Branched Structures of Softwood Celluloses - American Chemical

Downloaded by CITY UNIV OF HONG KONG on October 25, 2017 | http://pubs.acs.org Publication Date (Web): October 23, 2017 | doi: 10.1021/bk-2017-1251.ch008

Chapter 8

Branched Structures of Softwood Celluloses: Proof Based on Size-Exclusion Chromatography and Multi-Angle Laser-Light Scattering Yuko Ono, Tsuguyuki Saito, and Akira Isogai* Department of Biomaterials Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan *E-mail: [email protected].

Wood holocelluloses, softwood α-cellulose, bleached kraft pulps, and bacterial cellulose were dissolved in 8% (w/w) lithium chloride/N,N-dimethylacetamide (LiCl/DMAc) after ethylenediamine pretreatment and subsequent solvent exchange to DMAc. The obtained solutions were diluted to 1% (w/v) LiCl/DMAc and analyzed using size-exclusion chromatography and multi-angle laser-light scattering (SEC-MALLS). The holocelluloses and kraft pulps showed bimodal molecular-mass distributions because of the presence of high-molecular-mass (HMM) cellulose-rich and low-molecular-mass fractions. The neutral sugar compositions of the samples were also determined and compared with the data obtained by SEC-MALLS analysis. The results showed that hardwood holocelluloses and hardwood bleached kraft pulp had linear cellulose molecules in the HMM fractions and random coil conformations in the solution as in the case of bacterial cellulose. In contrast, softwood holocellulose, softwood α-cellulose, and softwood bleached kraft pulp were likely to have cellulose molecules partly branched with glucomannan molecules through lignin fragments in the HMM fractions.

© 2017 American Chemical Society Agarwal et al.; Nanocelluloses: Their Preparation, Properties, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Introduction Cellulose, hemicelluloses, and lignin, which are present in wood cell walls as natural composites, contribute to the high mechanical strengths of living trees and the timber produced from them. The formation and presence of lignin–carbohydrate complexes or chemical linkages between lignin and hemicelluloses in native wood cell walls have been discussed in the literature, although no direct evidence of these linkages has been reported (1–5). Probably, the amounts of such chemical linkages in wood components are too small to be detected or isolated using common analytical methods for determining chemical structures. A combination of size-exclusion chromatography and multi-angle laser-light scattering (SEC-MALLS) is a powerful tool for studying the conformations and branched structures of polymers dissolved in good solvents. SEC-MALLS analysis provides the molecular-mass parameters of polymers such as the weight-average and number-average molecular masses (Mw and Mn, respectively), molecular conformations, branched structures, and degrees of branching when polymers are dissolved in solvents at the individual molecular level without any aggregation. LiCl/N,N-dimethylacetamide (LiCl/DMAc) and LiCl/1,3-dimethyl-2-imidazolidinone (LiCl/DMI) are suitable cellulose solvents for SEC-MALLS analysis because they are transparent and dissolve pure celluloses without degradation, and the obtained solutions are stable for a long time at room temperature (6–11). Many studies of the molecular-mass parameters of cellulosic materials using SEC-MALLS have been reported (6–11). Most hardwood kraft pulps and some pure celluloses such as cotton and cotton linters celluloses, and microcrystalline cellulose are soluble in 8% (w/w) LiCl/DMAc after solvent exchange from water to DMAc through acetone. However, softwood kraft pulps, wood holocelluloses, and some pure celluloses with high crystallinities such as bacterial, tunicate, and algal celluloses cannot be dissolved in LiCl/DMAc even after solvent exchange from water to DMAc. In contrast, the LiCl/DMI system can dissolve most softwood kraft pulps and pure celluloses, and can therefore be used for their SEC-MALLS analyses (11–15). However, DMI is much more viscous than DMAc; this causes operational problems in SEC-MALLS analysis of cellulose solutions in LiCl/DMI and it is sometimes difficult to obtain reliable data. In our previous paper, we reported that ethylenediamine (EDA) pretreatment and subsequent solvent exchange to DMAc through methanol enables dissolution of almost all kraft pulps, wood holocelluloses containing large amounts of hemicelluloses, and pure celluloses in 8% (w/w) LiCl/DMAc (16–18). More reliable molecular-mass parameters can therefore be obtained for almost all cellulosic samples using LiCl/DMAc as the solvent and eluent in SEC-MALLS analysis (19). In this study, Japanese cedar, ginkgo, eucalyptus, and birch holocelluloses, Japanese cedar α-cellulose, and softwood and hardwood bleached kraft pulps (SBKP and HBKP, respectively) were dissolved in 8% (w/w) LiCl/DMAc after EDA pretreatment and subsequent solvent exchange to DMAc through methanol. The obtained solutions were diluted to 1% (w/v) LiCl/DMAc and subjected to 152 Agarwal et al.; Nanocelluloses: Their Preparation, Properties, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

SEC-MALLS analysis to study the structures and conformations of the cellulose molecules in the solvent. The neutral sugar compositions of the same samples were also determined. Bacterial cellulose was used as a pure standard cellulose without any branched structures.

Experimental Section


Materials Commercially available never-dried HBKP and SBKP were used (Nippon Paper Ind. Co., Japan). Freeze-dried bacterial cellulose fine particles were prepared from bacterial cellulose pellicles incubated in our laboratory by purification and disintegration in water (17, 20). Japanese cedar (Cryptomeria japonica), ginkgo (Ginkgo biloba), eucalyptus (Eucalyptus globulus), and birch (Betula maximowicziana) wood meals were prepared from the coppresponding wood chips using a high-speed mill (HS-20, Senion Ind. Ltd., Tokyo, Japan). LiCl, DMAc, EDA, and other chemicals and solvents used were laboratory grade (Wako Pure Chemicals Ind. Ltd., Japan) and used as received.

Preparation of Holocelluloses and α-Cellulose Wood meals were dewaxed by soaking in 90% aqueous acetone at room temperature for 1 d and air-dried. The dewaxed wood meals were delignified using the Wise method (21). One delignification cycle involved treatment in NaClO2-containing water at pH 4–5 and 70 °C for 1 h, and this cycle was repeated six times for Japanese cedar and ginkgo, and three times for eucalyptus and birch. Japanese cedar holocellulose was soaked in 17.5% NaOH at room temperature for 1 h to prepare α-cellulose. The yields of Japanese cedar, ginkgo, eucalyptus, and birch holocelluloses, and Japanese cedar α-cellulose were 79%, 77%, 83%, 68%, and 60%, respectively, based on the dry weights of the dewaxed wood meals.

Preparation of Sample Solutions in 8% LiCl/DMAc All the holocelluloses, pulps, Japanese cedar α-cellulose, and bacterial cellulose (0.04 g each) were stirred in EDA (10 mL) at room temperature for 4 d. The EDA-containing samples were solvent-exchanged to DMAc through methanol using centrifugation (17, 18). The EDA-pretreated and solvent-exchanged samples were suspended in 8% (w/w) LiCl/DMAc (10 g), and the mixtures were stirred at room temperature to obtain clear solutions. The kraft pulps, hardwood holocelluloses, bacterial cellulose, and α-cellulose dissolved in 8% (w/w) LiCl/DMAc within 1 month, and it took approximately 4 months to prepare clear solutions of Japanese cedar and gingko holocelluloses. The solutions were diluted with DMAc to 1% (w/v) LiCl/DMAc to prepare ~0.04% or ~0.02% (w/v) sample solutions for SEC-MALLS analysis. 153 Agarwal et al.; Nanocelluloses: Their Preparation, Properties, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


SEC-MALLS-UV Analysis The SEC-MALLS system consisted of a SEC column (KD-806M, Shodex, Japan), a photodiode array detector for determining the UV absorption at 280 nm of each SEC-elution volume (SPD-M20A; Shimadzu), a MALLS detector (DOWN HELEOS-II, λ = 658 nm, Wyatt Technologies, USA), and a refractive index (RI) detector (RID-10A, Shimadzu, Japan). These units were connected in this order after sample injection. The eluent and specific RI increment (dn/dc) used were 1% (w/v) LiCl/DMAc and 0.131 mL/g, respectively (18). The other SEC attachments and operating conditions have been described elsewhere (17, 18). Molecular-mass parameters such as Mw and Mn, and the number of branch units per molecule were calculated using ASTRA IV software attached to the MALLS system (Wyatt Technologies, USA). Neutral Sugar Composition Analysis Dried samples (50 mg each) were dissolved in 72% (w/w) H2SO4 (0.5 mL) for 2 h followed by hydrolysis with 3% (w/v) H2SO4 (14.5 mL) at 120 °C for 1 h. The internal standard was myo-inositol. Sulfate ions in the mixtures were removed with Ba(OH)2 and BaCO3, and the hydrolysates were then freeze-dried. The obtained solids were dissolved in a water/acetonitrile mixture (0.5 mL each), and the solutions were subjected to high-performance liquid chromatography (HPLC). The HPLC system consisted of guard and amino columns (NH2P-50G 4A and NH2P-50 4E, respectively, Shodex, Japan), and an RI detector (Optilab T-rEX, λ = 658 nm, Wyatt Technologies, USA). HPLC analysis was performed using an eluent consisting of a 75% (v/v) acetonitrile/water solution containing 250 mM phosphoric acid at 50 °C (17). Calibration lines for glucose (Glc), galactose (Gal), mannose (Man), arabinose (Ara), xylose (Xyl), and rhamnose (Rha) were prepared beforehand, and used for neutral sugar composition analysis of the hydrolysates.

Results and Discussion SEC-MALLS Analysis When pretreatment by EDA soaking and subsequent solvent-exchange treatment was used, all the wood holocelluloses, kraft pulps, α-cellulose, and bacterial cellulose had dissolved in 8% (w/w) LiCl/DMAc after stirring at room temperature within 4 months (17, 18). Small amounts of hemicelluloses in the holocelluloses (

Branched Structures of Softwood Celluloses - American Chemical

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