Purification, Structural Characterization, and Modification of

Article pubs.acs.org/JAFC

Purification, Structural Characterization, and Modification of Organosolv Wheat Straw Lignin Laurie Mbotchak, Clara Le Morvan, Khanh Linh Duong, Brigitte Rousseau, Martine Tessier, and Alain Fradet* Sorbonne Universités, UPMC Univ Paris 06, Institut Parisien de Chimie Moléculaire UMR 8232, Chimie des Polyméres, F-75005 Paris, France CNRS, IPCM UMR 8232, F-75005, Paris, France S Supporting Information *

ABSTRACT: Biolignin, a wheat straw lignin produced by acetic acid/formic acid/water hydrolysis, was characterized by 31P and 13 C−1H 2D NMR spectroscopy and by size-exclusion chromatography. Biolignin is a mixture of low molar mass compounds (Mn = 1660 g/mol) made up of S, G, and H units and of coumaric and ferulic acid units. β-5 and β-O-4 interunit linkages are partially acylated in the γ-position by acetate and p-coumarate groups. Deacylated samples with a low content of contaminants were obtained by combining alkaline hydrolysis and solvent extraction. The high phenolic OH content found by 31P NMR reflects the presence of condensed aromatic units, such as 5−5 units. Reaction of purified lignin with ethanol and ethane-1,2-diol yielded esterified lignins much more soluble than Biolignin in common organic solvents. During this reaction, the secondary OH of β-O4 linkages was simultaneously etherified. Phenol hydroxyethylation by 2-chloroethanol yielded samples containing only aliphatic hydroxyl groups. KEYWORDS: wheat straw lignin, NMR, esterification, etherification

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late).25 However, several issues have to be addressed before lignins can find extensive applications in polymeric materials: (1) Technical lignins are rarely recovered in pure form. While the major portion of hemicelluloses is removed during the fractionation step, the presence of residual carbohydrates in crude lignin fractions (i.e., obtained directly after the delignification process) cannot be excluded. Wax and sugar content in a lignin sample are closely related to the process and to the pre- or posttreatment steps used for lignin isolation. Their presence has to be carefully considered as it may influence lignin reactivity and, therefore, the properties of final materials. Treatment of straw by apolar/polar solvents mixtures prior to the fractionation process is used in some cases to produce dewaxed straw.3−5 On the other hand, basic hydrolysis is an efficient way to remove residual polysaccharides by cleavage of ester linkages between hemicellulose and lignin, yielding almost polysaccharide-free technical lignins.4,6,26,27 (2) As mentioned above, the structure of technical lignins depends heavily on the conditions used in the delignification and purification processes. In order to elaborate materials with controlled properties and to ensure synthesis repeatability, an indepth knowledge of lignin structure and reactivity is required. (3) With regard to polymer synthesis, the major drawbacks of technical lignins are their low solubility in organic medium, their functional group heterogeneity, and their relatively poor hydroxyl group accessibility.

INTRODUCTION

Conversion of lignin to value-added products is one of the challenges for the expanding biobased products industry. In this context, the utilization of lignin from grasses, and especially from crop residues, has attracted increasing research attention.1,2 Wheat straw lignin is a major industrial crop residue and its separation, purification, and structure have been extensively studied.3−12 Like lignins from other grasses, wheat straw lignin is a complex polymer formed of three types of aromatic units, syringyl (S), guaiacyl (G), and 4-hydroxyphenyl (H) units, linked to each other by short aliphatic “side chains” of various types through C−C or C−O−C bonds. It presents a relatively high content of acetate and p-hydroxycinnamate groups, predominantly bound at the γ-position of side chains.13 In plants, lignin is linked to polysaccharides, mainly hemicellulose, by ether or ester groups, thus ensuring structural cohesion and rigidity to cell walls. The delignification process, designed to cleave lignin−saccharide bonds, leads to more or less extensive modification of native lignin structure. Therefore, the composition of the resulting technical lignins, for example, the relative abundance of S/G/H units, the nature of side chains, the presence of cinnamate and acetate groups and the content in functional groups (alcohols, phenols, and carboxylic acids), is highly dependent on the delignification process conditions.14 Technical lignins are highly functionalized aromatic compounds and, as such, particularly attractive for use as comonomers in polymeric materials.15 Lignins have thus been used as rigid polyols in polyester,15−18 polyurethane,19−21 and epoxy22−24 thermosetting resins and, after modification, for the synthesis of starlike polystyrene and poly(methyl methacry© 2015 American Chemical Society

Received: Revised: Accepted: Published: 5178

February 11, 2015 May 9, 2015 May 10, 2015 May 11, 2015 DOI: 10.1021/acs.jafc.5b02071 J. Agric. Food Chem. 2015, 63, 5178−5188

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Journal of Agricultural and Food Chemistry

overnight at 50 °C under vacuum to yield 0.75 g of ethanol-soluble modified lignin (s-Lp-Et). Modification of Lignin with Ethane-1,2-diol. Lp (5 g), ethane1,2-diol (75 mL) and p-toluenesulfonic acid monohydrate as a catalyst (530 mg, 0.6 wt %) were heated at 90 °C for 20 h. The homogeneous mixture was poured into 500 mL of deionized water. The precipitate was filtered on sintered glass filter (porosity 5), washed with deionized water until neutral pH, and dried at 50 °C under vacuum for 24 h to give 4.95 g of ethane-1,2-diol-modified lignin (Lp-EG). Modification of Lignin with 2-Chloroethanol. Lp (3.1 g) was dissolved in 30 mL of 1 M aqueous NaOH at 90 °C. 2-Chloroethanol (6 mL, 84 mmol) was added dropwise and the mixture was heated at 90 °C for 20 h. The mixture was poured into 300 mL of water and neutralized with 2 M aqueous HCl. The precipitate was filtered on sintered glass filter (porosity 5) and dried at 50 °C under vacuum for 24 h to give 2.80 g of 2-chloroethanol-modified lignin (Lp-CE). Synthesis of 4-(2-Hydroxyethoxy)cinnamic Acids. 4-(2Hydroxyethoxy)cinnamic acids were synthesized by reacting the corresponding 4-hydroxycinnamic acids with 2-chloroethanol in alkaline medium according to a procedure described for hydroxyethoxylation of 4-hydroxybenzoic acid.35 (2E)-3-[4-(2-Hydroxyethoxy)phenyl]prop-2-enoic Acid, 20. A solution of p-coumaric acid (8.2 g, 50 mmol) in 40 mL of 3 M aqueous NaOH was heated to 50 °C. 2-Chloroethanol (5 mL, 75 mmol) was added slowly, and the mixture was heated at 90 °C for 6 h. After cooling, the mixture was poured into 50 mL of deionized water and acidified with 1 M aqueous HCl. The residue was filtered and washed with acetone to remove residual p-coumaric acid and ester byproducts. Product 20 was recrystallized from ethanol: yield 6.2 g (60%). 1H NMR (DMSO-d6) δ, ppm 7.61 (d, J = 8 Hz, H-2, H-6), 7.54 (d, J = 16 Hz, H-α), 6.96 (d, J = 8 Hz, H-3, H-5), 6.36 (d, J = 16 Hz, H-β), 4.02 (t, J = 6 Hz, −CH2−OAr), 3.72 (t, J = 6 Hz, −CH2−OH). 13C NMR (DMSO-d6) δ, ppm 167.7 (Cγ), 160.3 (C-4), 143.6 (C-α), 129.8 (C-2, C-6), 126.6 (C-1), 116.4 (Cβ), 114.8 (C-3, C-5), 69.6 (−CH2−OAr), 59.4 (−CH2−OH). (2E)-3-[4-(2-Hydroxyethoxy)-3-methoxyphenyl]prop-2-enoic Acid, 21. Compound 21 was synthesized from ferulic acid following the same procedure: yield 5.6 g (47%). 1H NMR (DMSO-d6) δ, ppm 7.51 (d, J = 16 Hz, H-α), 7.31 (d, J4 = 2 Hz, H-2), 7.17 (dd, J4 = 2 Hz, J3 = 8 Hz, H-6), 6.97 (d, J3 = 8 Hz, H-5), 6.44 (d, J = 16 Hz, H-β), 4.00 (t, J = 6 Hz, −CH2−OAr), 3.81 (s, −CH3-O−), 3.72 (t, J = 6 Hz, −CH2−OH). 13 C NMR (DMSO-d6) δ, ppm 167.9 (C-γ), 150.1 (C-4), 149.0 (C-3), 144.1 (C-α), 127.0 (C-1), 122.5 (C-6), 116.6 (C-β), 112.5 (C-5), 110.4 (C-2), 70.1 (−CH2−OAr), 59.4 (−CH2−OH), 55.5 (CH3O−). (2E)-3-[4-(2-Hydroxyethoxy)-3,5-dimethoxyphenyl]prop-2-enoic Acid, 22. A solution of sinapic acid (0.9 g, 4 mmol) in 5 mL of 3 M aqueous NaOH was heated to 50 °C. 2-Chloroethanol (0.65 mL, 9.5 mmol) was added slowly, and the mixture was heated at 90 °C for 6 h. After cooling, the mixture was poured into 20 mL of deionized water, acidified with 1 M aqueous HCl, and extracted with ethyl acetate (3 × 20 mL). Organic phases were washed with brine and concentrated under reduced pressure. Product 22 was recrystallized from ethyl acetate: yield 0.11 g (10%). 1H NMR (DMSO-d6) δ, ppm 7.52 (d, J = 16 Hz, H-α), 7.03 (d, J = 2 Hz, H-2, H-6), 6.53 (d, J = 16 Hz, H-β), 3.90 (t, J = 6 Hz, −CH2−OAr), 3.81 (s, CH3-O−), 3.61 (t, J = 6 Hz, −CH2−OH). 13C NMR (DMSO-d6) δ, ppm 167.6 (C-γ), 153.0 (C-3, C-5), 144.0 (C-α), 138.4 (C-4), 129.6 (C-1), 118.4 (C-β), 105.8 (C-2, C-6), 74.1 (−CH2− OAr), 60.1 (−CH2−OH), 56.0 (CH3O−). Ethyl (2E)-3-[4-(2-Hydroxyethoxy)-3-methoxyphenyl]prop-2enoate, 23. A mixture of 22 (4.02 g, 16.9 mmol), ethanol (60 mL), and 18 M sulfuric acid as a catalyst (310 mg, 0.6 wt %) was refluxed for 20 h. After cooling, the solution was concentrated under vacuum to yield an orange-colored viscous product. This product was dissolved in 100 mL of diethyl ether, and this solution was washed twice with 30 mL of 0.5 M aqueous solution of NaHCO3, twice with 30 mL of saturated NaCl solution, then twice with 30 mL of deionized water. The organic phase was dried on MgSO4, filtered, and evaporated to dryness under vacuum. The resulting white powder was dried at 60 °C under vacuum for 12 h: yield 3.25 g (72%). 1H NMR (DMSO-d6) δ, ppm 7.57 (d, J = 16 Hz, H-α), 7.35 (d, J4 = 2 Hz, H-2), 7.21 (dd, J4 = 2 Hz, J3 = 8 Hz, H-6), 6.98 (d, J3 = 8 Hz, H-5), 6.54 (d, J = 16 Hz, H-β), 4.86 (t, J = 5 Hz,

To overcome these issues, preliminary purification and chemical modification of technical lignins have been described: for example, esterification of hydroxyl groups by anhydrides,28 reaction with epichlorhydrin,22 or reaction with propylene oxide.29−31 While the characterization of oxypropylated lignin with modern methods has been reported,31,32 most modified technical lignins, however, have subsequently been used without detailed characterization. The aim of this study was to evaluate the influence of purification steps and of esterification and etherification chemical modifications of wheat straw lignin on its chemical and structural characteristics. This study was undertaken on wheat straw lignin produced by the CIMV (Compagnie Industrielle de la Matière Végétale) acetic acid/formic acid/water fractionation process,11,33 available under the trade name Biolignin. Lignin was first purified by hydrolysis and solvent extraction and then modified by esterification with ethanol or ethane-1,2-diol to mask acid groups or by etherification with 2-chloroethanol in alkaline conditions to convert phenol groups into more reactive primary alcohols. The modification reactions were expected to provide samples with enhanced solubility, homogenized functionality in terms of function types, and improved reactivity for future applications as comonomer in polymeric materials. These samples were thoroughly characterized by means of 31P and 13C−1H 2D NMR spectroscopy and size-exclusion chromatography (SEC).

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MATERIALS AND METHODS

Materials. Absolute ethanol, ethane-1,2-diol (99.8%), 2-chloroethanol (99%), p-coumaric acid (98%), ferulic acid (99%), sinapic acid (98%), p-toluenesulfonic acid monohydrate (98.5%), sulfuric acid (99%), 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (95%), anhydrous pyridine (99.8%), chromium(III) acetylacetonate (97%), and acetic anhydride (≥99%) were obtained from Sigma−Aldrich (Saint-Quentin-Fallavier, France). N-Hydroxy-6-norbornene-2,3-dicarboximide (>96%) was obtained from Fluka (Saint-Quentin-Fallavier, France). All reagents were used without further purification. Biolignin (Klason lignin content 89.8%)34 was obtained from CIMV (Compagnie Industrielle de la Matière Végétale, Neuilly, France). Wheat straw from Triticum aestivum L. subsp. aestivum, seeded in November 2011, was harvested in Champagne region (France) in July 2012. Biolignin was extracted by the CIMV process with acetic acid/ formic acid/water 30:55:15 v/v/v) at 105 °C under atmospheric pressure.12,33 Lignin Purification. Biolignin (20.19 g) was hydrolyzed by 1 M aqueous NaOH (300 mL) at 80 °C for 4 h. The reaction mixture was then acidified to pH 2 with 3 M aqueous HCl. The precipitate was filtered and washed thoroughly with deionized water until neutral pH, then dried at 50 °C under reduced pressure for 24 h to give 18.13 g of hydrolyzed Biolignin (Lh; yield 90 wt %). The acidic filtrate was concentrated under reduced pressure to afford a dried hydrolysis residue, Rh. Lh was further purified by repeated washing steps with CH2Cl2 (2×) and ethyl acetate (3×) as follows: Lh (17 g) was poured into 100 mL of solvent, heated to reflux for 1 h, and filtered. After drying under reduced pressure, 13.33 g of purified Biolignin (Lp) was obtained (overall yield from Biolignin 70.5 wt %). Organic phases were evaporated to yield the purification residue, Rp. Modification of Lignin with Ethanol. Lp (2 g), ethanol (30 mL), and p-toluenesulfonic acid monohydrate as a catalyst (200 mg, 0.8 wt %) were heated at 80 °C for 20 h. The insoluble fraction was filtered on sintered glass filter (porosity 5), washed with deionized water until neutral pH, and dried overnight at 50 °C under vacuum to give 1.25 g of ethanol-insoluble modified lignin (i-Lp-Et). The alcohol phase was concentrated under reduced pressure. The residue was precipitated in 200 mL of water, filtered on sintered glass filter (porosity 5), and dried 5179

DOI: 10.1021/acs.jafc.5b02071 J. Agric. Food Chem. 2015, 63, 5178−5188

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Journal of Agricultural and Food Chemistry −OH), 4.17 (q, J = 7 Hz, −CH2−OOC−), 4.01 (t, J = 5 Hz, −CH2− OAr), 3.81 (s, CH3−O−), 3.72 (q, J = 5 Hz, −CH2−OH), 1.25 (t, J = 7 Hz, −CH3). 13C NMR (DMSO-d6) δ, ppm 166.4 (C-γ), 150.3 (C-4), 149.0 (C-3), 144.5 (C-α), 126.8 (C-1), 122.8 (C-6), 115.5 (C-β), 112.4 (C-5), 110.5 (C-2), 70.05 (−CH2−OAr), 59.7 (−CH2−OOC−), 59.35 (−CH2−OH), 55.5 (CH3O−), 14.1 (−CH3). Spectroscopic Methods. One-dimensional (1D) 1H and 31P NMR spectra and two dimensional (2D) 13C−1H NMR correlation spectra were recorded on a Avance 500 spectrometer (Bruker, Karlsruhe, Germany), at 500 MHz (1H NMR), 125 MHz (13C NMR), or 202 MHz (31P NMR) with a 5 mm double-resonance broadband inverse probe (BBI). The 1D 13C NMR spectra were recorded on the same spectrometer at 125 MHz with a 10 mm double-resonance broadband observe probe (BBO). Lignin samples were dissolved in deuterated dimethyl sulfoxide, DMSO-d6 (1H, 13C, and 2D 13C−1H NMR), or CDCl3/pyridine mixture (31P NMR). The 2D 13C−1H correlation spectra were recorded through a phasesensitive gradient-enhanced 2D heteronuclear single quantum coherence (HSQC) with echo−antiecho experiment (HSQCETGP sequence). The 2D 13C−1H long-range correlation heteronuclear multiple bond correlation (HMBC) spectra were recorded via heteronuclear zero and double quantum coherence experiment (HMBCGPLPNDQF sequence). Chemical shifts were referenced to residual DMSO-d5 (δ = 2.50 ppm) for 1H NMR and to DMSO-d6 (δ = 39.43 ppm) for 13C NMR. 31 P NMR experiments were carried out following a slightly modified previously reported method:36 Lignin (30 mg) was dissolved in a CDCl3/pyridine mixture (1:1.6 v/v, 0.5 mL). The phosphitylation reagent, 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (150 μL), and the internal standard, N-hydroxy-6-norbornene-2,3-dicarboximide (100 μL of 0.1 M solution in 1:1.6 v/v CDCl3/pyridine mixture)37 were added successively. Chromium(III) acetylacetonate (100 μL of 0.014 M solution) in the same CDCl3/pyridine mixture was added to the solution in order to homogenize and accelerate phosphorus relaxation. Spectra were recorded with a 25 s relaxation time and an average number of 10 000 scans. The quantitative results shown are the average of duplicate NMR experiments. Chemical shifts are relative to the signal of the phospholane hydrolysis product at 132.2 ppm. The integral value of the internal standard was used for calculations of the absolute amount of each functional group. Size-Exclusion Chromatography. SEC analyses were performed on a 300 × 7.5 mm i.d., 5 μm Mixed-C (200−(2 × 106) Å) PLgel threecolumn set (Agilent Technologies, Les Ulis, France), connected to a VE5200 automatic injector (Malvern Instruments Ltd., Guyancourt, France) and a model 515 pump and a model 410 differential refractometer (Waters Corp., Guyancourt, France). Tetrahydrofuran (THF) was used as eluent (1 mL/min). Acetylated lignin solution in THF (100 μL, 5 g/L) was injected. Chromatograms were processed by use of OmniSEC software. Polystyrene standards (Polymer Laboratories) were used for the calibration. Lignin Acetylation. Lignin (220 mg) was reacted with acetic anhydride (5 mL) and pyridine (5 mL) at room temperature for 72 h.38 The resulting mixture was poured in water (100 mL). The precipitated acetylated lignin was filtered and dissolved in CH2Cl2. The solution was dried over MgSO4 and evaporated under reduced pressure to afford acetylated lignin (210 mg, 95% yield).

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RESULTS AND DISCUSSION Purification and Characterization of Biolignin. Biolignin was first hydrolyzed in 1 M aqueous NaOH solution for 4 h at 80 °C, leading to hydrolyzed Biolignin (Lh). This alkaline hydrolysis leads to the cleavage of ester bonds, including those involved in carbohydrate−lignin complexes, and allows the elimination of residual polysaccharides and esters together with some aromatic compounds.27 Moreover, traces of acetic and formic acids present in the starting material and resulting from the delignification process are also removed. In order to remove water-insoluble impurities, such as low molar mass aromatic

Figure 1. 2D 13C−1H HSQC NMR spectra of (A) Biolignin, (B) Lh, and (C) Lp: expansion of the lignin side-chain domain. Structure notations are given in Figure 3 and discussed in main text.

compounds and waxes, Lh was then extracted with dichloromethane and ethyl acetate, yielding a final, purified Biolignin (Lp) with highly reduced contaminant content. The purification steps were followed by 1H NMR. Analysis of the hydrolysis and purification residues confirmed the elimination of polysaccharides and of fatty acids, p-coumaric acid, and ferulic acid, 5180


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Figure 2. 2D 13C−1H HSQC NMR spectra of (A) Biolignin, (B) Lh, and (C) Lp: expansion of the aromatic domain. Structure notations are given in Figure 4 and discussed in main text.

Figure 3. Structures, atom numbering, and HSQC correlations (δC/δH, ppm) of interunit linkages found in wheat straw lignin (R1−4 = H, CH3O; R′ = H, CO).

in Biolignin, as shown by the correlations corresponding to their β- and γ- positions, the latter being more intense (Figure 1A). These two correlations are no longer present after hydrolysis in Lh and Lp spectra (Figure 1B,C). α-Oxidized alkyl−aryl ether structures, 3, are also detected by a correlation corresponding to the β-position. Correlations corresponding to the α-, β-, and γpositions of β-5 (phenylcoumaran), 4, and β−β (resinol) linkages, 5, are of much lower intensity as compared to those

respectively, together with various low molar mass aromatic compounds. 2D 13C−1H HSQC NMR Analyses of Biolignin, Lh, and Lp. 2D 13 C−1H HSQC NMR analyses were performed in DMSO-d6 (Figures 1 and 2). Correlations were assigned according to literature (Figures 3 and 4).7,27,38−40 The β-O-4 linkages (alkyl− aryl ether), 1, of H, G, and S units are easily detected by their α, β, and γ resonances. γ-Esterified β-O-4 linkages, 2, are also present 5181


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Figure 4. Structures, atom numbering, and 2D 13C−1H HSQC NMR correlations (δC/δH, ppm) of aromatic units found in wheat straw lignin.

of β-O-4, which, therefore, seem to be predominant. However, it is difficult to conclude on their relative abundance, since the NMR sequence used for these analyses does not allow cross-peak quantitation. In the aromatic domain (Figure 2A), the resonances of H, G, and S units are observed together with moieties derived from pcoumaric and ferulic acids. As previously reported in studies on wheat straw lignin,41 sinapic acid derivatives are not present, since their characteristic signal at 106.1/6.98 ppm (2,6positions) is not detected. In Biolignin, the presence of free pcoumaric and ferulic acids 7 and 9, and of their esters 8 and 10, is reflected by their 2,6-, α- and β-correlations. A cross-peak close to

Figure 5. Structure of model 4-(2-hydroxyethoxy)cinnamic derivatives.

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Table 2. Number- and Mass-Average Molar Masses and Molar-Mass Dispersities of Acetylated Lignin Samplesa

a

sample

Biolignin

Lp

i-Lp-Et

s-Lp-Et

Lp-EG

Lp-CE

Mw (g/mol) Mn (g/mol) ĐM

31890 1660 19.2

18400 1560 11.8

24270 2100 11.6

4160 1340 3.1

23520 1765 13.3

4633 1155 4.0

Determined by size-exclusion chromatography.

are not detected in Biolignin spectra. The acidic conditions of the CIMV process probably lead to the elimination of this type of compound. 31 P NMR Spectroscopy. 31P NMR spectroscopy was used to differentiate and quantitate the different types of free hydroxyl groups (aliphatic, phenolic, and carboxylic) in Biolignin and Lp, after phosphitylation by 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane, as described by Argyropoulos and co-workers.8,36 N-Hydroxy-5-norbornene-2,3-dicarboximide was used as internal standard instead of cyclohexanol,37 as its signal at 151.9 ppm is well separated from those of lignin. It should be noted that the un-, mono-, and disubstituted 4-hydroxyphenyl groups quantitated by this method correspond only to a fraction of the H, G, and S units present in lignin, that is, those bearing free phenol groups. Moreover, the disubstituted 4-hydroxyphenyl structures detected by this method comprise syringyl units with free phenol groups, as well as condensed phenolic units.36 There are two main differences between the spectra of Biolignin and Lp (Figure 6): (1) lower relative intensity of unconjugated carboxylic acid resonances with respect to conjugated ones (134.70 and 135.05 ppm,27 respectively) in the spectrum of Lp, due to the elimination of fatty acids and residual acetic and formic acids during the hydrolysis and purification steps; and (2) disappearance of the sharp peaks emerging from broad signals in the phenolic OH region of Biolignin spectrum (un-, mono-, and disubstituted 4-hydroxyphenyl groups at 137−144 ppm). The well-resolved peaks at 137.80 and 139.40 ppm are assigned to p-coumaric and ferulic acids, respectively.27 Aliphatic, phenolic, and acidic hydroxyl contents were determined by integration of the corresponding peaks with respect to the internal standard (Table 1). In Biolignin, phenolic OH groups are preponderant, with the aliphatic/phenolic OH ratio rOH being close to 0.8, and acidic OH groups represent about 10% of total OH content. Monosubstituted 4-hydroxyphenyl OH unit content is slightly higher than that of disubstituted units (mono/disubstituted = 1.2), while unsubstituted 4-hydroxyphenyl units represent ca. 15% of total phenolic groups. Hydrolysis and purification lead to a slight increase in total OH content (6.90 vs 5.61 mmol/g) without

Figure 6. 31P NMR spectra of (A) phosphitylated Biolignin and (B) phosphitylated purified Biolignin Lp. (*) Impurity; (**) internal standard; (a) disubstituted p-hydroxyphenyl structure as syringyl and condensed phenolic units; (b) monosubstituted p-hydroxyphenyl structure as guaiacyl units; (c) unsubstituted p-hydroxyphenyl structure.

the 6-correlation of 9 and 10 was assigned to p-etherified ferulic acid 11, with the help of a model of these moieties, namely, (2E)3-[4-(2-hydroxyethoxy)-3-methoxyphenyl]prop-2-enoic acid 21 (Figure 5). Low molar mass aromatic derivatives are also detected: syringic acid 14, vanillin 15, and syringaldehyde 16. Correlations at 122.9/7.54, 112.8/6.78, and 149.1/8.10 ppm were assigned to the CH groups of furaldehyde 18, a product of polysaccharide degradation, by comparison with the spectrum of an authentic sample. The CHO correlation of 18 was also observed at 178.3/9.61 ppm (region not shown). After hydrolysis (Figure 2B), p-coumarates and ferulates 8 and 10 are no longer detected, but free acids 7, 9, and 14 and aldehydes 15 and 16 are still present, and a correlation corresponding to acetosyringone 17 appears. These low molar mass compounds are removed during the solvent extraction steps, and the spectrum of Lp (Figure 2C) exhibits a very simplified pattern consisting mainly of signals relative to H, G, and S units and to p-etherified ferulic acid (11). As found for other lignin types,13 p-etherified ferulates are an integral part of the lignin backbone. These findings were confirmed by the 2D NMR study of hydrolysis and solvent extraction residues. Milled wood (MW) wheat straw lignin is also reported to contain flavonoids, such as tricin [5,7-dihydroxy-2-(4-hydroxy3,5-dimethoxyphenyl)-4H-chromen-4-one], which presents strong and well-resolved HSQC signals.7 However, these signals

Table 1. Hydroxyl Content of Biolignin, Lp, Lp-Et, Lp-EG, and Lp-CE Ligninsa OH content (mmol/g)

OH content (% of total OH + COOH)

sample

phenolic

aliphatic

COOH

(a)b

(b)b

(c)b

aliphatic

COOH

Biolignin Lp s-Lp-Etc Lp-EG Lp-CEd

2.84 3.49 3.12 2.08

2.24 2.57 1.13 2.67

0.53 0.84 0.04 0.04

19.40 22.50 32.40 20.0 3.30

23.35 21.60 30.70 17.40 3.40

7.85 6.50 9.50 6.00 0.90

39.95 37.25 26.30 55.70 87.80

9.45 12.15 1.10 0.90 4.60

a Determined by quantitative 31P NMR after phosphitylation with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane. b(a) Di-, (b) mono-, and (c) unsubstituted 4-hydroxyphenyl OH, respectively. cEthanol-soluble fraction of Lp-Et. dOH content (millimoles per gram) was not determined since Lp-CE is not completely soluble in phosphitylation medium. OH content (percent) was calculated on the soluble fraction.

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Figure 7. 2D 13C−1H HSQC NMR spectra of (A) s-Lp-Et and (B) Lp-EG: expansion of the lignin side-chain domain. Structure notations are given in Figure 8. EG, residual ethane-1,2-diol.

hydrolysis of p-coumarates present in lignin structure. A similar p-coumarate hydrolysis during the acidic CIMV delignification process could also be involved to explain the low unsubstituted 4hydroxyphenyl unit content of Biolignin. Size-Exclusion Chromatography. The impact of the hydrolysis and purification steps on average molar masses and molar mass distributions was evaluated by SEC (Table 2). The samples were acetylated, in order to improve their solubility in THF and to avoid the formation of aggregates.46 Biolignin is a mixture of oligomeric compounds (Mn = 1660 g/mol) exhibiting a very large molar-mass dispersity, close to ĐM = 19. The hydrolysis and purification steps do not affect molar mass since the Mn of Lp lies in the same range. The molar-mass dispersity of Lp is lower, but still quite high (ĐM = 11.8). The Mn values previously reported for various technical wheat straw lignins (1000−3780 g/mol)7,9,12,44 are close to those found for Biolignin in this work, while literature ĐM values vary between 1.79 and 10.6.44 However, it is difficult to compare these SEC values, since delignification and/or analysis conditions are different from each other. In this work, molar mass values are compared before and after chemical modification, in order to detect potential lignin backbone changes. Modification of Purified Biolignin. Modification of lignin prior to its use as macromonomer presents many advantages. Besides improving sample solubility in common organic solvents, this could allow selective masking of some functions and homogenizing of reactive group types. Lp was modified by three methods: esterification by (i) ethanol or (ii) ethane-1,2-diol to mask acid groups and (iii) etherification with 2-chloroethanol under alkaline conditions to replace phenol groups by more reactive aliphatic OH groups. Lignin Modification by Esterification. Both esterification reactions were catalyzed by p-toluenesulfonic acid. When esterifications were carried out in ethanol, the reaction mixture remained heterogeneous. After reaction, the insoluble fraction, iLp-Et, was isolated from the soluble fraction, s-Lp-Et, by filtration. Both samples were soluble in DMSO-d6 and were analyzed by 1H and 2D NMR spectroscopy. Spectra with very

much affecting the aliphatic/phenolic ratio and, therefore, lignin main structure. Although free aliphatic and cinnamic acids were removed by the hydrolysis and purification steps, COOH concentration increases, due to hydrolysis of the ester functions of 4-O-etherified ferulates. The high total phenolic group content indirectly reflects the presence of condensed phenolic units, such as 5−5 units, 6 (Figure 3). Shoulders at ca. 143 and 142 ppm in the 31P NMR spectra of phosphitylated lignins have been assigned to condensed phenolic structures.36 However, it is difficult to make any accurate quantitation in this region, even by deconvolution, due to the unknown number and width of signals that overlap in this region. Condensed phenolic units might also be detected in the aromatic region of HSQC spectra. On the basis of NMR literature data on model compounds,42,43 the 5−5 structure arising from coniferyl alcohol should give two signals (2- and 6-positions), close to the 2- and 6-correlations of G units. Unassigned correlations at 109.5/7.10 and 119.4/6.95 ppm are observed in the HSQC spectrum of Lp (Figure 2C). They could reflect the presence of 5−5 units, but this has yet to be confirmed. Comparison of 31P NMR literature data on technical wheat straw lignins highlights the importance of the fractionation method on the chemical composition of resulting lignin samples. When either bases, for example, soda wheat straw lignin (GreenValue SA),32 or organic acids44 are used, lignins samples exhibit aliphatic/phenolic OH ratio rOH ≤ 1, close to that found for Biolignin in the present work. On the other hand, rOH is close to 2.4−3 for MW wheat straw lignin8 and dioxane/HCl lignin45 samples. The un/mono/disubstituted 4-hydroxyphenyl unit distribution is also process-dependent, with MW samples exhibiting higher unsubstituted unit content as compared to samples obtained by alkaline or acidic processes. Crestini and Argyropoulos8 studied the effect of 2 M alkaline hydrolysis on MW wheat straw lignin. They observed a strong decrease in unsubstituted 4-hydroxyphenyl unit content, reaching values close to that found above for Biolignin and Lp. They showed that this was mainly due to release of p-coumaric acid by the basic 5184


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Figure 8. Structures, atom numbering and HSQC correlations (δC/δH, ppm) of modified lignin units found in Lp-Et, Lp-EG, and Lp-CE (R1−4 = H, CH3O; R′ = aliphatic chain).

were also confirmed by comparisons with the spectra of ethyl (2E)-3-[4-(2-hydroxyethoxy)-3-methoxyphenyl]prop-2-enoate 23, a model of p-etherified ethyl ferulate groups (Figure 5). The HSQC NMR spectrum also reveals that the secondary alcohols of β-O-4 structures were simultaneously etherified: The α-CH signal of β-O-4 linkages (1-α) is shifted to 79.70/4.48 ppm after reaction (24-α), that is, in the secondary ether resonance region. Moreover, new correlations are detected at 63.7/3.32 ppm (Figure 7A) and 14.80/1.08 ppm (not shown), which correspond, respectively, to methylene and methyl of the ethyl ether group of 24. Etherification of the secondary alcohol of β-O4 linkages has already been reported for Miscanthus giganteus lignin obtained by the ethanol−water−HCl process.47 Similar results were obtained when the reaction was carried out with ethane-1,2-diol. The −COO−CH2−CH2−OH meth-

similar profiles were obtained, so that only the spectra of s-Lp-Et will be further discussed. On the other hand, esterification with ethane-1,2-diol led very rapidly to a homogeneous reaction mixture and afforded a sample, Lp-EG, that was soluble in common organic solvents such as CH2Cl2, THF, and DMSO. The aliphatic domains of s-Lp-Et and Lp-EG HSQC NMR spectra are presented in Figure 7 with assignments in Figure 8. The spectrum of s-Lp-Et exhibits two distinct signals at 59.3/4.04 and 59.4/4.16 ppm (Figure 7A), assigned to the methylenes of ethyl ester groups of nonlignin aliphatic esters 25, and of petherified ethyl ferulate moieties 26, respectively. This was confirmed by long-range correlations between the corresponding carbonyl carbons and the methylene protons of ethyl esters at 172.45/4.04 and 166.25/4.16 ppm, respectively, observed in the HMBC 13C−1H NMR spectrum of s-Lp-Et. These assignments 5185


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unaffected by the chemical modification. A decrease in the aliphatic/phenolic OH ratio is observed for s-Lp-Et (rOH = 0.36), due to formation of ethyl ethers 24. In the case of Lp-EG, the aliphatic/aromatic OH ratio increases (rOH = 1.28). In this case, the formation of hydroxyethyl ethers 27 does not change total aliphatic hydroxyl group content, while the hydroxyethylation of 11 leads to etherified hydroxyethyl ferulates 29 and to hydroxyl group concentration increase. SEC analysis shows that s-Lp-Et exhibits lower Mn (1340 g/ mol) and dispersity (ĐM = 3.1) than Lp, while the Mn of the ethanol-insoluble fraction is slightly higher (2100 g/mol) and dispersity is similar (ĐM = 11.6). Lp and Lp-EG present very close Mn and molar-mass dispersity (Table 2). Cleavage of lignin β-O-4 linkages and their rearrangement into lignin-based polymeric materials was reported for beech MW lignin liquefaction with ethane-1,2-diol.48 The experimental hydroxyethylation conditions used in the present work (0.6% ptoluenesulfonic acid, 90 °C) are significantly milder than those used for wood liquefaction (3% p-toluenesulfonic acid, 150 °C) and appears to leave β-O-4 linkages unchanged, with the exception of the α-hydroxyethoxylation reaction discussed above. Lignin Modification by Etherification with 2-Chloroethanol. Etherification of Lp phenol groups with 2-chloroethanol under alkaline conditions yields a modified lignin, Lp-CE, that is insoluble in CH2Cl2 and only partially soluble in DMSO. Lp-CE characterization was, therefore, carried out on the soluble fraction. Analysis of the HSQC aliphatic domain (Figure 10A) shows that phenol groups have reacted, since three different signals are observed at 69−74/3.8−4.0 ppm, assigned to the Ar− O−CH2−CH2−OH methylene of 2-hydroxyethoxy groups in the 4-position of H, G, and S aromatic rings (Figure 8). Assignments of the resulting etherified units H′, G′, and S′ were made with the help of model compounds 20, 21, and 22 (Figure 5) obtained by reaction of coumaric, ferulic, and sinapic acids with 2-chloroethanol in similar conditions. The −CH2−OH methylenes of these groups and of β-O-4 linkages (1-γ) give overlapping cross-peaks. During the reaction, carboxylic acids are partly esterified into hydroxyethyl esters since signals corresponding to ferulate (29-9 and 29-10) and aliphatic esters (28-9 and 28-10) are also detected. On the other hand, β-O-4 aliphatic secondary hydroxyl groups remain unchanged, since no signal corresponding to aliphatic ethers (24-α) is detected at 79.7/4.48 ppm. Etherification of phenol groups by 2-chloroethanol is reflected by new correlations at 112.55/6.86 (G′-5) and 113.90/ 6.84 ppm (H′-3,5) (Figure 10B). Lp-CE was also analyzed by 31P NMR (Figure 9). Since the sample was not fully soluble in phosphitylation medium, quantitative determination of OH groups was not possible. The relative aliphatic/aromatic OH content was nevertheless determined on the soluble fraction, showing an almost 7-fold decrease in phenol content, with unchanged ratio of un-, mono-, and disubstituted 4-hydroxyphenyl units. Hydroxyethylation by 2-chloroethanol in basic medium, therefore, appears to be a very efficient modification method of lignin phenol groups. Since Lp-CE was only partially soluble in THF, even after acetylation, the SEC analyses were performed on the soluble fraction after filtration, leading to lower molar mass (Mn = 1155 g/mol) and dispersity (ĐM = 4.0) than Lp, as expected for a fractionated polymer sample. Finally, we can conclude that alkaline hydrolysis (1 M NaOH at 80 °C) and solvent extraction of Biolignin yields a purified, deacylated lignin sample (Lp) with very low content of

Figure 9. 31P NMR spectra of phosphitylated lignin samples: (A) Lp, (B) s-Lp-Et, (C) Lp-EG, and (D) Lp-CE. (*) Impurity; (**) internal standard; (a) disubstituted p-hydroxyphenyl structures as syringyl and condensed phenolic units; (b) monosubstituted p-hydroxyphenyl structures as guaiacyl units; (c) unsubstituted p-hydroxyphenyl structures.

Figure 10. 2D 13C−1H HSQC NMR spectra of Lp-CE: expansion of lignin (A) aliphatic and (B) aromatic domains. Structure notations are given in Figure 8. EG, residual ethane-1,2-diol.

ylenes of non-lignin aliphatic esters 28 and ferulates 29 are clearly identified at 65.20/4.01 and 65.25/4.14 ppm (Figure 7B). As discussed above in the case of modification by ethanol, αetherified β-O-4 structures 27 are also observed. No noticeable differences were observed in the aromatic domain, except a slight shift of the α- and β-resonances of feruloyl moieties after reaction, 29. Comparison of Lp, s-Lp-Et, and Lp-EG 13C NMR spectra confirmed the preceding conclusions, in particular with the 2 ppm upfield shift of −COO− resonances (esterification of acids) and the detection of new peaks assigned to −CH2−O− ether resonances of 24 and 27 at 63.7 and 70.1 ppm, respectively. The 31P NMR spectra (Figure 9) show that, as expected, carboxylic acid content strongly decreases, from 0.84 mmol/g in Lp to 0.04 mmol/g in s-Lp-Et and in Lp-EG (Table 1). The ratio of un-, mono-, and disubstituted 4-hydroxyphenyl units remains 5186


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(2-hydroxyethoxy)-3,5-dimethoxyphenyl unit; MW, milled wood

contaminants and that the purification does not significantly affect starting lignin main structure and molar masses. Lp contains primary and secondary alcohol groups, carboxylic acid groups, and phenolic OH groups. The large phenolic OH content reflects the existence of condensed aromatic structures, such as units of 5−5 type, which bring two additional phenolic OH groups per lignin molecule. Modification of Lp by esterification with ethanol or ethane1,2-diol in the presence of p-toluenesulfonic acid catalyst was efficient and, as expected, led to esterified lignin samples (Lp-Et and Lp-EG, respectively) with a very low carboxylic acid content. In these conditions, the secondary alcohols were also totally etherified, but no β-O-4 linkage cleavage was observed, leading to modified lignin containing only primary alcohols and phenols. With ethane-1,2-diol, simultaneous hydroxyethoxylation of secondary OH groups during COOH esterification resulted in an increased aliphatic OH/phenolic OH ratio with respect to LpEt. Etherification of phenolic OH groups with 2-chloroethanol yielded lignins containing only aliphatic alcohol groups. These modification reactions (ethylation, hydroxyethylation, ethoxylation) allow control of the nature and content of OH groups and, therefore, of the chemical functionality of lignins. This is a prerequisite for their use as comonomers in polycondensation reactions, where stoichiometry is a key parameter (e.g., polyesterifications), or as initiators in ringopening polymerizations and, after further tailored modification, in controlled radical polymerizations. The purified and modified lignins described in this work are, therefore, interesting macromonomers for the preparation of well-defined polymers or polymer blends.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra of Biolignin, Lh, Lp, Rh, and Rp; 2D 13C−1H HSQC NMR spectra of Rh and Rp side-chain and aromatic domains; 2D 13C−1H HMBC NMR spectrum of Rp; 2D 13 C−1H HMBC NMR spectrum of s-Lp-Et; and 13C NMR spectra of Lp, s-Lp-Et, and Lp-EG. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b02071.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Telephone +33-144273804; fax +33-144277089; e-mail alain. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Bouchra Benjelloun-Mlayah and Professor Michel Delmas (CIMV) for lignin samples and for helpful discussions. ABBREVIATIONS Lh, hydrolyzed Biolignin; Lp, purified Biolignin after hydrolysis and solvent extraction; Rh, hydrolysis residue; Rp, solvent extraction residue; Lp-Et, reaction product of purified Biolignin with ethanol; s-Lp-Et, ethanol-soluble fraction of Lp-Et; i-Lp-Et, ethanol-insoluble fraction of Lp-Et; Lp-EG, reaction product of purified Biolignin with ethane-1,2-diol; Lp-CE, reaction product of purified Biolignin with 2-chloroethanol; G, guaiacyl unit; H, 4hydroxyphenyl unit; S, syringyl unit; G′, 4-(2-hydroxyethoxy)-3methoxyphenyl unit; H′, 4-(2-hydroxyethoxy)phenyl unit; S′, 45187


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