Structural characterization and isolation of lignin and hemicelluloses from barley straw

Industrial Crops and Products 33 (2011) 588–598

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Structural characterization and isolation of lignin and hemicelluloses from barley straw Xiao-Feng Sun a,b,∗ , Zhanxin Jing a , Paul Fowler b , Yaoguo Wu a , M. Rajaratnam c a b c

Department of Applied Chemistry, College of Science, Northwestern Polytechnical University, Xi’an 710072, China Biocomposites Centre, University of Bangor, Gwynedd LL57 2UW, United Kingdom Division of Pharmaceutical Chemistry, Faculty of Pharmacy, FIN-00014, University of Helsinki, Finland

a r t i c l e

i n f o

Article history: Received 11 October 2010 Received in revised form 1 December 2010 Accepted 8 December 2010 Available online 8 January 2011 Keywords: Barley straw Treatment NMR Hemicelluloses Lignin

a b s t r a c t Structural characterization and isolation of lignin and hemicelluloses from crops are very important for industrial utilization. In this paper, the sequential treatments of barley straw using 90% dioxane, 80% acidic dioxane, 100% dimethyl sulfoxide, and 8% NaOH released total 93% of original lignin and 87% of original hemicelluloses. The extractions with acidic dioxane and dimethyl sulfoxide produced the original hemicelluloses and high-condensed lignin mainly from the middle lamella. FT-IR and NMR analyses show that the hemicelluloses of barley straw contain acidic arabinxylans as the major polysaccharides, which are substituted by ␣-l-arabinofuranose, 4-O-methyl-glucuronic acid, acetyl group (DS = 0.13), and xylose at O-3 and/or O-2 of xylan, and lignin contains ␤-O-4 as a predominant interunit linkage with high amounts of ␤-5 and ␤-1 . The guaiacyl and syringyl units are more etherified, and the proportion of erythro-␤-O-4 is slightly higher than that of threo-␤-O-4 in the lignin of barley straw. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Fractionation and bioconversion of agricultural residues have received much attention because of their important application such as efficient conversion of biomass to biofuels. Energy consumption has increased steadily over last century as the world population has grown and more countries have become industrialized (Sun and Cheng, 2002). There is a shortage of crude oil for many countries such as US, China, and Japan etc. However the bioconversion of corn into biofuel resulted in a serious problem, what will people eat in the future? Agricultural residues bring the world another route to solve this problem, but the efficient fractionation and bioconversion of agricultural residues must build on the information of the structures of agricultural residues. Structural characterization of original lignin and hemicelluloses become very important for better understanding of chemical process and bioconversion. However it is a challenge to isolate both original lignin and hemicelluloses from agricultural residues such as barley straw (Buranov and Mazza, 2008). A variety of methods have been developed in an attempt to isolate and identify lignin and hemicelluloses and, in some cases, to assess how their struc-

∗ Corresponding author at: Department of Applied Chemistry, College of Science, Northwestern Polytechnical University, You Yi West Road 127, Xi’an 710072, China. Tel.: +86 29 88431672; fax: +86 29 88431672. E-mail address: [email protected] (X.-F. Sun). 0926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2010.12.005

tures influence cell wall hydrolysis by chemical or enzymes. One of the most suitable methods for the extraction of original lignin is that proposed by Björkman (1956), based on extensive grinding of plant material in a non-swelling liquid. The lignin preparations obtained by this method, also referred to as milled wood lignin (MWL) or milled straw lignin (MSL), have largely been considered as the basic material for performing most chemical and biological studies upon this complex polymer (Terron et al., 1996). However the Björkman procedure gives low yields and ignores the extraction of original hemicelluloses. Hemicelluloses, the second most common polysaccharides in nature, represent 30–40% of agricultural residues. Characterization of original hemicellulose is very important study in its bioconversion into biofuel. Isolation of hemicelluloses is usually accomplished by extraction with alkali after delignification or directly with alkaline peroxide (Sun et al., 2003). However, hemicelluloses from graminacous plants contain 1–2% of O-acetyl groups and a small amount of uronic acid as well as phenolic acids substituted by ester and ether linkages, which are accessible to action with dilute acids and alkali (Puls and Schuseil, 1993). In previous studies (Sun et al., 2005, 2010, in press), we have developed methods to extract the original hemicelluloses and lignin from wheat straw and maize stem. In this study, the original hemicelluloses and lignin of barley straw were isolated by using 90% dioxane, 80% acidic dioxane, 100% dimethyl sulfoxide, and 8% NaOH. Structure of the lignin and hemicellulosic fractions isolated was studied using a combination of several destructive and non-

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589

Dewaxed sample (15.0g) Ball milling treatment for 4 days Ball-milled sample Extraction with 90% dioxane at 85 °C for 3 h

Residues Filtrate (H 1 and L1) Extraction with 80% dioxane using 0.05 M HCl at 75 °C for 3 h.

Filtrate (H2 and L2)

Residues

Extraction with dimethyl sulfoxide at 85 °C for 3 h Residues Filtrate (H3 and L3) Extraction with 8% NaOH at 50 °C for 3 h

Residues

Filtrate (Separation for hemicelluloses and lignin) Neutralization with 6 M HCl to pH 5.5, concentration at reduced pressure, and then precipitation in 3 volumes of ethanol

Filtrate

Pellet Washed with 70% ethanol and air dried. Hemicelluloses (H4) Evaporation of ethanol, concentration, and precipitation of lignin at pH 1.5, adjusted by 6M HCl Solid Washed with acidified water (pH 2.0) and then freeze-dried Acid-insoluble lignin (L4) Fig. 1. Scheme for extractions of original hemicelluloses and lignins from barley straw.

destructive techniques. Advanced NMR techniques such as HSQC NMR, 13 C NMR and 1 H NMR were employed for structural characterization of hemicelluloses and lignin. 2. Experimental 2.1. Plant materials Barley straw was obtained from the Silsoe Research Institute (Silsoe, Bedfordshire). All weights and calculations were made on oven-dried material (60 ◦ C, 16 h). The composition (w/w) of the straw used was cellulose 37.5%, hemicelluloses 37.1%, lignin 15.8%, protein 2.6%, wax 2.6%, and ash 4.2% on a dry weight basis (Sun and Sun, 2002). All chemicals used were of analytical or reagent grade.

a rotary mill for a period of 4 days. Ball milled substrate was directly suspended in dioxane–water (90:10, v/v) with a ratio of solid to liquid of 1:20 and refluxed under nitrogen at 85 ◦ C for 3 h. The resulting mixture was filtered and collected. The solid residue was washed with fresh dioxane. After drying in a vacuum oven at 60 ◦ C overnight, the solid residue was then treated with acidic dioxane–water (80:20, v/v) with 0.05 mol/L HCl and refluxed at 85 ◦ C for 3 h, and then solid residue and filtrate were collected. In order to extract original hemicelluloses, the collected solid residue was treated with 100% DMSO at 85 ◦ C for 2 h. The final step is the treatment with 8% NaOH solution at 50 ◦ C for 3 h, and the alkali treatment favors the separation of residual hemicelluloses and lignin from barley straw. The four steps were used for the extraction of hemicelluloses and lignin from all cell walls of barley straw. The scheme for sequential treatments of barley straws is illustrated in Fig. 1.

2.2. General experimental procedures 2.2.1. Sequential treatments of barley straw Barley straw (15 g, dry material) was ground in a 4.5 L porcelain jar with the addition of 5 mL toluene. The jar was placed on

2.2.2. Separation of hemicelluloses and lignin The four filtrate solutions collected from each treatment, was neutralized to pH 5.5 with 6 M HCl, and evaporated nearly to dryness. Then water (20 mL) was added into each. The solubi-

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lized hemicellulosic fractions were isolated by precipitation of each water solution in three volumes of 95% ethanol. After filtration, the isolated hemicellulosic fractions were thoroughly washed with 70% ethanol and then dried in a vacuum oven. After evaporation of ethanol, the lignin fractions were obtained from corresponding supernatants by reprecipitation at pH 1.5 adjusted with 6 M HCl. The isolated lignin preparations were purified by washing with acidified water (pH 2.0). The scheme for separation of hemicelluloses and lignins from the filtrate solution is also illustrated in Fig. 1. All the yields of hemicelluloses and lignins represent the mean of at least triplicate analyses. 2.3. Characterization of lignin and hemicellulosic preparations To determine the neutral sugar composition, the hemicellulosic preparations (H1 , H2 , H3 , and H4 ) and lignin preparations (L1 , L2 , and L3 ) were hydrolyzed with 2 M trifluoroacetic acid at 120 ◦ C for 2 h. The hydrolysates were reduced, acetylated and analyzed as their alditol acetates by gas chromatography (GC) according to the method of Blakeney et al. (1983). The chemical composition of phenolic acids and aldehydes, liberated from alkaline nitrobenzene oxidation of the lignin and hemicellulosic fractions at 180 ◦ C for 2.5 h, were determined by Hewlett-Packard 1050 HPLC system with UV detector on a Hichrom H5ODS column (250 mm × 4.6 mm, Phenomenex Co., England). The individual compounds were detected at 280 nm by computer comparison of retention times and peak areas with the authentic phenolics (Lawther et al., 1995). The content of uronic acids in hemicellulosic preparations was determined colorimetrically using the 3-phenylphenol reagent according to the procedure outlined in a previous paper (Lawther et al., 1995). Analysis method of the molecular weights of hemicellulosic and lignin preparations by GPC was described in the previous paper (Lawther et al., 1995). FT-IR spectra were obtained on a FT-IR spectro-photometer (Nicolet 510) with KBr discs containing 1% finely ground samples. 13 C and 1 H NMR spectra of hemicellulosic and lignin samples were recorded on a Bruker Avance 500 MHz spectrometer from 200 mg and 80 mg of sample dissolved in DMSO-d6 (1.0 mL), respectively. The HSQC (Heteronuclear Single Quantum Coherence) NMR spectrum was acquired by applying a 90◦ pulse width, a 0.2 s acquisition time, a 2.0 s pulse delay, and 1 JC–H of 150 Hz. In particular, 30,000 scans were used for the acquisition of 13 C NMR spectra of hemicellulosic and lignin preparations. 3. Results and discussion 3.1. Yields and chemical composition As shown in Table 1, the treatment of ball milled barley straw with 90% dioxane–water solution for 3 h at 85 ◦ C solubilized 2.1% lignin and 4.0% hemicelluloses (% dry matter), respectively. The Table 1 Yields of the hemicellulosic and lignin fractions (% dry matter) solubilized during the treatments of barley straw. Fractions

Table 2 The content of the neutral sugars (relatively % dry weight, w/w) in the isolated hemicellulosic and lignin preparations. Sugars (%)

Yield (%) Hemicellulose (H)

Fractions (H1 and L1 ) solubilized by treatment with 90% dioxane Fractions (H2 and L2 ) solubilized by treatment with 80% acidic dixane Fractions (H3 and L3 ) solubilized by treatment with 100% DMSO Fractions (H4 and L4 ) solubilized by treatment with 8% NaOH Total

high yield of 8.7% lignin and 12% hemicelluloses was obtained by following extraction with 80% acidic dioxane–water solution, respectively. This indicated that both dioxane treatments lead to the substantial dissolution of both lignin and hemicelluloses from the cell walls. The two-stage treatments with both dioxane and acidic dioxane yielded 10.8% original lignin from barley straw (% dry matter). The relatively low yield of 7.4% hemicelluloses and 1.5% lignin was obtained by the extraction with 100% DMSO, and the final alkali treatment resulted in the dissolution of 8.7% hemicelluloses and 2.4% lignin. This sequential treatment totally released 93% of original lignin and 87% of original hemicelluloses from barley straw. The data on neutral sugar composition in the recovered hemicellulosic fractions (H1 , H2 , H3 , and H4 ) and lignin preparations (L1 , L2 , L3 , and L4 ) are given in Table 2. Obviously, the hemicellulosic fraction (H1 ) extracted by using 90% dioxane contains arabinose (16.5%), xylose (15.7%) and glucose (35.2%) as main sugar constituents. The arabinose to xylose ratio in hemicellulosic fraction (H1 ) is 1.05. However, a relatively high quantity of glucose (35.2%) indicates that there are some mixed-linked glucans associated with xylans, and these polysaccharides are easily extracted due to their location in cell walls, particularly in the primary cell wall and middle lamella, indicating that the glucans are less or not associated with lignins. Xylose is a predominant sugar constituent in three hemicellulosic preparations (H2 , H3 and H4 ), comprising 64.6%, 64.1% and 58.2% of the total sugars, respectively, while arabinose (10.4%, 9.7% and 11.5%) and glucose (9.3%, 7.0% and 16.3%) present in smaller amounts. Galactose, rhamnose, and mannose were observed as minor sugar constituents in four hemicellulosic fractions. In addition, as can be seen in Table 2, all the hemicellulosic fractions contained small proportions of uronic acids, ranging between 7.3 and 10.1%. This monosaccharide analysis reveals that the three hemicellulosic fractions (H2 , H3 and H4 ) contain acidic arabinoxylans as the major polysaccharides. The current results were consistent with the studies of hemicelluloses from cell walls of barley plants by Kato et al. (1981a,b, 1987). The authors reported that the acidic arabinoxylan had a linear backbone chain of ␤-1,4-d-xylose residues, about 50% of which were substituted at the O-2 and/or O-3 position, mainly with arabinofuranose and glucuronic acid residues. They also revealed that most of the glucose residues in the alkali extract were found to be derived from the ␤-d-glucan, and methylation analysis and enzyme degradation studies indicated that the glucan had ␤-(1 → 3)- and (1 → 4)-linked d-glucopyranosyl residues in an approximate molar ratio of 1.0:2.3. Similarly, in view of these facts, the occurrences of glucose-containing hemicellulose in this study imply that the hemicellulosic preparation may also contain a small portion of ␤-dglucan because the non-cellulose ␤-d-glucan is widely distributed in cell walls of various monocotyledons.

Lignin (L)

4.0

2.1

12.0

8.7

7.4

1.5

8.7

2.4

32.1

14.7

Rhamnose Arabinose Xylose Mannose Galactose Glucose Total Uronic acids Ara/Xyl a b

Preparationsa H1

H2

H3

H4

L1

L2

L3

9.1 16.5 15.7 2.2 8.5 35.2 87.1 7.6 1.05

2.3 10.4 64.6 0.4 1.2 9.3 88.2 10.1 0.16

1.6 9.7 64.1 0.2 0.2 7.0 82.8 8.5 0.15

1.5 11.5 58.2 0.3 0.3 16.3 88.1 7.3 0.20

0.5 4.0 2.8 0.1 0.2 1.4 9.0 NDb 1.43

0.9 5.4 5.9 0.1 0.1 0.4 12.8 NDb 0.92

0.4 4.2 5.0 0.1 0.2 0.6 10.5 NDb 0.84

Corresponding to the preparations in Table 1. ND = not detected.

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Table 3 The yields (% sample, w/w) of the phenolic acids and aldehydes from alkaline nitrobenzene oxidation of the isolated hemicellulosic and lignin preparations. Phenolic acids and aldehydes

p-Hydroxybenzoic acid p-Hydroxybenzaldehyde Vanillic acid Vanillin Syringic acid Syringaldehyde Acetovanillin Acetosyringone p-Coumaric acid Ferulic acid Cinnamic acid Total a b c

Preparationsa H1

H2

H3

H4

L1

L2

L3

L4

0.09 0.38 0.19 1.09 Tb 0.72 0.22 T T T 0.09 2.68

0.15 0.26 0.16 1.07 0.07 0.60 0.32 0.11 T T 0.09 2.74

0.36 0.52 0.69 2.31 0.21 1.62 0.11 0.11 T T 0.16 5.93

0.20 0.29 0.64 1.28 0.39 1.05 0.61 0.30 0.04 0.03 0.07 4.77

2.70 4.45 3.32 20.74 4.19 24.75 2.27 2.04 0.51 T 0.76 65.71

0.50 1.90 1.28 11.81 0.42 10.80 1.23 0.84 T T 0.69 29.45

0.45 2.70 3.06 12.00 0.70 11.93 1.51 0.84 T T 0.90 34.08

3.20 5.40 5.60 28.30 2.80 38.30 6.60 3.20 NDc ND 0.97 93.50

Corresponding to the preparations in Table 1. T = trace. ND = not detected.

The contents of total neutral sugars in the isolated lignin fractions are 9.0% (L1 ), 12.8% (L2 ) and 10.5% (L3 ), respectively, in which arabinose and xylose, decomposed from associated hemicelluloses, are predominant sugars. Interestingly, arabinose to xylose ratio in lignin preparations (L2 and L3 ) is higher than that in the hemicellulosic preparations (H2 and H3 ) as shown in Table 2, indicating that hemicellulose is linked to lignin mainly by its arabinosyl sidechains.

namic acid is identified to be present in a minor quantity, in lignin fractions. Results concerning the composition of lignin bound to the four hemicellulosic preparations indicated that a substantial cleavage of covalent linkages between lignin and hemicelluloses occurred as shown by the rather low amounts of bound lignin (2.68–5.93%) in isolated hemicellulosic fractions (H1 , H2 , H3 , and H4 ). The results show that the hemicelluloses are more linked to non-condensed guaiacyl lignins than to non-condensed syringyl lignins. The contents of ferulic and p-coumaric acids were not detected in H1 , H2 , and H3 , due to their complete oxidation during alkaline nitrobenzene oxidation at 180 ◦ C.

3.2. Content of phenolic acids and aldehydes The composition of lignin preparation and the associated lignin in hemicelulosic fractions were characterized by alkaline nitrobenzene oxidation following HPLC analysis. According to Table 3, the low yields of non-condensed phenolic compounds suggest that the lignin fractions (L2 and L3 ) isolated with acidic dioxane and DMSO treatments have a high degree of condensation, unlike the lignin fractions (L1 and L4 ) isolated by 90% dioxane and alkali treatments (Sun et al., 2003), and the phenomenon suggests that the lignin fractions (L2 and L3 ) are mainly from the middle lamella. The predominant oxidation products from lignin preparations were identified to be vanillin (11.81–28.30%) and syringaldehyde (10.80–24.75%), resulting from the oxidation of guaiacyl (G) and syringyl (S) units involved in the non-condensed structure of lignin, respectively (14). The presence of relatively higher amount of phydroxylbenzaldehyde (4.45–5.40%) and p-hydroxybenzoic acid (2.70–3.20%), indicative of non-condensed p-hydroxyphenyl (H) units, confirms that the lignin preparations (L1 and L4 ) have a low degree of condensation and incorporation of p-hydroxyphenyl alcohol in initial lignification. The molar ratios of G:S:H in lignin preparations are 5:4:1 and 4.5:3:1 for L2 and L3 , but 3:3:1 and 4:4:1 for L1 and L4 , respectively. It is found that lignin preparations (L2 and L3 ) with high condensation have high amount of G units and low amount of H units in barley straw. The lignin fractions (L1 and L4 ) with low condensation, extracted with mild dioxane and alkali solution, contain nearly equal of vanillin (11.81% and 12.00%) and syringaldehyde (10.80% and 11.93%). In addition, cin-

3.3. Average molecular weights As shown in Table 4, the lignin fractions (L1 , L2 , and L4 ) have a relatively same of weight-average molecular weight (Mw , 3600–3800 g mol−1 ), but different number-average molecular weights (Mn , 1400–2100 g mol−1 ) and polydispersity (1.84–2.57). The low weight-average molecular weight (Mw , 3000 g mol−1 ) and low yield (1.5%) of lignin fraction (L3 ) could suggest that the dissolution of lignin by DMSO is limited from the cell walls. The values of the weight-average molecular weight (Mw , 12,600 and 16,700 g mol−1 ) of the solubilized hemicellulosic fractions (H2 and H4 ) are relatively lower in comparison with that (Mn , 28,800 g mol−1 and 20,200 g mol−1 ) of the hemicellulosic fractions (H1 and H3 ) extracted with 90% dioxane and DMSO from barley straw. That is because acidic or alkali condition causes some degradation of hemicelluloses. However acidic or alkali treatment did not result in the degradation of lignin as shown in Table 4. 3.4. FT-IR spectra The FT-IR spectra of hemicellulosic fractions (H1 , H2 , H3 , and H4 ) are illustrated in Fig. 2, and clearly show the typical signal pattern expected for a hemicellulosic moiety, except for H1 containing some glucans. The broad band at 3427 cm−1 is attributed

Table 4 Weight-average (Mw ) and number-average (Mn ) molecular weights and polydispersity (Mw /Mn ) of the isolated hemicellulosic and lignin preparations. Preparationsa H1 Mw Mn Mw /Mn a

28,800 10,000 2.88

H2

H3

H4

L1

L2

L3

L4

12,600 7300 1.73

20,200 6800 2.97

16,700 7200 2.32

3700 1800 2.06

3600 1400 2.57

3000 1600 1.88

3800 2100 1.81

Corresponding to the preparations in Table 1.

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Fig. 2. FT-IR spectra of the hemicellulosic fractions extracted with 90% dioxane (spectrum H1 ), 80% acidic dioxane (spectrum H2 ), 100% DMSO (spectrum H3 ), and 8% NaOH (spectrum H4 ).

to hydroxyl groups, and the behaviour of these spectra in the 2850–3000 cm−1 region shows the C–H stretch in methyl and methylene groups. The wavenumber characteristic for typical xylan is 1044 cm−1 , which is assigned to the C–O and C–C stretching and the glycosidic linkage ␯ (C–O–C) contributions. A sharp band at 896 cm−1 , corresponding to the C1 group frequency or ring frequency, is attributed to the ␤-glycosidic linkages (1 → 4) between xylose units in hemicelluloses (Sun et al., 1996; Sun and Sun, 2002). The absorption at 1642 cm−1 is principally related to the absorbed water. The four small bands at 1463, 1421, 1378, and 1322 cm−1 represent the C–H and C–O bending or stretching frequencies. The two adsorptions at 1739 and 1251 cm−1 in H1 , H2 , and H3 are due to the carbonyl groups in uronic acids or acetyl groups attached to hemicelluloses, particularly in H3 . The two lowintensity shoulders at 1166 and 953 cm−1 in H3 show the presence of the arabinosyl side-chains. The two rather weaker adsorptions at 1512 and 831 cm−1 in H1 than H2 , H3 and H4 are originated from

aromatic skeletal vibrations in associated lignin, indicating that the hemicellulosic fraction is slightly contaminated with minimal amounts of bound lignin (Vazquez et al., 1997). This is in agreement with the results obtained from the alkaline nitrobenzene oxidation of linked lignin in isolated hemicelluloses. The structural features in lignin fractions (L1 , L2 , L3 , and L4 ) were also analyzed by FT-IR spectroscopy (Fig. 3). The major peaks show up in the spectra are the broad band at 3415 cm−1 , as attributed to hydroxyl groups in aliphatic and phenolic structures, and the bands between 2859 and 3000 cm−1 predominantly arising from C–H stretching in methoxyl group and methylene group. The shoulders at 1719 and 1653 cm−1 originate from unconjugated and conjugated carbonyl stretches, however, the intensity of the two shoulders decreased in the spectra of L2 and L4 , particularly the shoulder at 1653 cm−1 , indicating that acidic and alkali conditions resulted in the degradation of hydroxycinnamic moieties in lignin. Aromatic skeletal vibrations give three strong peaks at 1597, 1509,

Fig. 3. FT-IR spectra of the lignin fractions extracted with 90% dioxane (spectrum L1 ), 80% acidic dioxane (spectrum L2 ), 100% DMSO (spectrum L3 ), and 8% NaOH (spectrum L4 ).

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Fig. 4.

13

C NMR spectrum of the hemicellulosic fraction (H3 ) extracted with 100% DMSO at 85 ◦ C for 3 h.

and 1421 cm−1 (Sakakibara, 1991; Vazquez et al., 1997). Further bands are located at 1461 (asymmetric C–H deformations), 1361 (symmetric C–H bending), 1324 (syringyl ring breathing with C–O stretching), 1265 (guaiacyl ring breathing with C O stretching), 1236 (aromatic ring breathing with C–O and C O stretching), 1126 (aromatic C–H in-plane deformation, syringyl type), 1089 (C–O deformation, secondary alcohol and aliphatic ethers), 1024 (aromatic C–H in-plane deformation plus C–O in primary alcohols, guaiacyl type), 953 (C–H out of plane in aromatic rings), and the band at 839 cm−1 (aromatic C–H out of plane deformation). The weak signal at 1163 cm−1 shows the presence of p-coumaric ester group, typical for GSH lignins (Galkin et al., 1997; Vazquez et al., 1997). In short, the FTIR spectrum shows that lignin is of GSH type with a small content of hydroxycinnamic acids. It is indicated in the spectra that the content of guaiacyl unit is higher than that of syringyl unit in L3 with high condensation, as shown by the band at 1024 cm−1 stronger than that band at 1126 cm−1 , whereas L1 and L4 with low condensation have a higher content of syringyl units than guaiacyl units. 3.5.

13 C

593

NMR spectrum of hemicellulosic preparation

The hemicellulosic preparation (H3 ) isolated with DMSO was investigated using 13 C NMR spectroscopy (Fig. 4). The important features found in the spectrum are the two signals at 171.14 and 23.42 ppm assigned to acetyl group and signals of 8-O-4 diferulates by these signals at 168.44 (C-9), 129.99 and 129.55 (C-7 ), 142.26 (C-7), 119.3 (C-5), 131.83 (C-1), and 57.5 ppm (OMe) (Bunzel et al., 2003). The spectrum shows five strong signals at 102.3 (C-1), 74.9 (C-2), 75.9 (C-3), 76.3 (C-4), and 63.3 ppm (C-5) corresponding to (1 → 4)-linked ␤-d-xyl residues. The signals at 81.0, 78.21, 82.4, and 61.6 ppm correspond to C-2, C-3, C-4, and C-5 of (1 → 3) linked ␣-l-arabinofuranosyl residues (Sun et al., 1996; Izydorczyk

and Biliaderis, 1995), respectively. Among others, signals observed at 180.2, 73.3, 72.9, 83.2, 74.6 and 58.8 ppm, respectively, are characteristic of C-6, C-3, C-2, C-4, C-5, and the methoxy group of (1 → 3) linked 4-O-methyl-d-glucuronic acid residues (Evtuguin et al., 2003; Sun et al., 2002). The signals at 66.1 and 66.7 for O-5 of ␣-l-arabinofuranosyl residues verify that ␣-l-arabinofuranosyl side chain is esterfied to ferulic acid (168.44 ppm, C-␥) (Kato et al., 1987). 3.6. HSQC and 1 H NMR spectra of hemicellulosic preparation HSQC and 1 H NMR spectra clearly show the typical signal pattern expected for a hemicellulosic moiety (Figs. 5 and 6). The anhydroxylose units of hemicellulose preparation (H3 ) are indicated by signals at ıC /ıH 102.5/4.23 (C1–H), 63.1/3.85 and 3.17 (C5–H2 ), 76.0/3.59 (C4–H), 75.1/3.28 (C3–H), and 73.2/3.07 (C2–H) in HSQC NMR spectrum, in which the chemical shifts of 3.85 and 3.17 ppm are assigned to the equatorial and axial protons linked at C-5, respectively. The xylan substituted at O-2 and O-3 by acetyl groups is detected by the signals at ıC /ıH 63.2/3.17/3.70 and 63.2/3.20/3.80, which correspond to C5–H2 of anhydroxylose. The acetyl group attached to the hydroxyl group of hemicelluloses is seen by a signal at ıC /ıH 23.2/1.83 ppm. The signal at ıC /ıH 38.6/2.62 corresponds to DMSO-d6. The methoxyl group of 4-O-methylglucouronic acid is detected by the signals at ıC /ıH 57.2/3.45/3.33. Compared to all of the chromatography-based techniques, the 1 H NMR method is an attractive alternative because it provides excellent resolution of sugars, including minor components, does not require sample derivatization or neutralization, and is very fast (instrument time 15–30 min). The well-resolved double peaks at 4.38 and 4.35 ppm are assigned to the ␤ anomeric C1 protons of the anhydroxylose units of hemicelluloses. The spectral region from 3.2 to 4.0 ppm corresponds to the protons attached to C5, C2, C3, and C4

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Fig. 5. HSQC NMR spectrum of the hemicellulosic fraction (H3 ) extracted with 100% DMSO at 85 ◦ C for 3 h.

Fig. 6.

1

H NMR spectrum of the hemicellulosic fraction (H3 ) extracted with 100% DMSO at 85 ◦ C for 3 h.

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Table 5 Carbon and proton chemical shifts (ı, ppm) of hemicellulosic preparation (H3 ) isolated with dimethyl sulfoxide. Assignmentsb

Structural unitsa

␤-d-Xylc Xyl (r.e.) Xyl-3Ac Xyl-2Ac Xyl-2, 3 Ac Xyl-2GlcA Xyl-3Ac-2GlcA ␣-l-Araf (1 → 2) ␣-l-Araf (1 → 3) ␣-d-Xyl (1 → 3) MeGlcA (1 → 2) MeGlcA (1 → 3)

C-1/H-1

C-2/H-2

C-3/H-3

C-4/H-4

C-5/H2 -5

102.5/4.23 101.9 102.3 4.71d 101.5 101.7 102.8 5.23d 5.39/5.36d 5.18d 98.4 98.3

73.2/3.07 73.9 72.5 74.8 74.8 77.8 75.9 81.0 80.8 72.9 72.5 72.9

75.1/3.28 76.0 76.3 76.9 76.3 73.3 76.3 78.21 78.27 74.6 73.3 73.3

76.0/3.59 69.5/3.95 c NDe 77.3 78.2 78.2 77.8 82.4 82.4 76.8 83.2 83.2

63.1/3.17 and 3.85 61.8 63.2/3.20 and 3.80c 63.2/3.17 and 3.70c 66.0 63.3 67.2/3.45/3.33c 61.6 66.1 and 66.7f 61.3 74.7 74.6

a Abbreviations: Xyl (r.e.), reduced end xyloses; Xyl-3Ac, xylans substituted at O-3 by acetyl groups; Xyl-2,3Ac, xylans substituted at both O-2 and O-3 by acetyl groups; Xyl-2GlcA, xylans substitued at O-2 by 4-O-methyl-glucuronic acid; Xyl-3Ac-2GlcA, xylans substituted at O-3 by acetyl group and O-2 by 4-O-methyl-glucuronic acid; ␣-l-Araf (1 → 3), ␣-l-arabinofuranose residuses attached to xylans at O-3; MeGlcA (1 → 2), 4-O-methyl-glucuronic acid attached to xylans at O-2; MeGlcA (1 → 3), 4-Omethyl-glucuronic acid attached to xylans at O-3. b The assignments are based on the previous studies (Evtuguin et al., 2003; Kabel et al., 2003). c It belongs to the signals of backbone of xylans obtained in HSQC NMR spectrum and has some solvent-induced shifts caused by DMSO-d6 . d Obtained in 1 H NMR spectrum (DMSO-d6 ). DMSO causes some solvent-induced shifts, and signal is broad, however, it allows to the detection of acetyl groups and lignin structure contaminated in hemicelluloses. e Not detected. f Arabinofuranose esterified at O-5 by ferulates.

of the anhydroxylose units. The result on the distribution of acetyl groups per 100 xylose residues is calculated by 1 H NMR spectrum. The degree of substitution (DS) of anhydroxylose by acetyl groups is found to be ca. 0.13. The anomeric protons of terminal arabinofuranose linked to O-3 of xylans are indicated by two weak resonances at 5.39 and 5.36 ppm in the 1 H NMR spectrum (Cyran et al., 2003). 2-O-Acetylated non-reducing end xylose residues gave a signal at

Fig. 7.

13

4.63 (Kabel et al., 2003). The small peak at 8.36 ppm is attributed to the carboxyl group of uronic acids. Expected, the 1 H NMR spectrum displays clear resonance in the region of CH CH and phenolic moieties, 5.6–7.4 ppm, showing that hydroxycinnamic acids constitute a significant proportion of straw hemicelluloses. As shown in Table 5, HSQC, 1 H and 13 C NMR analysis clearly show that the hemicelluloses isolated from barley straw are sub-

C NMR spectrum of the lignin fraction (L2 ) extracted with 80% acidic dioxane at 85 ◦ C for 3 h.

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Fig. 8.

1

H NMR spectrum of the lignin fraction (L2 ) extracted with 80% acidic dioxane at 85 ◦ C for 3 h.

stituted by ␣-l-arabinofuranose, 4-O-methyl-d-glucuronic acid, ␣-d-xylopyranose, and acetyl group at O-3 and/or O-2 of xylan. 3.7.

13 C

and 1 H NMR spectra of lignin preparation

The lignin preparation extracted with acidic dioxane (L2 ) was investigated by both 13 C and 1 H NMR spectroscopy (Figs. 7 and 8). According to the literature (Sakakibara, 1991; Ralph et al., 1996), we assigned the signals in two NMR spectra as shown in Table 6.

The most striking characteristic of the 13 C NMR spectrum is the presence of p-coumarate ester (C-␥, 166.5 ppm; C-␤, 115.3 ppm) and etherified ferulates (C-␥, 163.0 ppm) (Sun et al., 1996). Diferulate and p-coumarate ester in ␤-O-4 are also identified by two signals at 164.3, and 161.4 ppm, respectively, assigned to C-␥ and C -4 (Bunzel et al., 2003; Ralph et al., 1996). The occurrence of acetyl groups showed as three signals at 172.1, 22.0 and 20.9 ppm implies that some monolignols were acetylated during lignification of barley straw, which has been studied by Lu and Ralph (2002). A signal

Table 6 Carbon chemical shifts (ı, ppm) in 13 C NMR spectrum of lignin isolated with acidic dioxane–water. ıC -ppm (intensity)a

Assignments

ıC -ppm (intensity)

Assignments

181.8 (m) 177.2 (w) 172.1 (m) 166.5 (m) 164.3 (w) 163.0 (w) 161.4 (s) 159.8 (w) 157.4 (m) 152.9 (s) 152.1 (s) 149.1 (w) 148.2 (w) 147.2 (m) 146.96 (s) 145.4 (m) 138.1 (w) 134.5 (w) 134.2 (w) 132.97 (w) 130.3 (m) 127.9 (w) 125.0 (w) 120.4 (w) 119.5 (m) 119.4 (m) 119.3 (m) 115.8 (s) 115.3 (s) 114.7 (s) 110.96 (m)

Carboxyl group C-␥ in uronic acids Acetyl group in aliphatic chain C-␥ in p-coumarate ester, in ␥-ester C-␥ in 8-O-4 diferulate C-␥ in etherified ferulic acid C-4 in p-coumarate ester, in ␤-O-4 C-4 in p-coumarate ester C-4 in p-hydroxyphenyl units C-3 in guaiacyl units with ␣-ether C-3/C-5 in etherified syringyl unit C-3 in etherified guaiacyl unit C-3 in non-etherified guaiacyl unit C-3/C-5 in non-etherified syringyl unit C-4 in etherified guaiacyl unit, in ␤-5 C-4 in non-etherified guaiacyl units C-4 in etherified syringyl unit C-1 in etherified guaiacyl unit C-1 in etherified syringyl unit C-1 in non-etherified syringyl unit CH-2/CH-6 in p-coumarate ester CH-2/CH-6 in p-hydroxyphenyl units C-1 in p-coumarate ester C-6 in guaiacyl units, in 5-5 type CH-6 in guaiacyl units CH-6 in guaiacyl units CH-6 in guaiacyl units CH-3/CH-5 in p-hydroxyphenyl units C-␤ in p-coumarate ester CH-5 in guaiacyl unit, in ␤-1 units CH-2 in guaiacyl unit

104.2 (s) 102.0 (s) 98.95 (w) 97.5 (w) 96.1 (w) 94.3 (w) 87.0 (m) 86.0 (m) 82.6 (s) 79.6 (m) 77.2 (m) 77.0 (m) 75.4 (m) 74.7 (m) 72.2 (m) 71.6 (m) 70.9 (w) 69.5 (m) 65.8 (w) 64.4 (w) 63.1 (w) 62.6 (w) 60.1 (s) 56.3 (s) 54.0 (w) 53.0 (w) 48.6 (w) 31.2 and 28.9 (m) 22.0 (m) 20.9 (s) 13.9 (s)

CH-2/CH-6 in syringyl units CH-1 in xylans CH-1 in MeGlcA C-1 in reducing xlyose, ␤-anomer C-, unkown CH-1, in xylose, ␣-anomer CH-␤, in syringyl ␤-O-4 (erythro) CH-␣, in syringyl units CH-␤, in ␤-O-4 CH-3 in arabinofuranose CH-4 in xylans with MeGlcA CH-4 in xylose internal unit CH-␣, in ␤-1 C-␣, in ␤-O-4 CH-␣, in ␤-O-4 (erythro) guaicyl CH-␥, in ␤-␤ units, and CH-␣ in ␤-O-4 (threo) guaicyl CH-␥ in p-hydroxyphenyl units CH-4 in xylose non reducing end unit CH2 -5 in esterified arabinose CH2 -␥, in ␤-1 CH2 -␥, in ␤-5 , C5-H2 in xylans CH2 -␥, in ␤-O-4 with C␣ O or ␤-1 CH2 -␥, in ␤-O-4 OCH3 CH-␤, in ␤-␤ unit CH-␤, in ␤-5 unit C-␤, in cyclic unit CH2 -␣ and ␤, in dihydroconiferyl alcohol Acetyl- (CH3 ) Acetyl- (CH3 ) Long chain CH2 and CH

a

Intensity abbreviations: s, strong; m, mean; w, weak.

X.-F. Sun et al. / Industrial Crops and Products 33 (2011) 588–598

at 177.2 ppm arises from uronic acids, indicating that uronic acids could be esterified to lignin. Characteristic aromatic carbon signals of etherified and nonetherified syringyl, guaciacyl, and p-hydroxylphenyl residues were detected in 13 C NMR spectra, as shown in Table 6. It can be found that more guaiacyl units do join in the construction of ␤-5 and ␤-1 . The 13 C NMR spectrum give three resonances at 82.6, 74.7, and 60.1 ppm (very strong), assigned to C-␤ in ␤-O-4 , C-␣ in ␤-O4, and C-␥ in ␤-O-4 , respectively. These signals indicate that the treatment with acidic dioxane under the conditions given did not significantly attack the ␤-aryl ether structure. By comparison of the intensity of signals, the content of ␤-aryl syringyl ethers decreased, and this may be due to its easier cleavage than ␤-aryl guaiacyl ethers during treatment (Sugimoto et al., 2002), and the proportion of erythro-␤-O-4 is slightly higher than that of threo-␤-O-4 . ␤-O-4 linkages with ␣-carbonyl groups were also detected by the signal at 62.6 ppm assigned to CH2 -␥ (Sakakibara, 1991; Ralph et al., 1996). In this study, many structural features of barley straw lignin are similar to that of wheat straw lignin (Sun et al., 2005). However, it is found that the guaiacyl and syringyl units are more etherified in the lignin of barley straw. In the 1 H NMR spectrum (Fig. 8), the two signals at 2.50 and 3.46 ppm arise from DMSO-d6 and HDO, respectively. A broad peak centered at 3.73 ppm is assigned to the resonance of the methoxy and side-chain protons in various structures, such as ␤-5 and ␤-1 forms, as well as aliphatic hydroxyl groups. The signals from 6.2 to 7.7 ppm were attributed to the aromatic protons of the lignin. The broad signal at 5.39 ppm (data not shown) corresponds to H-␣ of ␤-5 structures (Galkin et al., 1997; Vazquez et al., 1997). H-␤, H-␣ and OH in ␤-O-4 are documented by the peak at 4.26 and signals between 4.87 and 5.25 ppm, and the signal of H-␥ in ␤-O-4 was overlapped with that of HDO. The phenolic hydroxyl group (Li and Lundquist, 2001) is detected by a weak signal at 8.81 ppm (data not shown). The small peak at 10.08 ppm (data not shown) is due to p-coumarate units (Li and Lundquist, 2001), and the resonance of aliphatic acetyl groups is shown by the signals between 1.9 and 2.0 ppm, and the small peak at 1.23 ppm corresponds to methylene groups of aliphatic chain.

4. Conclusion This sequential extraction using mild dioxane, acidic dioxane, DMSO, and alkali treatments gave both lignin and hemicellulosic preparations with higher yield and important features. The dioxane treatments led to the substantial dissolution of both lignin and hemicelluloses from the cell walls, however, DMSO treatment yielded mainly hemicelluloses, and the hemicelluloses and lignin fractions extracted with dixoane and DMSO have high purity. It is found that the first step with mild dioxane extraction resulted the dissolution of substantial polysaccharides and lignin from the primary cell wall and middle lamella, and both acidic dioxane and DMSO treatments produced original hemicelluloses and high-condensed lignin mainly from the middle lamella, and alkali treatment resulted in the removal of residual hemicelluloses and lignin from the secondary cell wall. By the analysis of the lignin and hemicellulosic fractions, important structural features and relationships between lignin and hemicellulose are also obtained. Hemicelluloses of barley straw contain acidic arabinoxylans as the major polysaccharides, which are substituted by ␣-l-arabinofuranose, 4-O-methyl-d-glucuronic acid, acetyl group (DS = 0.13), and xylose at O-3 and/or O-2 of xylan. Lignin contains ␤-O-4 as a predominant interunit linkage with high amounts of ␤-5 and ␤-1 . The guaiacyl and syringyl units are more etherified, and the proportion of erythro-␤-O-4 is slightly

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