Successive Solvent Fractionation of Bamboo Formic Acid Lignin for Value-Added Application

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1 August 27-, Beijing, CHINA Successive Solvent Fractionation of Bamboo Formic Acid Lignin for Value-Added Application Ming-Fei LI, Shao-Ni SUN, Feng XU, *, Run-Cang SUN, 2, * Institute of Biomass Chemistry and Technology, Beijing Forestry University, 0008, Beijing, China Corresponding author. Tel./fax: address: xfx5@bjfu.edu.cn (Feng Xu); rcsun@bjfu.edu.cn (Run- Cang Sun). 2 State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, 50640, Guangzhou, China Abstract Formic acid delignification process is a promising organosolv technique, which is used to separate lignocelluloses for the production of bioethanol as well as cellulose-based materials. To achieve a full volatilization of lignocelluloses in the concept of biorefinery, the dissolved lignin, a major by-product in the process, should be fully exploited and utilized. However, the value-added utilization of lignin is strongly affected by its heterogeneous nature. In this case, bamboo formic acid lignin was successively fractionated with organic solvents of increasing dissolving capacity (i.e., ether, ethyl acetate, methanol, acetone, and dioxane/water) to obtain homogeneous preparations. The starting lignin and the fractions obtained were compared in terms of molecular weight and functional groups by a set of chemistry and spectroscopy technologies. It was found that the yield of the five fractions obtained was 2.80, 9.85, 8.64, 2.8, and.0%, respectively. The lignin fraction extracted with ethyl acetate contained homogeneous materials of low molecular weight whereas the lignin fraction extracted with acetone was composed of a mixture of medium and high molecular weight materials. As evidenced by sugar analysis, there was strong association between hemicelluloses and lignin in the preparations with different molecular weights. Spectroscopy analysis indicates that with increasing the dissolving capacity of solvent, the contents of methoxyl, phenolic, and aliphatic hydroxyl groups in the extracted lignin fractions were decreased. The lignin fraction extracted with ethyl acetate, having a high radical scavenging index (RSI), will be a good feedstock as stabilizer. The results above suggest that the sequential solvent fractionation provides a promising way to prepare lignin with homogeneous structure and good functional properties for potential application. Keywords: Bamboo, Formic acid lignin, Solvent fractionation, Antioxidant capacity Paper PS-77 of 9

2 August 27-, Beijing, CHINA Introduction The value-added utilization of lignin is strongly affected by its heterogeneous nature. Lignin shows different physicochemical properties depending on the sources, processing as well as post-treatments [, 2]. Due to the heterogeneous feature of lignin, it is very difficult to control and standardize the properties and qualities of the derived products from lignin; thus, several techniques have been developed to homogenize the macromolecule. Lignin can be fractionated into sub-fractions by gel permeation chromatography [], ultrafiltration [4, 5], selective precipitation [6], etc. However, these methods have high cost in large scales [7]. Recently, increasing attention has been attracted in fractionation lignin into homogeneous preparations with organic solvents in a successive manner. Successive fractionation of lignin with increasing hydrogen-bonding solvents is mainly based on the pioneering work of Schuech [8], who found that the salvation power of solvent depended on both the cohesive energy and hydrogen-bonding capacity of the solvent. Sun et al. [9] fractionated sodaanthraquinone (AQ) lignin from oil palm empty fruit bunch by successive extractions and found that the high molecular weight fraction had a high thermal stability. Similar conclusions have also been drawn in the subsequent fractionation investigation, in which kraft-aq lignin from Eucalyptus pellita was sequentially extracted with different solvents [0, ]. In addition, fractionation provides an attractive approach to refine lignin for a variety of potential applications. For instance, commercial Alcell lignin, birch organosolv lignin, as well as steam-exploded pine and eucalypt lignins were sequentially fractionated with organic solvents, and the obtained fractions with high homogeneity were further modified by laccases via polymerization [2]. The phenoxyl radicals obtained can be used for further enzymatic or chemical functionalization. Lignin-starch composites were prepared by casting fractionated preparations from pine kraft lignin with starch, in which the low molecular weight fractions of lignin seemed to be principally involved in plasticization or compatibilization []. Several solvent fractionated preparations from Alcell lignin were applied to manufacture matrix granules with bromacil as a model compound through a melt process [4]. It was speculated that the heterogeneity of lignin played an important part in altering the release rate of the active ingredient for the matrix granules. In this case, the organosolv lignin from bamboo was successively fractionated by organic solvents with increasing dissolving capacity for lignin, and the starting lignin and its subfractions were compared in terms of yield, molecular weight distribution, and functional groups. Additionally, their antioxidant activity and thermostability were also investigated owning to the importance for value-added application. Materials and Methods Raw material and preparation of organosolv lignin. Air-dried Bamboo (Phyllostachys sulphurea) (70 g) together with formic acid was placed in a L flask equipped with a condenser, and delignified by using formic acid with a concentration of 88%, a temperature of 00.5 C, a cooking time of 20 min and a liquid to solid ratio of 0 ml/g. Subsequently, the cellulosic pulp was filtered and washed with 88% formic acid followed by water and then dried. The filtrate and washing liquor (88% formic acid-soluble) were combined and concentrated under reduced pressure. After 0 volumes of water was added into the concentrated liquor, the solid material was recovered by filtration and washed thoroughly with water. Subsequently, it was freeze-dried and extracted with pentane in a Soxhlet Paper PS-77 2 of 9

3 August 27-, Beijing, CHINA apparatus to remove extractives. The specimen was dried and noted as crude bamboo organosolv lignin. Solvent fractionation of crude lignin. The crude lignin was sequentially fractionated with ether, ethyl acetate, methanol, acetone, and dioxane/water (9/, v/v). 8 g lignin was suspended in 60 ml specified solvent in a 250 ml volumetric flask. After that, the mixture was agitated for 2 h using a magnetic stirrer at room temperature. The undissolved material was filtered over a filter paper. Subsequently, the solid fraction was suspended for an identical extraction until filtrates were colorless. Then the filtrates were combined and evaporated under reduced pressure to obtain lignin fractions. The lignin fractions were suspended in water and then freezing-dried to ensure complete removal of the rest of the solvents. The lignin fractions extracted with ether, ethyl acetate, methanol, acetone, and dioxane/water (9/, v/v) were noted as F0, F, F2, F, and F4, respectively. Component analysis, structural characterization and antioxidant activity determination were conducted according to a previous report [5]. Results And Discussion Fractionation of organosolv lignin. The organosolv lignin prepared from bamboo was sequentially extracted with five organic solvents with increasing dissolving capacity for lignin according to our preliminary experiments, i.e., ether, ethyl acetate, methanol, acetone, and dioxane/water (9/, v/v); and the yields of the five fractions obtained were 2.80, 9.85, 8.64, 2.8, and.0%, respectively (Table ). The ether-soluble fraction of organosolv lignin was a sticky residue, rather than free-flowing powders in the case of the other fractions. In addition, the FTIR spectroscopy analysis in a preliminary investigation indicates that it was extractives (such as fatty acids and resinous materials). For this reason, together with its low yield (2.80%), this preparation was discarded as unmeaning fraction and further analyses were focused on the fractions extracted with ethyl acetate (F), methanol (F2), acetone (F), and dioxane/water (F4). Table Yield, weight-average ( M w ) and number-average ( M n ) molecular weights and polydispersity ( M w / M n ) of bamboo organosolv lignin and fractionated lignins (%). Lignin sample a Yield M w (g/mol) M n (g/mol) M w / M n FL F F F F a FL represents the starting lignin; F, F2, F, and F4 represent lignin preparations extracted with ethyl acetate, methanol, acetone, and dioxane/water, respectively. To investigate the effect of solvent extraction on the molecular weight of the lignin, the samples were investigated with GPC. The weight-average and number-average molecular weights and polydispersity of the lignin specimens, calculated based on the curves of GPC, are listed in Table. From F to F, a steady increase of both the molecular weight and polydispersity strongly suggests that F contained materials of relatively homogeneous low molecular weight whereas F was composed of a mixture of medium and high molecular Paper PS-77 of 9

4 August 27-, Beijing, CHINA weight materials. With respect to F, a significantly high amount of low molecular weight lignin in organosolv lignin (FL) was extracted with ethyl acetate. In addition, F2 was a medium molecular weight material, basically depleting of materials with low and high molecular weights. verall, the solvents used in the present study showed good selectivity for fractionation organosolv lignin, evidenced by the increasing molecular weight from F to F. Sugar analysis. Sugar content of lignin and its sub-fractions is listed in Table 2. verall, the bound sugar contents were below 4.0%, indicating that the organosolv lignin separated by formic acid delignification technology produced lignin by-products with a relatively low amount of impurities. Clearly, there were no marked differences among the sugar contents of the fractionated specimens and the starting material. This suggests that the strong association between hemicelluloses and lignin existed in the organosolv lignin samples with different molecular weights. For all preparations, xylose was the predominate sugar followed by arabinose and glucose. This was in well agreement with the fact that arabinoxylan was the main sugar impurity in lignin being linked with arabinose side chains of xylan via ether/ester bonds in grass [6]. Table 2 Sugar analysis of bamboo organosolv lignin and fractionated lignins (%). Lignin sample a Total Arabinose Galactose Glucose Xylose Glucuronic acid FL F F F F a Corresponding to the lignin samples in Table. Paper PS-77 4 of 9

5 August 27-, Beijing, CHINA Fig. HSQC spectra of bamboo organosolv lignin (FL) and fractionated lignins (F and F). See Fig. 2 for the main lignin substructures. Spectral analysis. The HSQC spectra of bamboo organosolv lignin (FL) and the lignin fractions were recorded and typical spectra of lignin samples (FL, F, and F) are illustrated in Fig.. and the main substructures identified are shown in Fig. 2. The signal assignments of HSQC spectra are shown as follows. In the side religion, the prominent correlation signals corresponding to --4 substructures (A) were observed for - and γ-c positions at δ C /δ H 7.2/4.8 and 59.6/.25.85, and for -C positions at δ C /δ H 8.4/4.2 in G and H type lignins. γ-acylated --4 aryl ether linkages (A ) were observed with their correlations at δ C /δ H 6.9/ for the γ-c-position. Additionally, signals corresponding to other linkages were also observed. Strong signals for resinol structures (- /--γ /γ-- linkages, B) were observed with their C-H correlations for -, -, and γ-c positions at δ C /δ H 84.6/4.65, 5.4/.07, and 70.9/.80, 70.9/4.6. Phenyl coumaran substructures (C) were observed by C-H correlations for - and γ-c positions at δ C /δ H 86.8/5.46 and 62.2/.75. Cinnamate units (E) were characterized by their C -H correlations at δ C /δ H 45.0/7.58 and C -H correlations at δ C /δ H 4.4/6.0, due to p-coumarate and ferulate [7]. In the aromatic region, signals from G S H units were clearly observed. The S units showed a prominent signal for the C 2, 6 -H 2, 6 units at δ C /δ H 04.0/6.68, whereas the G units showed different correlations for C 2 -H 2 (δ C /δ H 0./6.92), C 6 -H 6 (δ C /δ H 8.5/6.77), and C 5 (δ C /δ H 5.0/6.80). In addition, signals from the oxidized (-ketone) structure of syringyl lignin (S ) were presented at δ C /δ H 06.2/7.0 [8]. A high amount of p-hydroxyphenyl (H) units was identified by the correlations at δ C /δ H 27.8/7.2. Paper PS-77 5 of 9

6 August 27-, Beijing, CHINA (H C) H H γ H C 5' 4' CH 6' ' 2' ' (CH ) H 5' 6' 4' ' ' 2' (H C) H ' ' γ' γ H C 5' 4' CH 6' ' 2' ' (CH ) (H C) γ ' ' CH 2' ' 4' ' 5' 6' γ' (CH ) A A' CH B H γ H C CH C ' 6' 2' 5' 4' ' CH 5 4 G H CH H C 5 4 S H CH H C S' CH 5 4 H H H E (CH ) (H C) Fig. 2 Main substructures of bamboo organosolv lignin involving different side-chain linkages and aromatic units identified by HSQC. The functional groups in lignin were determined by H NMR spectroscopy, and the data are presented in Fig.. A decrease of the contents of methoxyl, phenolic and aliphatic hydroxyl groups in the lignin fraction was observed from F to F4. Generally, a higher phenolic hydroxyl content indicates more serious degradation of the lignin macromolecule. Among these fractions, the highest content of phenolic hydroxyl group in F was in well agreement with its low molecular weight due to the serious degradation. For the preparation of novel materials, the most important functional group in lignin structure is free phenolic group, which is preferentially attacked by chemicals. The highest phenolic hydroxyl group in F suggested that it had the greatest potential to react with oxyalkylating reagents (i.e., ethylene oxide and propylene oxide). The compatibility between lignin and polyolefins would be greatly improved and the lignin would disperse more homogeneously [9]. The high content of phenolic hydroxyl group in F, suggesting its good reactivity with formalde. J H CH Paper PS-77 6 of 9

7 Inhabitation (%) RSI index Content (mmol/g) Proceedings of the 55th International Convention of Society of Wood Science and Technology August 27-, Beijing, CHINA 6 Phenolic hydroxyl group Aliphatic hydroxyl group Methoxyl group FL F F2 F F4 Lignin sample Fig. Contents of phenolic and aliphatic hydroxyl groups in bamboo organosolv lignin and fractionated lignins FL F F2 F F Concentration (mg/ml) (a) FL F F2 F F4 Lignin sample (b) Fig. 4 Antioxidant activity against DPPH of bamboo organosolv lignin and fractionated lignins. (a) DPPH inhibitory effect; and (b) RSI value. Antioxidant capacity. Many studies have demonstrated that the extracts from lignocelluloses, mainly composed of phenolic structures, have antioxidant and antimicrobial capacity [20, 2]. To investigate the effect of the fractionation on the antioxidant capacity of lignin, the samples were tested against DPPH since oxidation is usually occurred resulted from free radical attack. The profiles of DPPH inhibitory effects of the lignin fractions are illustrated in Fig. 4a and the corresponding radical scavenging index (RSI) values are calculated as shown in Fig. 4b. From Fig. 4a, the inhibitory effect increased with increasing the concentration of lignin for all samples. The calculated RSI values were.27, 0.7, 0.50, and 0.05 for F, F2, F, and F4, respectively, as compared to 0.82 for FL. bviously, the antioxidant capacity of the lignin fraction showed a decreasing trend with increasing the dissolving capacity of solvent. The antioxidant ability of lignin is mainly contributed by the free phenolic hydroxyl group content. In addition, other groups, such as aliphatic hydroxyl group and methoxyl group, also showed effects on the antioxidant ability. The most powerful radical scavenger of F may be explained as follows. The high content of phenolic hydroxyl Paper PS-77 7 of 9 0.0

8 August 27-, Beijing, CHINA group was essential for the formation of large amounts of phenoxyl radicals (i.e., hydrogen atom abstraction) [22]. The high amount of the methoxyl group in the lignin largely stabilized the phenoxyl radicals formed. The lignin fraction F with the highest high RSI value would be a promising material as stabilizer to prevent polymer aging [2]. Conclusions Bamboo organosolv lignin (FL) was fractionated into five preparations by sequential solvent extractions with ether (F0), ethyl acetate (F), methanol (F2), acetone (F), and dioxane/water (F4). A significantly high amount (9.85%) of low molecular weight fraction in organosolv lignin was extracted with ethyl acetate. Sugar analysis suggests that the strong association between hemicelluloses and lignin existed in the organosolv lignin samples with different molecular weights. The contents of methoxyl, phenolic,and aliphatic hydroxyl groups in the lignin fractions were decreased from F to F4. The antioxidant capacity of the lignin fractions showed a decreasing trend with increasing the dissolving capacity of solvent. The lignin fraction extracted with ethyl acetate, having a high RSI value, would be a promising material as stabilizer to prevent polymer aging. References [] S. Sahoo, M.. Seydibeyoglu, A.K. Mohanty, M. Misra, Characterization of industrial lignins for their utilization in future value added applications, Biomass Bioenerg. 5 (20) [2] A. Casas, M. liet, M.V. Alonso, F. Rodríguez, Dissolution of Pinus radiata and Eucalyptus globulus woods in ionic liquids under microwave radiation: Lignin regeneration and characterization, Sep. Purif. Technol. doi:0.06/j.seppur [] T.K. Kirk, W. Brown, E.B. Cowling, Preparative fractionation of lignin by gel-permeation chromatography, Biopolymers. 7 (969) 5 5. [4] A. Toledano, A. Garcia, I. Mondragon, J. Labidi, Lignin separation and fractionation by ultrafiltration, Sep. Purif. Technol. 7 (200) 8 4. [5] C. Bhattacharjee, P.K. Bhattacharya, Ultrafiltration of black liquor using rotating disk membrane module, Sep. Purif. Technol. 49 (2006) [6] A. Garcia, A. Toledano, L. Serrano, I. Egues, M. Gonzalez, F. Marin, J. Labidi, Characterization of lignins obtained by selective precipitation, Sep. Purif. Technol. 68 (2009) [7] R.W. Thring, M.N. Vanderlaan, S.L. Griffin, Fractionation of Alcell R lignin by sequential solvent extraction, J. Wood Chem. Technol. 6 (996) [8] C. Schuerch, The solvent properties of liquids and their relation to the solubility, swelling, isolation and fractionation of lignin, J. Am. Chem. Soc. 74 (952) [9] R.C. Sun, J. Tomkinson, S. Griffiths, Fractional and physico-chemical analysis of soda- AQ lignin by successive extraction with organic solvents from oil palm EFB fiber, Int. J. Polym. Anal. Ch. 5 (2000) [0] K. Wang, F. Xu, R. Sun, Molecular characteristics of Kraft-AQ pulping lignin fractionated by sequential organic solvent extraction, Int. J. Mol. Sci. (200) [] T.Q. Yuan, J. He, F. Xu, R.C. Sun, Fractionation and physico-chemical analysis of degraded lignins from the black liquor of Eucalyptus pellita KP-AQ pulping, Polym. Degrad. Stabil. 94 (2009) Paper PS-77 8 of 9

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