of oak fiber acetylated MWL was very similar to that of oak crill acetylated MWL. Both spectra were also similar to that reported for beech wood MWL (Nimz and Ludemann 1976), indicating that the residual lignin in the high-yield pulp was changed little from that in the wood. However, the acetylated MWLs from birch fiber and crill gave significantly different cmr spectra (Fig. 2) under identical acquisition conditions. Birch fiber MWL appeared to have more syringyl character than the crill MWL. After the spectra were expanded and integrated (Fig. 3), the integral ratios of syringyl (aryl 2,6) to guaiacyl (aryl 2,5,6) tertiary carbon resonances were determined and compared (Table 1). Except for spectra acquired without NOE and with pulsing delay times at least five times the relaxation time (T 1) of the slowest relaxing carbon, no exact correlation can be drawn between integrated peak areas and the number of carbon nuclei under each peak. Thus, the experimentally Fig. 3. Expanded 13C nuclear magnetic resonance spectrum of acetylated milled wood lignin from red oak fibers indicating the signals integrated for determining the syringyl/guaiacyl ratio (Table 1). determined cmr syringyl/guaiacyl ratio does not represent the actual ratio of monomeric types present; the value of this ratio is only as a comparative measure among isolated lignins. For example, the cmr syringyl/ guaiacyl ratio of birch fiber MWL was twice that of the birch crill MWL and indicated a significantly greater syringyl content. White birch ray cell acetylated MWL was also prepared and its cmr spectrum gave a syringyl/ guaiacyl ratio between that of acetylated MWL from white birch fiber and crill (Table 1). Therefore, on the basis of comparative cmr spectroscopy, red oak lignin appears to be relatively homogeneous with regard to monomer composition (Table 1) while white birch lignin appears to be syringyl-rich in the fiber wall and guaiacyl-rich in the middle lamella. Although no experimental evidence has ever been presented to show from which part of the wood MWL originated, it has been widely assumed that the bulk of MWL came from the middle lamella (Lai and Sarkanen 1971; Chang et al. 1975). Recently, however, Whiting and Goring (1981) determined the yields of Fig C nuclear magnetic resonance spectra of acetylated milled wood lignin from white birch (a) fibers and (b) crill.

4 300 John R. Obst, and John Ralph Holzforschung Table C nuclear magnetic resonance syringyl/guaiacyl integrated peak area (A) ratios, methoxyl content, and yields of red oak and white birch lignins Lignin type 1 CNMR 2 Methoxyl Methoxyl on Yield (A syringy/ Klason lignin 3 A guaiacyl) from corresponding lignins % % % Red oak Fiber MWL Klason lignin MWEL 1.57 n.d Crill MWL Klason lignin White birch Fiber MWL Klason lignin MWEL 1.77 n.d Crill MWL Klason lignin MWEL 0.82 n.d Ray cells MWL Klason lignin MWEL 1.33 n.d MWL = milled wood lignin; MWEL = milled wood enzyme lignin. 2 From 13 C magnetic resonance of acetylated lignins; areas integrated indicated in Fig Obtained in 85-90% yields. MWL from fractions of black spruce wood enriched in middle lamella and fiber cell wall and concluded that approximately 93% of the lignin in MWL originated from the S 2 layer of the fiber cell wall. If hardwood MWL is mainly cell wall lignin, then it should be syringyl-rich based upon the results of UV microscopy (Musha and Goring 1975). However, as reviewed by Adler (1977), MWLs of hardwoods actually have a slightly lower syringyl/guaiacyl ratio than that of the total lignin in the wood although this difference was considered insignificant. MWLs isolated from birch wood after short milling times had lower methoxyl contents than MWLs obtained after longer milling times (Lee et al. 1981). Similarly, cmr indicated that MWL from poplar had a lower syringyl/guaiacyl ratio than that of the whole lignin (Lapierre et al. 1982). Although these studies indicate lignin heterogeneity with respect to monomer composition, they are the opposite of the expected results if the lignin in MWL originates mainly from the syringyl-rich fiber cell wall (Musha and Goring 1975; Whiting and Goring 1981). The yields of MWL from oak fiber and crill and birch fiber were about the same while that from birch crill was much higher (Table 1). These results do not agree with those from black spruce wood (Whiting and Goring 1981). However, there were marked differences between the two studies including the use of different mills, hardwoods rather than spruce, and high-yield pulps instead of wood. Because the hardwood crill MWL yields were high, these samples should contain a greater proportion of middle lamella lignin than that in the fiber MWLs. Interestingly, we obtained an MWL in only 15% yield from the crill fraction (42% Klason lignin) of a highyield loblolly pine kraft pulp. Because MWL represents only a fraction of the total lignin, the residues from MWL extraction were digested with polysaccharidases. The resulting lignin residue was acetylated, and the portion soluble in acetone, Ac-MWEL, was used for cmr analysis. In this manner, the yield of soluble acetylated lignin analyzed by cmr was more than half of the total lignin in each fraction (Table 1). For white birch, the cmr-determined syringyl/guaiacyl ratio was significantly lower for the MWEL than that of the corresponding MWL (Table 1). This appeared to agree with the supposition of syringyl-rich cell wall lignin removed in the MWLs leaving a guaiacyl-enriched MWEL. However, oak fiber MWL and MWEL were similar (Table 1), again indicating a relatively homogeneous oak lignin. Methoxyl Contents Since the validity of the cmr method for determining the relative syringyl/guaiacyl composition of lignin was untested, an independent measure of the syringyl content of the various MWLs and MWELs was required. Methoxyl analysis should provide such a measure (Obst 1982b). The MWLs from red oak fibers and crill had similar syringyl/guaiacyl ratios, judging from the cmr data, and also had the same methoxyl contents (Table 1). The methoxyl contents of the MWLs from white birch fibers, crill, and ray cells were similar, varying only 1.9, 0.5, and 1.4%, respectively, from their average. The cmr syringyl/guaiacyl ratios of white birch MWLs were significantly different. Therefore, if methoxyl content is a reliable indicator of syringyl content, the cmr method as performed by all investigators to date does not yield a meaningful syringyl/guaiacyl comparison. Klason lignins were prepared from red oak fiber and crill. Their methoxyl contents were similar to those of Klason lignins prepared from the corresponding MWLs and acetylated MWELs (Table 1). All methoxyl analyses for the Klason lignins from red oak were higher than the methoxyl analyses of the MWLs, but the differences are not considered significant. This supports the hypothesis that acid-soluble lignin is not syringyl-rich and that the Klason lignin methoxyl is representative of the methoxyl content of the total lignin (Obst 1982b). The methoxyl content of the Klason lignin from the white birch fibers agreed with that of the corresponding Klason lignins from the acetylated MWL and MWEL (Table 1). The methoxyl content of the Klason lignin from white birch crill was the same as that of the Klason lignin from the corresponding acetylated MWL, but that of the acetylated MWEL Klason lignin was lower. The white birch ray cell Klason lignins gave the lowest methoxyl contents as had been also observed earlier (Obst 1982b). This was not in agreement with the UV microscopic results (Musha and Goring 1975) of syringyl-rich ray cell wall lignin, especially if this MWL originated mainly from the cell wall. These results do not

5 Bd. 37 (1983) H. 6 Characterization of Hardwood Lignin: Investigation of Syringyl/Guaiacyl Composition by 13 C NMR 301 necessarily indicate discrete syringyl and discrete guaiacyl lignins; a possible explanation is that birch ray cells contained significant amounts of nonlignin material which was carried over into the Klason lignin preparations. UV Absorptivities of MWLs The absorptivities of MWLs from red oak fiber and crill were similar before and after borohydride reduction and in the range typical for hardwood MWLs (Table 2). The absorptivities of MWLs from white birch were different (Table 2). Although the MWL from white birch crill had a higher absorptivity after borohydride reduction, it was not high enough to be considered representative of a guaiacyl lignin; MWLs of softwoods have absorptivities in the range of 20 to 24. However, this difference from the absorptivity of the MWL from birch fibers was significant and may have contributed to the difference observed in the UV microscopic study of birch wood (Musha and Goring 1975), especially considering the higher absorptivity of the nonreduced white birch crill MWL. Table 2. Ultraviolet absorptivities of milled wood lignins Milled wood lignin Absorptivity (280 nm) Fiber Crill Ray cells Red oak borohydride reduced White birch borohydride reduced Molecular Weight Distributions of MWLs The molecular weight distributions of the white birch fiber and white birch wood MWLs were similar (Fig. 4). The molecular weight distribution of the white birch crill MWL indicated a significantly greater pro- M Fig. 4. Molecular weight distribution of milled wood lignins from white birch wood, fibers, crill, and ray cells (Sephadex, 0.1N NaOH). portion of higher molecular weight lignin (Fig. 4). The molecular weight distribution of the ray cell MWL was between that of the MWLs from fiber and crill (Fig. 4). In contrast, the molecular weight distributions of MWLs from red oak fiber and crill were similar (Fig. 5). Red oak MWLs had a greater proportion of higher M Fig. 5. Molecular weight distribution of milled wood lignins from red oak fiber and crill (Sephadex, 0.1 N NaOH). molecular weight lignin than did the white birch fiber MWL but not as much as the white birch crill MWL. As reviewed by Goring (1971), the molecular weight of lignin has been investigated mainly with degraded lignins such as lignosulfonates and kraft lignin. More recently, determinations of molecular weight distributions of MWLs have allowed comparisons among different wood species (Faix, Lange, and Salud 1981), but little may be deduced about the nature of lignin in situ. Because separation of hardwood cell types and cell fractions has only recently been reported, a unique opportunity has arisen to gain better insight into the molecular weight of lignin in different morphological regions. However, because all of the MWLs were obtained in varying yields from high-yield kraft pulps, precise correlation of these results to the molecular weight of lignin in wood is tenuous. If it is assumed that these MWLs are somewhat representative of the lignin in the wood, it is apparent that distinct species differences occur between red oak and white birch. Moreover, unlike red oak, white birch contained lignin of varying molecular weight distribution in different morphological regions. However, differences in grinding efficiency, perhaps caused by uncontrolled variations in the milling process or the physical differences among the samples, may affect the observed molecular weight distributions. Additional studies are in progress to resolve this uncertainty. Molecular weight distributions of the birch acetylated MWELs are shown in Figure 6. For molecular weight distributions of the corresponding acetylated MWLs were also run (Fig. 6). As expected, a higher molecular weight distribution was observed for "MWEL" than for MWL. However, while both white birch fiber and ray cell MWELs had large excluded peaks, the crill MWEL did not. Correlation of Molecular Weight Distribution and cmr Syringyl/Guaiacyl Ratio Generally, the experimentally determined cmr syringyl/guaiacyl ratio of the MWLs (Table 1) decreased as the molecular weight distribution shifted to higher molecular weights (white birch, Figs. 4 and 6), while similar molecular weight distributions (red oak, Fig. 5) gave similar cmr syringyl/guaiacyl ratios. Although the spin-lattice relaxation times (T 1) of the guaiacyl tertiary aryl carbons were about twice those of the syringyl tertiary aryl carbons in monomeric models (Ralph 1982), it was expected that the relaxa-

6 302 John R. Obst, and John Ralph Holzforschung Fig. 6. Molecular weight distribution of acetylated milled wood lignin (Ac-MWL) and acetylated milled wood enzyme lignin (Ac MWEL ) from white birch (a) fibers, (b) ray cells, and (c) crill (octyl sepharose, dimethylformamide/acetic acid/water). tion times of all tertiary carbons in polymeric lignins would be equal (Doddrell, Glushko, and Allerhand 1972). However, the apparent correlation of molecular weight to the cmr syringyl/guaiacyl integral ratio suggests that some of the MWLs are not of high enough molecular weight to give equivalent tertiary carbon T 1 values. Thus the assumption made for relative quantitation of polymers by cmr may not be valid for all MWLs. Those MWLs with a larger proportion of lower molecular weight components, for example white birch fiber MWL, gave significantly greater syringyl cmr signals, which was analogous to the difference the T 1 s of the monomers. Additional experiments are required to resolve whether cmrsyringyl/guaiacyl ratios determined for lignins are causally or coincidentally related to molecular weight. In particular, fully integrable spectra must be obtained, that is, spectra without NOE and with pulse delays of at least five times the relaxation time of the slowest relaxing carbon. Conclusions Lignin carbon-13 nuclear magnetic resonance spectra obtained in this study with typical acquisition parameters were not adequate for quantitative structure comparisons among lignins. The molecular weight distribution of the lignin may influence the cmr-determined syringyl/guaiacyl ratio. Acknowledgment The authors are grateful to G.R. Henseler and D.M. Crawford for technical assistance, to M.F. Wesolowski and L.C. Zank for methoxyl determinations, to M.E. Effland for Klason lignins, to D. Hillebrand for discussion of carbon magnetic resonance, to J.M. Harkin for discussion of this manuscript, and to the members of the New Zealand Research Advisory Council for their partial funding of this project. References Adler, E. Lignin chemistry past, present, and future. Wood Sci. Technol. 11, Allerhand, A. and R.K. Hailstone. Carbon-13 fourier transform nuclear magnetic resonance. X. Effect of molecular weight on 13 C spin-lattice relaxation times of polystyrene in solution. J. Chem. Phys. 56, Chang, H.-M., E.B. Cowling, W. Brown, E. Adler, and G. Miksche. Comparative studies on cellulolytic enzyme lignin and milled wood lignin of sweetgum and spruce. Holzforschung 29, Cho, N.S., J.Y. Lee, G. Meshitsuka, and J. Nakano. On the characteristics of hardwood compound middle lamella lignin. Mokuzai Gakkaishi 26, Clark, E.P Studies on Gossypol. VI. The action of boiling hydriodic acid as used in the Zeisel method upon Gossypol and some of its derivatives. A semi-micro Zeisel methoxyl method. J. Am. Chem. Soc. 51, Doddrell, D., V. Glushko, and A. Allerhand. Theory of nuclear Overhauser enhancement and 13 C- 1 H dipolar relaxation in protondecoupled carbon-13 nmr spectra of macromolecules. J. Chem. Phys. 56, Effland, M.E. Modified procedure to determine acid-insoluble lignin in wood and pulp. 60 (10), Faix, O., W. Lange, and E.C. Salud. The use of HPLC for the determination of average molecular weight distributions of milled wood lignins from Shorea polysperma (Blco.). Holzforschung 35, 3-9. Goring, D.A.I. Polymer properties of lignin and lignin derivatives. In Lignins: Occurrence, Formation, Structure, and Reactions. (Eds., K.V. Sarkanen and C.H. Ludwig.) Wiley- Interscience, New York. Hardell, H.-L., G.J. Leary, M. Stoll, and U. Westermark. Variations in lignin structure in defined morphological parts of birch. Svensk Papperstidning 83, Y.Z. and K.V. Sarkanen. Isolation and structural studies. In Lignins: Occurrence, Formation, Structure, and Reactions. (Eds., K.V. Sarkanen and C.H. Ludwig.) Wiley-Interscience, New York. Lapierre, C., J.Y. Lallemand, and Monties. Evidence of poplar lignin heterogeneity by combination of 13 C and 1 H NMR spectroscopy. Holzforschung 36, Lee, Z.Z., G. Meshitsuka, N.S. Cho, and J. Nakano. Characteristics of milled wood lignins isolated with different milling times, Mokuzai Gakkaishi 27, Musha, Y. and D.A.I. Goring. Distribution of syringyl and guaiacyl moieties in hardwoods as indicated by ultraviolet microscopy. Wood Sci. Technol. 9, Nimz, H. and H.-D. Lüdemann. Kohlenstoff-13-NMR Spektren von Ligninen, 6. Lignin- und DHP-Acetate. Holzforschung 30, Nimz, H., D. Robert, O. Faix, and M. Nemr. Carbon-13 NMR spectra of lignins, 8. Holzforschung 35, Obst, J.R. 1982a. Frequency and alkali resistance of lignin-carbohydrate bonds in wood. 65, Obst, J.R. 1982b. Guaiacyl and syringyl lignin composition in hardwood cell components. Holzforschung 36, Ralph, J. Ph. D. Thesis. University of Wisconsin-Madison, Madison, Wis. Whiting, P. and D.A.I. Goring. The morphological origin of milled wood lignin. Svensk Papperstidning 84, R120-R122.