Article pubs.acs.org/Biomac
Milled Wood Lignin: A Linear Oligomer Claudia Crestini,*,† Federica Melone,† Marco Sette,† and Raffaele Saladino‡ †
Dipartimento di Scienze e Tecnologie Chimiche, Tor Vergata University, Via della Ricerca Scientifica, 00133, Rome, Italy Dipartimento di Agrobiologia e Agrochimica, University of Tuscia, via San Camillo de Lellis, 01100, Viterbo, Italy
‡
ABSTRACT: The degree of polymerization (DP) of softwood and hardwood milled wood lignin samples and their branching degrees were quantitatively evaluated by a novel end-group titration approach composed of QQ-HSQC, 31P NMR, and DFRC coupled with 31P NMR analysis techniques. The DP of lignin can be calculated when the C9 formula, the amounts of phenolic groups, pinoresinol (β-β), diphenylethane (β-1), and phenolic diphenyl (55′) lignin subunits have been determined. Data on the degree of polymerization of lignin obtained by NMR techniques were not affected by supramolecular aggregation processes. 31P NMR analysis coupled with DFRC and QQ-HSQC allowed a detailed evaluation of the occurrence of condensed units in lignin and showed the terminal nature of diphenyl ether and diphenyl subunits. The resulting data unequivocally show that milled wood lignin is a linear oligomer.
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molecular weight distribution is an illuminating paradigm. Several analytical techniques have been employed in order to characterize lignin average molecular weight, ranging from vapor osmometry,5,6 crioscopy,7 isopiestic methods,8 ultrafiltration,9 to GPC,10 MALDI-ToF MS,11,12 and light scattering analysis;13 however, different analytical techniques yield discordant results. The determination of Mn, Mw, and their distributions, far from being of univocal acceptance, is today still object of debate. Early determinations mainly based on colligative properties gave rise to results ranging for milled wood lignin (MWL) from 500 to 5000 Da. This would account for an average DP of MWL ranging from about 3 to 25. It was clear also in early studies that association phenomena were operative.5,6 The solvent system used to determine the MW distribution is of crucial importance since non-hydrogenbonding solvents such as dioxane and THF can undergo large self-aggregation in lignin and hence results highly dependent on concentration that interfere with Mn determinations. Vapor osmometry can give informations related to the number-average molecular weight, while light scattering data provide weight-average molecular weight of lignins. These techniques suffer from the disadvantage of being affected by supramolecular aggregation processes. Lignin in fact shows a high tendency to aggregate and the results of such analyses are strongly dependent upon the freshness of the lignin preparation studied.14,15 Sarkanen concluded that the intermolecular associative effects are apparently governed by nonbonded orbital interactions presumably among the aromatic moieties in the components.16
INTRODUCTION Lignin is a phenyl-propanoid (C9) polyphenol mainly linked by arylglycerol ether bonds between the monomeric phenolic pcoumaryl (H) coniferyl (G) and sinapyl alcohol (S) units.1 Lignin structure is the result of a biosynthetic pathway which occurs via oxidative radicalization of monolignols, followed by radical coupling of two monomer radicals that form a dehydrodimer (Scheme 1). Coupling is favored at monolignol β positions resulting in arylglycerol-β-aryl ether (β-O-4′), pinoresinol (β-β′), phenylcoumaran (β-5′), spirodienone (SD), and diphenylethane (β-1′) dimers formation.1 In a subsequent step the dimer is newly dehydrogenated to phenoxy radical and then can couple with another monomer radical in an end-wise coupling mode. Coupling of two lignin oligomers at the positions 4 and 5 yields diaryl ether (4-O-5′) and diphenyl (5− 5′) lignin subunits formation (Scheme 1). In turn, 5-5′ subunits can undergo α-β-O-4-4′ coupling to dibenzodioxocine units (DBDO).1 Both DBDO and 4-O-5′ coupling modes constitute branching points in lignins. The phenylpropane (C9) units are thus attached to one another by a series of characteristic linkages (β-O-4′, β-5′, β-β′, β-1′, SD, 5-5′, DBDO, and 4-O-5′). Although not exhaustively proven by experimental evidence, lignin has been extensively reported to be a cross-linked network polymer.2 Higuchi in 1993 wrote: “Lignins are aromatic polymers of methoxylated phenylpropanoids”.3 In 2004 Ralph et al. reported that: “Lignins are complex natural polymers resulting from oxidative coupling of, primarily, 4hydroxyphenylpropanoids”.1 Despite the huge amount of studies performed, lignin structure and polymerization degrees are to date not fully understood. This is largely due to lack of suitable analytical tools to reliably and extensively characterize such a complex system.4 From this viewpoint the determination of lignin © 2011 American Chemical Society
Received: July 8, 2011 Revised: September 2, 2011 Published: September 19, 2011 3928
dx.doi.org/10.1021/bm200948r | Biomacromolecules 2011, 12, 3928−3935
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Scheme 1. Lignin Biosynthesis: Coupling Mode for Monolignols and Oligomeric Lignin Chains
Ultrafiltration measurements yielded systematically higher values than vapor osmometry determinations.9 More recently, size exclusion chromatography and light scattering techniques have been employed more frequently. Such techniques showed a large dependence of the detected Mn and Mn values on lignin concentration, nature of the solvent system, pH, and lignin isolation methodology. For this reason, for example, light scattering was used to investigate lignin supramolecular aggregation processes. 17 Zimm plots from using Zimm, Debye, and Berry formalism showed high MW dependence on the lignin solution pH of analysis and significantly change upon liophylization of the samples, thus showing extensive aggregation processes.17 MALDI ToF MS analysis shows interesting peaks also at oligomeric level and gives values comparable to vapor osmometry determination of low Mn.18 Although GPC gives results depending on a number of different factors as for example the fact that UV absorbance detection does not detect aggregation, it is currently the most widespread analytical method currently in use for Mn lignin determinations due to its ease of use and facility of the experimental determinations. Gellerstat critically reviewed this analytical technique applied to lignin.10 One reason for the scarce data reproducibility is the lack of MW standards with a hydrodynamic volume comparable to lignin. The second reason that accounts for the variability of
the results obtained by GPC analysis of lignins is the variety of eluant systems and derivatization processes employed.10 An analytical method suitable for determination of DP of oligomers and small polymers is the end groups titration. It consists in the identification and quantification of a specific polymer end groups. Once the molecular formula of the monomeric unit is known, it is possible to calculate the average DP of the polymer as the ratio between the monomeric units and the end groups. This method, although not applicable to high molecular weight polymers, is completely independent of supramolecular association phenomena and leads to unequivocal determination of average DP and Mn values. NMR spectroscopy is a technique that allows to determine the average degree of polymerization of a polymer avoiding interferences due to supramolecular aggregation, provided the knowledge of monomer formula weight and the possibility to quantitatively integrate the amount of end groups and monomers of the polymer studied. Because of lignin heterogeneity, it is not possible to identify a specific monomer formula weight. However, based on the monomeric composition and more specifically on the relative abundance of G, S, and H groups, the average monomeric lignin subunit can be calculated. Since the average monomeric constituents of MWL, the so-called C9 unit,19 can be easily determined by elemental analysis and determination of the methoxy groups content, the identification and quantitative 3929
dx.doi.org/10.1021/bm200948r | Biomacromolecules 2011, 12, 3928−3935
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Chart 1. Lignin Interunit Bondingsa
a
Phenolic end groups in lignin highlighted in red and aliphatic end groups in blue.
evaluation of lignin end units is sufficient to determine the lignin average DP. During the past years the development of advanced NMR techniques both homo- and hetero-correlated has provided a new tool for the elucidation of lignin structure. More specifically, 31P NMR allows an accurate and reliable quantification of the phenolic OH groups.20 (2D) 13C−1Hcorrelated (HSQC, HMQC) spectroscopy, which combines the sensitivity of 1H NMR with the higher resolution of 13C NMR, continues to be the best method to reveal the frequencies of the different lignin units and the interunit bonding patterns and has allowed their use as a valid analytical technique in the analysis of complex samples.21−25 HSQC experiments allow the identification of lignin structural features that cannot be analyzed by alternative structural analytical techniques such as DBDO and SD interunit bondings.26,27 More recently, the development of quantitative HSQC sequences allowed to quantify different interunit bondings present in lignins.28−30 For example, the QQ-HSQC NMR pulse sequence can be successfully applied to lignin structural elucidation. It provides good resolution and signal-to-noise ratio in a reasonable analysis time. The standard deviations of the results were found ranging from 0.01 to 2 bondings per 100 C9 units.28 Because of MWL complex structure, the determination of its DP and branching cannot be directly accomplished by routine NMR experiments. However, the joint use of quantitative 31P NMR and QQ-HSQC analysis allowed us to develop a new analytical tool for the determination of the average degree of polymerization of lignin that does not suffer from interferences due to polymer branching or supramolecular aggregations. An other accepted paradigma of lignin structure is branching. Lignin branching can be associated with diaryl ether and diphenyl subunits.19 Wet chemistry degradative methods identified both 4-O-5 and 5-5′ bondings in MWL.31−34 Lindberg35 and co-workers considered the lignin molecule in solution to consist of a strongly immobilized, tight network core and a looser surface region. However, to date detailed
studies on lignin branching and their quantitative evaluation have not been reported. This is due mainly to the difficulty of quantitatively evaluate etherified 4-O-5′ and 5-5′ lignin subunits by wet chemistry methods.4 During the 1970s, Miksche and Erikson marginally approached the problem in the frame of a structural study of lignin based on the KMnO4 oxidation protocol. Although this degradative characterization technique is affected by severe experimental errors due to poor mass balance of the finally recovered products and to the occurrence of side reactions, it was possible to show that 4-O-5 etherified subunits were absent or present in low amount.36 Coupled to the spread of advanced NMR techniques, during the past decade lignin structural inquiries have been greatly facilitated by the development of various degradative protocols, such as DFRC (derivatization followed by reductive cleavage) 37 which efficiently cleaves the β-aryl ethers in lignins. The combination of DFRC with quantitative 31P NMR was shown to have significant potential for the determination of arylglycerol−α-aryl ether and other linkages.38,39 In the present work we have developed an experimental protocol aimed at elucidating the nature and quantitative occurrence of branching units in lignin. By combining quantitative data arising from 31P NMR and QQ-HSQC analyses of different softwood and hardwood milled wood lignin samples with data collected after DFRC treatment followed by 31P NMR of the resulting lignin fragments, it was possible to clearly evaluate the degree of branching in MWL.
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RESULTS AND DISCUSSION Toward the Determination of Lignin Degree of Polymerization (DP). The average degree of polymerization is defined as the amount of average monomeric C9 units/ polymeric unit. The amount of monomer units in lignin can be easily determined by elemental analysis and methoxy groups determination according to the Zeisel method.19 In this way it is possible to develop a C9 formula to calculate the average
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Table 2. Average Degree of Polymerization of Different MWL Samples As Evaluated from QQ-HSQC and 31P NMR Analysis lignin sample
chains/100C9
BS-MWL NS-MWL NS-EMAL Beech-MWL
20 12 13 78
± ± ± ±
3 3 3 3
DP
DP max error
5.7 9.4 7.2 12.1
0.7 2.2 2.0 5.9
Table 3. Number-Average Molecular Weight (M n ) Determined for Different MWL Samples by GPC and NMR End-Groups Titration
Figure 1. QQ-HSQC of Norway spruce MWL.
lignin sample
Mn (GPC)
Mn (NMR end-group titration)
NS-MWL NS-EMAL Beech-MWL
14200 7300 9600
1800 1400 2600
Figure 2. 31P NMR of Norway spruce MWL. Figure 3. Possible branching lignin subunits.
amount of monomeric units present in a specific lignin sample. Entry 1 of Table 1 shows the Mw of the average C9 unit in different softwood and hardwood milled wood lignin samples. Nature and Determination of Lignin End Units and Lignin Interunit Bondings. The number of lignin polymeric units is identified by its end groups. The amount of end units in lignin is complex to be determined. We focused our attention on phenolic end units since the aliphatic ones are less characterized and more widespread in an array of different structures, i.e., aldehydes, COOH, and cinnamyl and aliphatic OH groups that cannot be correctly quantified.1,19 If lignin were a linear polymer simply connected by β-O-4 aryl ether and phenyl coumaran (β-5′) interunit linkages, each polymer chain would contain a single phenolic unit (Chart 1A). Phenolic OH groups could be considered as lignin end groups; thus, the amount of phenolic OH would reflect directly the amount of polymer chains. However, since lignin is constituted by a complex array of interunit bondings, the end units of lignin chains can be unequivocally determined only taking into consideration all the relevant interunit bondings.
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Table 4. Lignin Subunits Amount Evaluated by QQ-HSQC, and 31P NMR after DFRC entry 1 2 3 4
5
interunit bonding/C9 unit phenolic 5-5′b (3a) phenolic 4-O5b (1a) DBDOa (2a + 2b) non-phenolic 4-O-5′ (1b) + DBDOc (2a, 2b) etherified 5-5′c (3b)
P NMR,
NS-MWL
NS-EMAL
Beech MWL
0.034 ± 0.005
0.057 ± 0.005
0.005
0.022 ± 0.005
0.016 ± 0.005
