Efficient separation and physico-chemical characterization of lignin from eucalyptus using ionic liquid–organic solvent and alkaline ethanol solvent

Industrial Crops and Products 47 (2013) 277–285

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Efficient separation and physico-chemical characterization of lignin from eucalyptus using ionic liquid–organic solvent and alkaline ethanol solvent Yong-Chang Sun a , Ji-Kun Xu a , Feng Xu a , Run-Cang Sun a,b,∗ a b

Institute of Biomass Chemistry and Technology, Beijing Forestry University, Beijing 100083, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China

a r t i c l e

i n f o

Article history: Received 9 January 2013 Received in revised form 12 March 2013 Accepted 15 March 2013 Keywords: Eucalyptus Lignin Ionic liquid Organic solvent HSQC

a b s t r a c t Ionic liquid (IL) 1-butyl-3-methylimidazolium acesulfamate ([BMIM]Ace)-organic solvents mixtures were utilized in the dissolution and extraction of lignin from eucalyptus. The resulting carbohydrateenriched materials were subsequently extracted with 70% ethanol containing 1 M NaOH. The results showed that the yield of alkaline ethanol lignin (AEL, 8.0–23.3%) was relatively higher than that of IL–organic solvents lignin (IOL, 11.5–15.4%, based on the Klason lignin content). The chemical structures of the lignin fractions obtained were characterized by carbohydrate analysis, gel permeation chromatography (GPC), Fourier transform infrared (FT-IR), and 2 D HSQC nuclear magnetic resonance (NMR) spectroscopy. The IOL fractions exhibited a larger molecular weight (Mw , 9050–6590 g/mol) and a higher polydispersity compared to the AEL fractions (Mw , 3820–2440 g/mol) except for IL–dioxane lignin (Mw , 1005 g/mol). Moreover, 2 D NMR spectra of ILL and AEL fractions showed a predominance of ˇ-O4 aryl ether linkages (77.4 and 79.0% of total side chains, respectively), followed by ˇ–ˇ resinol-type linkages (12.1 and 13.8%), and lower amounts of ˇ-5 (4.2 and 3.3%) and spirodienone linkages. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Lignocellulosic biomass is hypothesized to be a sustainable source of low-carbon biofuels capable of displacing a significant fraction of current petroleum supplies. However, lignocellulose is a porous micro-structured composite mainly consisting of cellulose, hemicelluloses and lignin, and these complex and rigid structure of the biopolymers makes it highly resistant to biological and chemical degradation (Yang and Wyman, 2008). Lignin has a high degree of aromaticity, and can be used to produce fuels and bulk chemicals (Alonso et al., 2010; FitzPatrick et al., 2010). Many pretreatment strategies have been developed to isolated lignin with a high yield. These processes included several physical and chemical pretreatment methods such as steam explosion (Cara et al., 2008), dilute acid (Lloyd and Wyman, 2005), hot water (Mosier et al., 2005), ammonia fiber expansion (Murnen et al., 2007), and organic solvent (Holtzapple and Humphrey, 1984). Recently, a new technology for pretreatment of biomass using ionic liquids (ILs) has been explored. ILs is a molten salt composed

∗ Corresponding author at: Institute of Biomass Chemistry and Technology, Beijing Forestry University, Beijing 100083, China. Tel.: +86 10 62336903; fax: +86 10 62336903. E-mail address: [email protected] (R.-C. Sun). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.03.025

of organic cations and anions with low melting temperature (<373 K) and unusual solvent properties. ILs can effectively dissolve cellulose and wood at moderate temperatures to a homogeneous solution (Heinze et al., 2005; Zavrel et al., 2009). ILs pretreatment has been shown to reduce the cellulose crystallinity, the hemicelluloses, and lignin content of biomass, and to increase its surface area, enzymatic hydrolysis kinetics, and the yield of fermentable sugars (Fort et al., 2007; Samayam and Schall, 2010; Sun et al., 2009). Wood, agricultural waste, grass can be dissolved in certain ILs. The dissolution of wood using 1-n-butyl-3-methylimidazolium chloride ([C4 mim]Cl) (Fig. 1a) has also been reported (Fort et al., 2007; Kilpelainen et al., 2007), only a part of wood can be dissolved at certain particle size and temperature. The dissolution requires the virtual absence of water, which necessitates extensive drying of the wood and ionic liquid under inert atmosphere. Sun et al. (2009) reported softwood (southern yellow pine) and hardwood (red oak) can be completely dissolved in the IL 1-ethyl-3-methylimidazolium acetate ([C2 mim]OAc) (Fig. 1c) after mild grinding. It was shown that ([C2 mim]OAc) is a better solvent for wood, increasing basicity of the acetate anion, which makes it more efficient at disrupting the inter- and intramolecular hydrogen bonding in biopolymers than Cl− . The drawbacks were long dissolution time (48 h) and relatively high content of carbohydrate. It has been reported that high amounts of lignin was removed after IL treatment, resulting in cellulose in wood flour to be hydrolyzed by cellulose

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Fig. 1. Structures of the ILs mentioned in this article: (a) 1-butyl-3-methylimidazolium chloride ([C4mim]Cl); (b) 1-ethyl-3-methylimidazolium xylenesulfonate ([C2mim][XS]); (c) 1-ethyl-methylimidazolium acetate ([C2mim]OAc); (d) 1-butyl-3methylimidazolium acesulfamate ([Bmim]Ace).

(Lee et al., 2009). An ionic liquid 1-ethyl-3-methylimidazolium xylenesulfonate [C2mim][XS] (Fig. 1b) was used to extract lignin from bagasse at high temperatures (170–190 ◦ C) and atmospheric pressure, with remarkable lignin extraction yields of up to 93% (mass fraction) (Tan et al., 2009). However, the high recovered lignin mass was due to the reaction of xylenesulfonate anions and lignin. The other polymeric products, which commonly produced from the reaction of the degraded polysaccharides (such as glucose and fructose) were also reacted with lignin (Tan et al., 2009). Pinkert et al. (2011) investigated 1-butyl-3methylimidazolium acesulfamate ([Bmim]Ace) for separating wood lignin. This IL can be readily synthesized from the sugar acesulfamate potassium (Ace K), and with good property of thermal stability, increased viscosities, and inability to dissolve crystalline cellulose. In addition, it is of low cost, commercial available, and nontoxic. Unfortunately, only a few studies have focused on the comparative study of the structural difference and physico-chemical properties of the lignin extracted by ILs and other solvents. Consequently, in this study, we present a more extensive study of the lignin extracted by the imidazolium acesulfamate IL [Bmim]Aceorganic solvents and alkaline ethanol solvent, aiming to determine the effect of ILs-organic solvents on lignin extraction and comparatively study the structural features of the lignin obtained. The chemical properties of the lignin fractions were elucidated by gel permeation chromatography (GPC), carbohydrate analysis. Fourier transform infrared (FT-IR), and heteronuclear single

quantum coherence (HSQC) NMR spectroscopes were also used to characterize the lignin structures. These findings were important for the industries of value-added utilization of lignin. 2. Methods 2.1. Materials and chemicals Eucalyptus is an important species for pulpwood production, and can be used in essential oil industry. Eucalyptus was received from Guangdong province, China. The stems were chipped into small pieces (1–3 cm) and drying at 60 ◦ C for 16 h in an oven. The chips were ground using a mortar for 30 min and sieved through a 40–60 mm mesh. The sawdust (50 g) was then milled using a planetary ball mill (Yuan et al., 2011). The chemical composition of the wood flour was analyzed according to the analytical procedure of the National Renewable Energy Laboratory (NREL/TP-510-42618, TP-510-42622) (Sluiter et al., 2005, 2008), US Department of Energy. The contents of acid-insoluble lignin, acid-soluble lignin, and ash were 30.1 ± 0.3%, 2.4 ± 0.6%, and 1.2 ± 0.5%, respectively. IL 1-n-butyl-3-methylimidazolium chloride ([C4 mim]Cl) (≥99%, purity) was purchased from Chemer, Hangzhou, China. The sugar acesulfamate potassium (Ace K) known as food additive was purchased from the Suzhou Hope Technology Co., Ltd., China. IL 1-butyl-3methylimidazolium acesulfamate ([Bmim]Ace) was synthesised according to the literature (Tan et al., 2009). The typical

Fig. 2. The scheme for the process of extracting wood lignin with IL–organic solvents mixtures.

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Fig. 3. Isolation procedure for alkaline ethanol lignin (AEL) from the carbohydrate-enriched materials (CEMs).

procedure for preparation of [Bmim]Ace was followed by Pinkert et al. (2011). The chemicals used were of analytical or reagent grade and directly used as purchased without further purification.

The lignin fraction obtained was named alkaline ethanol extracted lignin (AEL). 2.3. Characterization of extracted lignin

2.2. Isolation of lignin Fig. 2 shows the flowchart scheme for extracting lignin from wood flour with IL–organic solvents mixtures. The extraction method was described in a previously report (Pinkert et al., 2011). In the dissolution trail, 2.0 g of ground wood was added to 30.0 g of IL. The mixture was then placed into an oil bath and heated at 120 ◦ C for 3 h. At the same time, organic solvents (10 mL) were added to the mixture. Afterwards, the solution was filtered by gentle vacuum filtration, and the undissolved wood particles were obtained from the hot mixture. The filtrate above was evaporated to remove water and poured into a beaker filled with 30 mL of anhydrous acetone, and then the flocculent precipitate was formed. The precipitated IL–organic solvents lignin (IOL) was separated by centrifugation. The IL was recovered after distillation of acetone and organic solvents under reduced pressure. Each experiment was done in duplicate. The residues were carbohydrate-enriched materials (CEMs), which will be extracted by the alkaline ethanol solvent for the subsequent delignification. The extraction yield of lignin was calculated according to Eq. (1): Lignin yield (%) =

mel × 100% mail

(1)

where mel is defined as the content of the extracted lignin (after purification), mail is the content of acid-insoluble lignin of the eucalyptus. As shown in Fig. 3, the dried CEMs were further extracted with 70% ethanol containing 1 M NaOH at 70 ◦ C for 3 h. The isolation and purification were achieved by the methods of Sun et al. (2003).

The neutral monosaccharide components of the lignin fractions, CEMs, and residues were analyzed by hydrolysis with dilute sulfuric acid according to the procedure described in a previous paper (Sun et al., 2003). The weight-average (Mw ) and number-average (Mn ) molecular weights of the lignin fractions were determined by gel permeation chromatography (GPC) with a refraction index detector (RID) on a PL aquagel 10 mm Mixed-B 7.5 mm ID column according to a previous report (Sun et al., 2010). The Fourier-transform infra-red (FT-IR) spectra of the samples were recorded using a Thermo Scientific Nicolet iN10-MX FT-IR chemical imaging microscope (Thermo Scientific, America) fitted with a narrow-band liquid nitrogen cooled MCT detector. Spectra were recorded with 64 scans at a resolution of 4 cm−1 between the wave numbers of 4000 and 800 cm−1 . The HSQC experiments were conducted according to a previous study (Sun et al., 2010). HSQC cross-signals were assigned by comparing with some literatures (Villaverde et al., 2009; Lu and Ralph, 2003; Rencoret et al., 2009). 3. Results and discussion 3.1. Lignin extraction efficiency Fig. 4 shows the yield of lignin extracted from eucalyptus with IL–organic solvents mixtures and alkaline ethanol solvent. The mass of recovered lignin was calculated without the sugar content based on the Klason lignin content of the untreated biomass. It is obvious that the use of organic solvents played an important role in the lignin extraction. The maximum lignin

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Y.-C. Sun et al. / Industrial Crops and Products 47 (2013) 277–285 Table 1 Neutral sugars and uronic acids composition (±0.2%,a % dry sample, w/w) of the lignin fractions isolated by ionic liquid–organic solvents and fresh IL. Sugar

Arabinose Galactose Glucose Xylose Mannose Uronic acids Total

Lignin fraction ILL-1

IOL-2

IOL-3

IOL-4

IOL-5

1.7 5.1 2.8 7.6 2.9 4.3 24.4

0.2 2.2 2.0 2.4 0.7 1.8 9.3

1.4 6.2 3.2 8.7 2.8 3.8 26.1

3.6 8.5 7.5 12.2 3.6 4.5 39.9

1.7 5.2 3.1 10.3 2.9 5.6 28.8

a The average standard deviation of the fresh ionic liquid lignin (ILL) and ionic liquid–organic solvent lignin (IOL).

Fig. 4. Yield of lignin released in the treatment with IL–organic solvents mixtures and alkaline ethanol solvent.

extraction corresponds to a recovered lignin mass of 15.4% with the IL–toluene mixture at 120 ◦ C for 3 h. By contrast, the fresh IL extraction released 11.5% of lignin. Results indicated that the extraction efficiency order was IL–toluene > IL–dioxane > IL–ethyl acetate > IL–N,N-dimethylacetamide (DMAc) > fresh IL. It was proved that small amounts of some organic solvents can increase the extraction efficiency. The reasonable explanation for the behavior of the co-solvents may owe to the following reason. The conductivity of the co-solvents was different owing to the different dielectric constants (ε) of the organic solvents (Table A.1 in the Supplementary data). As the higher dielectric constant results in the higher critical aggregation concentration of the IL (Li et al., 2006), which means larger aggregates of the IL exist in the mixtures. As a consequence, the anion becomes less delocalized, thus weakening nucleophilic attack on the ether carbon atoms of lignin by acesulfamate anion. However, this hypothesis needs further investigation. Moreover, the decrease of the overall viscosity of the mixture could increase the interaction of wood with the IL. After extracted with IL–organic solvents mixtures, the CEMs obtained were extracted with alkaline ethanol solvent for the subsequent delignification. The alkaline treatment of lignocelluloses has been reported to cleave the alkali-labile linkages between lignin and carbohydrates, such as ester bonds between lignin and/or hemicelluloses (Spencer and Akin, 1980). As shown in Fig. 4, in general, the yield of lignin (8.0–23.3%) by alkaline ethanol extraction was higher than the IL–organic solvents treatments (11.5–15.4%). High yield of lignin AEL-3 (23.3%, w/w, based on the amount of Klason lignin) was obtained from the CEMs which were pretreated by IL–dioxane mixture. However, the AEL-1 (8.0%) was lower than other fractions, corresponding to the CEMs pretreated by the fresh IL. Results indicated that IL–organic solvents mixtures have the advantage of causing the cell wall to swell up to a large content beyond the swelling in the fresh IL. 3.2. Lignin purity analysis An analysis of the carbohydrate content and composition of the extracted lignin has been carried out by acid-hydrolysis before lignin purification. Tables 1 and 2 suggest the monosaccharide of pentose and hexose, as well as uronic acids (mainly glucuronic acid) of both the IOL and AEL fractions. The IOL fractions contained associated carbohydrate ranging from 9.3% to 39.9%. Xylose,

galactose, and glucose were the major sugars in the IOL fractions, indicating that hemicelluloses were associated with the IOL fractions. IL–DMAc extraction resulted in low amounts of carbohydrate (9.3%). By comparison, the IL–toluene and IL–ethyl acetate mixtures released high amounts of carbohydrate, amounting to 28.8 and 39.9%, respectively. The fresh IL extraction released 24.4% of carbohydrate associated with the lignin fractions. Moreover, small amounts of arabinose and mannose were detected in all the IOL fractions. However, all the total sugar contents are lower than the carbohydrate reported in the literature (Tan et al., 2009; Pinkert et al., 2011). Interestingly, the amount of glucose was almost the same of xylose in the IL–DMAc lignin fraction. This suggested that glucan may be degraded in the extraction mixture. Xylose was the major sugar in all IOL fractions, indicating that hemicelluloses were degraded in the processes. The carbohydrate content of the AEL fractions was relatively lower than that of the IOL fractions. As shown in Table 2, low content of total carbohydrate was ranged from 1.0 to 13.0%. In addition, xylose (0.2–7.2%) and glucose (0.1–1.3%) were demonstrated to be the two main sugars in the lignin fractions. However, AEL4 and AEL-5, which was isolated from the CEMs after extraction of IL–ethyl acetate and IL–toluene, had very low carbohydrate content (1.2% and 1.0%, respectively). This phenomenon might be explained by the cleavage of alkali-labile linkages between lignin and carbohydrates during alkali treatment, especially the ester linkages between lignin and hemicelluloses. 3.3. Molar mass distribution of the extracted lignin The weight-average (Mw ) and number-average (Mn ) molecular weights of the lignin fractions were calculated from the GPC curves. The polydispersity (Mw /Mn ) of the IOL, ILL, and AEL fractions are illustrated in Table 3. As shown in Table 3, the IOL fractions exhibited high Mw except for the IL–dioxane lignin (Mw , 1005 g/mol), ranging from 6590 to 9050 g/mol. The low Mw of IL–dioxane lignin farction indicated that small molecules of lignin were extracted. The IL–DMAc lignin has a higher molecular weight (9050 g/mol) Table 2 Neutral sugars and uronic acids composition (±0.2%,a % dry sample, w/w) of the lignin fractions isolated by alkaline ethanol solvent. Sugar

Arabinose Galactose Glucose Xylose Mannose Uronic acids Total a

Lignin fraction AEL-1

AEL-2

AEL-3

AEL-4

AEL-5

0.6 1.5 0.7 3.7 0.4 0.5 7.4

1.5 1.7 1.3 7.2 0.6 0.7 13.0

0.7 0.8 0.6 1.9 0.1 0.4 4.5

0.1 0.1 0.1 0.3 0.5 0.1 1.2

0.1 0.1 0.3 0.2 0.1 0.2 1.0

The average standard deviation of the alkaline ethanol lignin (AEL) fractions.

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Table 3 Weight-average (Mw ), number-average (Mn ) molecular weights and polydispersity (Mw /Mn ) of the ILL, IOL, and AEL fractions. Lignin fraction

Mw Mn Mw /Mn

ILL-1

AEL-1

IOL-2

AEL-2

IOL-3

AEL-3

IOL-4

AEL-4

IOL-5

AEL-5

3700 720 5.14

2560 1730 1.48

9050 1160 7.80

3590 1520 2.36

1005 850 1.18

3820 890 4.29

7315 870 8.41

3395 765 4.44

6590 1170 5.63

2440 760 3.21

and polydispersity (7.80) than other lignin fractions. Moreover, the lignin fraction (ILL-1) which was extracted with fresh IL has a low Mw (3700 g/mol) and Mn (720 g/mol), indicated that the addition of organic solvent could result in relatively large molecular of lignin fraction. Table 3 shows the molecular weights and polydispersity of the lignin fractions extracted from the CEMs by alkaline ethanol solvent. The results represent a more uniform and small value of molecular weights (Mw , 2440–3820 g/mol) and narrow polydispersity (1.48–4.44). The AEL-5 showed a low molecular weight (2440 g/mol), suggesting that the IL–toluene pretreatment could access to the cell wall to a large content. The molecular weight distribution curves showed that the ionic liquid–organic solvents lignin fractions have a relatively high and wide peak in the area of high molar mass distribution except for IOL-3, indicating a large lignin fragments of IOL structure (Fig. A.1 in the Supplementary data). The low molecular weights of AEL fractions indicated that the ether bond between the large lignin fragments might break down under alkaline conditions. Clearly, the molecular weights of IOL fractions were higher than that of AEL fractions. This result was also probably due to the reactions between lignin and hemicelluloses breakdown products. These were in good agreement with the previous report, which demonstrated that the carbohydrate linked to lignin can increase the molar mass of the lignin measured by GPC (Jääskeläinen et al., 2003). This was in line with the results of carbohydrate analysis as shown in Tables 1 and 2. In addition, organic solvents could affect the molecular weight and polydispersity of the lignin fractions. Similar effects have also been observed by Qi et al. (2008).

in conjugated p-substituted aryl ketone in lignin molecules. The bands at 1462–1457 cm−1 might arise from the asymmetric bending in C H deformation of methyl and methylene groups. Syringyl and condensed guaiacyl absorptions were obviously observed at 1313 cm−1 , whereas guaiacyl ring breathing with C O stretching appears at 1263 cm−1 (Faix and Beinhoff, 1988). The absorption bands at 1122 and 853 cm−1 in these spectra indicated that all lignin fractions are GSH-type lignins (Faix, 1991). Moreover, the peaks at 1358–1030 and 853–827 cm−1 are due to C O(H) stretching of first order aliphatic OH and ether groups and aromatic C H out of plane bending, respectively (Tejado et al., 2007; Xiao et al., 2001). Interestingly, the bands at 1170–1164 cm−1 were observed in the spectra of IOL the fractions; whereas they were absent in the AEL fractions. This indicates the presence of sulfur, suggesting that Ace anion was potentially incorporated in the lignin fractions. The band at 1164 cm−1 can be assigned to symmetric SO2 stretching (Tan et al., 2009). The different intensity was probably due to the chemical environment of the extraction mixture. The obvious shoulder absorption bands at 1152 cm−1 are attributed to the association of xyloglucan. In IOL spectra, the xyloglucan absorption is probably assigned at 1164–1177 cm−1 . The absorption at 1055 cm−1 is assigned to the C O C mode of the pyranose ring (Kaˇcuráková et al., 2000). In addition, the band at 1023–1036 cm−1 is indicative of the aromatic C H in-plane deformation. The strong intensity of the peak at 938 cm−1 is probably due to the C N stretch of the IL incorporated. All those peaks indicated that lignin was successfully extracted from eucalyptus with IL–organic solvents mixtures and alkaline ethanol solvent, and the wide and intensive peaks of AEL than IOL indicating a pure and high yield of lignin.

3.4. FT-IR spectra 3.5. NMR spectra FT-IR spectra of the IOL and AEL fractions are compared in Fig. 5. It was clear that the spectra of IOL and AEL were distinct in terms of locations and intensities of the absorption bands. All the spectra are dominated by a wide band at 3396–3381 cm−1 owing to aliphatic and phenolic OH-groups, followed by bands for C H stretching in methyl and methylene groups (2962–2874 cm−1 ). In the spectra of IOL fractions, the bands at 1570, 1502, and 1437 cm−1 are attributed to the aromatic skeletal vibrations, which are characteristic peaks of lignin. The absorptions at 1739 cm−1 are assigned to C O stretching of unconjugated ketone, carbonyl, and ester groups, indicating the presence of hydroxycinnamates, such as p-coumarate and ferulate (Sun et al., 2000). However, these absorption bands were almost disappeared in AEL fractions. The fresh IL extracted lignin (ILL-1) has a high intensity of this band, suggesting the increased content of hydroxycinnamates in the isolated lignin fraction. Ferulate esterified with an arabinosyl unit in hemicelluloses was attached to lignin with an ether bond to form a carbohydrates–ferulate–lignin bridge structure (Sun et al., 2001). The obvious differences indicated that the IL–organic solvents extraction mixtures could not disrupt this bridge structure thoroughly, while the alkaline ethanol extraction could cleave the linkages significantly. This was in accordance with the results obtained from the GPC analysis, which indicated lower molecular weights for AEL than IOL fractions. Another reason might be the high content of carbohydrate in the IOL fractions than that in the AEL fractions, which could affect the absorption bands of lignin. Absorption bands at 1652 cm−1 are from C O stretch

To explore the detailed structures of these lignin fractions, lignin fractions were characterized by HSQC NMR techniques. The IOL and ILL fractions exhibited similar structural features and contents of lignin linkages. The typical lignin fraction ILL-1 was characterized by HSQC NMR techniques for comparison with AEL fractions. The spectra show three regions corresponding to aliphatic, side-chain, and aromatic 1 H–13 C correlations. The aliphatic (nonoxygenated) region shows signals with no structural information and therefore is not discussed here. The side chain (ıH /ıC 2.7–5.1/47–91) and the aromatic (ıH /ıC 6.0–7.5/99–126) regions of the HSQC spectra of these lignin fractions are shown in Fig. 6. The main lignin crosssignals assigned in the HSQC spectra are listed in Table 4, and the main substructures are depicted in Fig. 7. In the side chain of the two lignin fractions, cross-signals of methoxyls (ıH /ıC 3.74/56.2) and side chain in ˇ-O-4 aryl ether linkages were the most prominent. The H␥ –C␥ correlations in ˇO-4 structures were observed at ıH /ıC 3.98/63.5 (structure A and A ). Moreover, a strong correlation of H␥ –C␥ of structure A and A was observed in Fig. 6c than in Fig. 6a. The H␥ –C␥ correlations of structure A was observed at ıH /ıC 3.48/60.1. This confirmed that both the lignin fractions from eucalyptus were highly acylated at the -carbon in ˇ-O-4 aryl ether linkages of side chains. It can be observed that the ILL fraction was relatively high acylated at the carbon in ˇ-O-4 aryl ether linkages of side chains than AEL fraction. This result is in accordance with FT-IR analysis. These differences

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Fig. 5. FT-IR spectra of lignin: (a) the IL–organic solvents lignin (IOL) and fresh IL–lignin (ILL) fractions; (b) alkaline ethanol lignin (AEL) fractions.

Table 4 Assignment of main lignin 1 H–13 C cross-signals in the HSQC spectra of the lignin fractions. Labels

ıH /ıC (ppm)

Assignment

B␤ –OMe A␥ (A , A  )␥

3.13/54.1 3.74/56.2 3.48/60.1 3.98/63.5

B␥ (A, A , A )␣

4.18/71.6 and 3.82/71.6 4.89/72.3

H␤ –C␤ in ˇ–ˇ (resinol) substructures (B) H–C in methoxyls H␥ –C␥ in ˇ-O-4 substructures (A) H␥ –C␥ in -acetylated ˇ-O-4 substructures (A /A  ) H␥ –C␥ in ˇ–ˇ (resinol) substructures (B)

C␥

3.42/63.2

B␣ A␤(S)

4.64/85.4 4.22/85.8 and 4.03/86.4 6.72/104.7 7.28/106.7



S2,6 S 2,6 G2 G5 G6



6.96/111.7 6.80/113.3 and 6.76/115.1 6.89/119.6

H␣ –C␣ in ˇ-O-4 substructures linked to a S unit (A/A /A  ) H␥ –C␥ in ˇ-5 (phenylcoumaran) substructures (C) H␣ –C␣ in ˇ–ˇ (resinol) substructures (B) H␤ –C␤ in ˇ-O-4 substructures linked to a S unit (A) H2,6 –C2,6 in syringyl units (S) H2,6 –C2,6 in C␣ -oxidized (C␣ O) phenolic syringyl units (S ) H2 –C2 in guaiacyl units (G) H5 –C5 in guaiacyl units (G) H6 –C6 in guaiacyl units (G)

might be due to the different isolation methods used for preparing the lignin fractions. The signals for the H␣ –C␣ correlations in the structures of A, A , and A was observed at ıH /ıC 4.89/72.3, while this signal was small in ILL fraction (Fig. 6c). In addition, the H␤ –C␤ corresponding to the erythro and threo forms of the S-type ˇ-O-4 substructures can be distinguished at ıH /ıC 4.22/85.8 and 4.03/86.4, respectively. Besides ˇ-O-4 ether structures, the other linkages such as ˇ–ˇ (resinol, B) was also observed. Signals for resinol substructures B was observed with their H␣ –C␣ , H␤ –C␤ , and the double H␥ –C␥ correlations at ıH /ıC 4.64/85.4, 3.13/54.1, and 3.82 and 4.18/71.6, respectively. However, the signal of H␣ –C␣ of B substructure could only be seen at lower contour levels in the spectrum of ILL fraction (Fig. 6c). Phenylcoumaran substructures C were also found in these lignin fractions, which were observed with H␥ –C␥ correlation at ıH /ıC 3.42/63.2, whereas the H␤ –C␤ correlations were overlapped with other signals. In addition to these linkages, the signals from ˇ-D-Xylp were evidently noted, with its H2 –C2 , H3 –C3 , and H4 –C4 correlations at ıH /ıC 3.12/73.1, 3.35/47.2, and 3.58/75.8, respectively. Its corresponding anomeric correlation (H1 –C1 ) was found at ıH /ıC 4.18/102.9 (data not shown). As shown, more signals corresponding to the sugar can be observed in Fig. 6c than in Fig. 6a, indicating that lower amounts of monosaccharides linked

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Fig. 6. Side-chain (top row) and aromatic regions (bottom row) in the 2D HSQC NMR spectrum, ıH /ıC 2.7–5.1/47–91 and ıH /ıC 6.0–7.5/99–126, respectively: (a and b) alkaline ethanol lignin (AEL); (c and d) ionic liquid lignin (ILL). See Table 4 for signal assignment.

to the AEL fractions. The ionic liquid–organic solvents extraction released more monosaccharides which attached to lignin fragment. This result observed by HSQC herein was also well consistent with these obtained in sugar analysis. In the aromatic regions of the HSQC spectra of ILL and AEL fractions (Fig. 6b and d, respectively), the signals for syringyl (S) and guaiacyl (G) lignin units could be observed. The S-lignin units showed a prominent signal for the H2,6 –C2,6 correlation at ıH /ıC 6.72/104.7. While in C␣ -oxidized S units (S ), the H2,6 –C2,6 correlation was observed at ıH /ıC 7.28/106.7. Moreover, G-units showed different correlations for H2 –C2 , H5 –C5 , and H6 –C6 at ıH /ıC 6.96/111.7, 6.80/113.3 and6.76/115.1, and 6.89/119.6, respectively. The small signals from p-hydroxyphenyl (H) could also be observed at higher contour levels (not shown). The different structural features of these lignin fractions were quantitatively investigated by S/G ratios. The S/G ratios obtained from the HSQC spectra were estimated to be 3.25 and 1.20 for AEL and ILL fractions, respectively. Evidently, the AEL fraction has a higher S/G ratio than the ILL fraction. This result potentially suggested that the lignin fragment rich in S-units was easily to be released under alkaline ethanol extraction. By contrast, the ionic liquid isolation released almost same amount of S- and G-units. In addition, the relative amounts of ˇ-O-4 linkage (A, A , A ) in AEL (79.0%) was higher than that

of ILL fraction (77.4%). The ˇ–ˇ substructure (B) appeared to be secondary major substructure, comprising about 13.8% and 12.1%, respectively. In addition, the ˇ-5 substructure (C) was calculated as minor amounts (3.3 and 4.2%, respectively). Moreover, trace amount of spirodienone was observed in the spectra when increase the contour line. 3.6. Effect of ionic liquid recycling To achieve environmentally friendly biomass processing, the recovery and reuse of ILs is one of the main challenges. The recycling experiments were performed at 120 ◦ C for 3 h with 2.0 g of ground wood added to 30 g of [Bmim]Ace. It was easily recycled with the yield of average yield over 94.6%. Clearly, the colorless IL became reddish brown after recycling up to three times. The coloration of the IL was probably due to the exposure to elevated temperatures and contaminants present in [Bmim]Ace, which could be degraded from IL and lignocellulosic biomass as well as the carbohydrate content (Li et al., 2010; Liebner et al., 2010). The results in Fig. A.2 (in the Supplementary data) showed a slightly decrease of lignin yield by recycled IL. In order to check the IL s purity, the 1 H NMR spectra of fresh and recycled [Bmim]Ace was shown in Fig. A.3 (in the Supplementary data). It

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Fig. 7. Main classical and acetylated substructures, involving different side-chain linkages, and aromatic units identified by 2D NMR of alkaline ethanol lignin (AEL) and ionic liquid–organic solvent lignin (IOL): (A) ˇ-O-4 linkages; (A ) ˇ-O-4 linkages with acetylated -carbon; (A  ) ˇ-O-4 linkages with p-coumaroylated -carbon; (B) resinol structures formed by ˇ–ˇ , ˛-O-  and -O-˛ linkages; (C) phenylcoumarance structures formed by ˇ-5 and ˛-O-4 linkages; (H) p-hydroxyphenyl unit; (G) guaiacyl unit; (S) syringyl unit; (S ) oxidized syringyl unit bearing a carbonyl group at C␣ (phenolic). See Table 4 for signal assignment.

was found that there was no obvious difference between the fresh and recycled IL. Moreover, the effect between the degraded IL and biomass components in the recycled IL should be further investigated.

4. Conclusions In this study, it could be concluded that the low-cost and environmental benign method using ionic liquid–organic solvents mixtures and alkaline ethanol solvent were effective to separate lignin from eucalyptus. It was found that the presence of organic solvent in IL can swell up the cell wall to a large content and increase the extraction efficiency of lignin. Especially, IL–toluene mixture resulted in a high yield of lignin (15.4%). Lignin extracted with alkaline ethanol solvent has a relatively low molecular weight (Mw , 3820–2440 g/mol) and carbohydrates (1.0–13.0%). HSQC results indicated that the AEL fraction has a higher S/G ratio (3.25) than the ILL fraction (1.20). In addition, IL could be recycled and reused easily with high yield.

Acknowledgements The authors are extremely grateful for the financial supports from the Specific Programs in Graduate Science and Technology Innovation of Beijing Forestry University (no. BLYJ201213), Major State Basic Research Projects of China (973, 2010CB732204), National Natural Science Foundation of China (31110103902) and China Ministry of Education (111).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop. 2013.03.025.

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