Revealing Structural Differences between Alkaline and Kraft Lignins

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Revealing Structural Differences between Alkaline and Kraft Lignins by HSQC NMR Chengke Zhao, Jingtao Huang, Linjie Yang, Fengxia Yue, and Fachuang Lu Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.9b00499 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 25, 2019

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Industrial & Engineering Chemistry Research

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Revealing Structural Differences between Alkaline and Kraft Lignins by HSQC

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NMR

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Chengke Zhao,† Jingtao Huang,† Linjie Yang† Fengxia Yue† and Fachuang Lu✲,†, ‡

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†

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Guangzhou, 510640, China

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‡

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✲ Corresponding

State Key Laboratory of Pulp and Paper Engineering, South China University of Technology,

Guangdong Engineering Research Center for Green Fine Chemicals, Guangzhou 510640, China. author: Fachuang Lu, E-mail: [email protected], Tel: +86 2-87113953

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ACS Paragon Plus Environment

Industrial & Engineering Chemistry Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT:

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Technical lignins, mostly generated as by-products from pulping industry, are highly abundant

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aromatic sources. However, they are underutilized due to their complexity, as well as the structural

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alteration during chemical pulping processes. In-depth elucidation of technical lignins is becoming

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essential for their valorization in view of lacking understanding of technical lignins structures. In

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this work, Alkaline and Kraft lignins were prepared and comparatively characterized by 2D HSQC

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NMR. By applying authenticated reference compounds, the phenylglycerol structures, characteristic

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of the cleavage of non-phenolic β-aryl ether by Soda pulping, in the alkaline lignin were identified

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and quantified by 2D HSQC NMR. Phenylglycerol structures in alkaline lignin were estimated to

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be 8% - 14%, which was much higher than that in Kraft lignin. This finding was supported by the

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results from model studies, i.e., the yield of phenylglycerol product was 40% that obtained from

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soda pulping treatment of non-phenolic β-aryl ether compound while the yield of that from kraft

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treatment was lower than 5%. In addition, styryl ether structures from phenolic β-aryl ethers of

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alkaline lignin were also revealed by 2D NMR. These new findings will benefit to mechanistic

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understanding of lignin reactions and structural elucidation of technical lignins, which will provide

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useful information (or new insights) for the development of lignin valorization strategies.

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KEYWORDS: Alkaline lignin; Kraft lignin; Structural elucidation; 2D HSQC; β-O-4 linkage; Aryl

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glycerol

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1. INTRODUCTION

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Lignins are abundant biopolymers present in plants accounting for approximately 30% of non-

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fossil organic carbon on the earth.1-2 Owing to their high carbon content and aromatic structural

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units, lignins have great potential to be used for producing chemicals, biofuels and materials.3-7

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Lignin is made mainly from three monolignols: p-coumaryl, coniferyl and sinapyl alcohols, which

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make up lignin polymer through end-wise radical coupling reactions forming aryl ether and carbon-

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carbon (C-C) bonds between units. In principle, β-aryl ether linkage (β-O-4) is the most abundant

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interunit linkage type, which constitutes of over 50% of all interunit linkages in lignin.1-2

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Technical lignins are primarily obtained from lignocellulosic biomass pulping processes for

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cellulosic pulps. During a pulping process, complicated reactions happen to lignin, which lead to

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the cleavage of large number of aryl ether bonds and generation of many uncertain condensed

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structures bearing stable C-C bonds.8-10 Such reactions cause severe structural alterations of the

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resultant technical lignins. That makes structural analysis and the valorization of technical lignins

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very challenging.4, 11-12 In paper industries, a large amount of technical lignins can be produced,

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while they are underutilized and usually used as fuel to burn or even discharged with other

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components in spent liquors.13-14

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Nowadays, NMR spectroscopy is becoming more and more useful tool for the structural

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elucidation of lignins. The application of advanced heteronuclear 2D NMR in the lignin analysis

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has greatly improved the structural understanding of these materials. Many efforts have been

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focused on the characterization of various technical lignins by HSQC (heteronuclear single-quantum

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coherence) NMR technology,15-21 and have provided many insights into their structural features,

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which not only are significant for the understanding of lignin fundamental reaction during 3



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processing, but also helpful for developing efficient process for lignin valorization. Recently, with

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the aids of advanced lignin model compounds, the structure of some technical lignins, both in terms

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of interunit linkages and functional groups, has been revealed. Structures with styryl/enol ether,

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stilbene, epiresinol, diarylmethane, and end-groups (arylglycerol, aryl acetic acid, aryl propanol, β-

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hydroxy acid) have been identified by 2D HSQC in kraft lignins.10, 12, 22-25 However, structures of

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kraft lignins were various,10, 26 and highly dependent on pulping processes, conditions, and even

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their separation methods. Some structures being minor or ignored in one case could become

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significant in another one. For instances, the aryl glycerol/phenyl glycerol structure, a major product

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from cleavage of β-O-4 compound, has chronically been suggested be produced under alkaline

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pulping conditions.16, 27-29 However, it has not been reported in technical lignins until recently, in

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which kraft lignins were found to contain small quantity of arylglycerol structures. 12

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Recently, an alkaline/soda lignin (AL) produced from eucalyptus soda pulping process with

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addition of a deresinator (a surfactant) drawn our attention because of three strong correlation

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signals at: δC/δH 74.4/4.43, 75.8/3.56 and 63.4/3.21-3.85 (partial spectrum is shown in Figure 1A,

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full spectrum is shown in Figure S1) in 2D HSQC NMR of the AL. Alkaline pulping without adding

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the deresinator also produced lignin with similar NMR characteristics. Although these signals were

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very close and potential to be the C-H correlations of phenylglycerol’s side chain, they were

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suspicious since such strong signals have not been reported in many technical lignins. This

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observation promoted us to revisit the β-O-4 lignin model reactions in such alkaline treatments,

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performing careful assignment of these signals and structural comparison of ALs with kraft lignins

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(KLs) aimed to identify or confirm the produced structures.

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Herein, we report the isolation and 2D HSQC comparative characterization of the ALs and 4


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KLs from spruce and eucalyptus wood, with focus on quantitation of phenylglycerol and styryl

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structures produced from cleavages of β-O-4 linkages in lignin models and lignins under Soda or

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Kraft pulping conditions. Our findjngs will provide with a deeper understanding on the features of

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technical lignins (ALs and KLs) for better lignin utilizations.

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2. EXPERIMENTAL

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2.1. Materials

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Spruce (softwood) and eucalyptus (hardwood) wood chips with about 3 cm length and 2 cm

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width, obtained from local pulping mills, were used as pulping feedstocks. The wood chips were

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thoroughly washed with water and dried at 80 °C for 12 h in oven. Lignin model compound, 4-

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benzyloxy-3-methoxyphenyl glycerol-β-aryl ether (seen in Scheme 1), were synthesized according

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to published methods.30 The method for synthetizing styryl ether compounds (Z/E-isomers) was

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described in supplementary materials. Two types of phenyl glycerol compounds, 3-methoxy 4-

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hydroxyl phenylglycerol and 3,5-dimethoxy-4-hydroxyl phenylglycerol were synthesized as

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previous reported.31 The corresponding benzylated and acetylated products were synthesized, and

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the structures were confirmed by NMR (Figure S4 - S11). Other chemicals used in this study were

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purchased from Macklin Biochemical Co., Ltd. (Shanghai, China).

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2.2 Quantitative analysis of products from reaction of model compounds

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The reactions of lignin model compounds under pulping conditions were performed in a 20-

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mL hydrothermal reactor.

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solution (8 mL) or 1M NaOH and 0.25 M Na2S solution (8 mL) for alkaline treatment or Kraft

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treatment, respectively. The hydrothermal reactor was put in an oven and the temperature was kept

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at 170 °C for 2 h. Then, the reactor was cooled down with tap water. The pH value of the reaction

20 mg of non-phenolic dimeric compound were added into a 1 M NaOH

5



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liquid was adjusted to below 3 by adding 3% HCl. The acidic turbid liquid was extracted with

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CH2Cl2 (4×10 mL). The combined organic layers was washed with saturated NaCl and dried with

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anhydrous MgSO4. After filtration, the filtrate was collected into a flask and evaporated under

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reduced pressure.

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The reaction products were analyzed and quantified by GC/MS after trimethylsilyl

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derivatization by using of standard compounds. The products in flask were dissolved into 4 mL

128

CH2Cl2 from which 1 mL of solution was transferred into a GC vial. After pyridine (150 μL) and

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N, O-bis trimethylsilyl trifluoroacetamide (BSTFA, 98%, 150 μL) were added into the vial, the

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mixture was heated to 60 °C and kept for 40 min. The TMS-derivatized products were analyzed by

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GCMS-TQ-instrument (Shimadzu GCMS-TQ8040 triple quadrupole GC/MS/MS) equipped with a

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SH-Rxi-5Sil MS column (Shimadzu, 30 m × 0.25 mm × 0.25 μm). The solutions with different

133

concentration of reference compounds (phenylglycerols, styryl ethers and other compounds) were

134

prepared and analyzed by GC/MS under the same conditions. The content of reaction products was

135

determined by plotting the calibration curve (peak area vs. reference compound concentration). The

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product yield was calculated based on the amount of reacted dimeric compound, as follows:

137

Yield % 

n product n0  n

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Where n product is the molar amount of product, n0 and n are the molar amount of the initial β-O-4

139

dimer used and the remained β-O-4 dimer, respectively.

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2.3 Cellulolytic enzyme lignin preparation

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The cellulolytic enzyme lignin (CEL) was prepared according to the method described in the

142

literature.32 Briefly, the wood chips were smashed to obtain the meal sample between 30 - 80 mesh,

6


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and then extracted with 80% ethanol. The extractive-free sample (3 g) was subjected to ball-milling

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by using a PM 100 mill (Retsch, Germany). The ball-milled sample (8 g) was incubated at 40 °C,

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in a 20 mM NaOAc buffer (100 mL, pH 4.8) containing 300 mg Celluclast 1.5 L (a cellulase with

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enzymatic activity value of 700 EGU/g, Novozymes) and 400 mg Viscozyme L (a cell wall

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degrading enzyme complex with enzymatic activity value of 100 FBG/g, Novozymes) for 48 h (2

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times). Then, the suspension was centrifuged, and the solid residue was freeze-dried, extracted with

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96% dioxane (100 mL, 12 h, 2 times). After centrifugation, the supernatant was collected and

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concentrated to ~3 mL, then transferred into 45 mL cold water in a centrifuge tube. The precipitated

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solid products (CEL) were collected by centrifugation. The CEL was finally obtained after freeze-

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drying. The CEL yields for spruce and eucalyptus were 38% and 44% respectively, based on the

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klason lignin of these two kinds of wood materials.

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2.4 Alkaline lignin and Kraft lignin preparations

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The alkaline and Kraft pulping processes of wood feedstocks were conducted in a 1 L digester.

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The dosage of chemicals and the liquor ratio are listed in Table S1. More drastic conditions are

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generally used for spruce pulping process than those used for eucalyptus due to more condensed

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lignins of spruce.33 After pulping, the pulp yields from alkaline and kraft processes were all over

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50%, and over 90% lignin was dissolved in the pulping black liquor --- the residual lignin content

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in pulp was less than 4% for eucalyptus, and about 5% for spruce. The lignin was precipitated out

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by blowing CO2 into the stirred spent liquor (100 mL) to lower its pH to 8.5. The crude lignin (70%

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- 80%, based on the lignin removed from feedstocks) was collected after centrifugation and air-

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drying. Then the crude lignin was washed with deionized water (2 × 100 mL, pH 5) to remove

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some salts and saccharides. After centrifugation and freeze-drying, small molecular fraction was 7



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removed by extraction with CH2Cl2 (3×50 mL). Finally, the purified lignins (28 - 46% yield based

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on the lignin removed from feedstocks, Table S2) were obtained.

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2.5 Acetylated derivatization of lignins

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Lignin sample (200 mg) was dissolved in pyridine/acetic anhydride (1:1, v/v) solution. The

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reaction mixture was kept at room temperature for 12 h. The product solution was dried under

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reduced pressure at 45 ° C, co-evaporating with additional ethanol several times to remove the

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pyridine and acetic acid.

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2.6 2D NMR HSQC experiments

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NMR spectra were recorded on a Bruker AVANCE Ⅲ HD 600 MHz spectrometer. Lignin

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model compound (~10 mg) or lignin preparation (80 mg) was dissolved in 0.5 mL DMSO-d6 in a

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NMR tube, respectively. The Bruker program of “hsqcedetgpsisp 2.3” was selected for HSQC

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(heteronuclear single quantum coherence) experiments. HSQC experiments for the lignins were

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performed using the following parameters: acquired from 10 to 0 ppm in F2 (1H) with 2048 data

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points and a 1s recycle delay, 160 to 0 ppm in F1 (13C) with 256 increments of 64 scans. The total

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acquisition time for a sample was 5 h. The experiments for the model compounds or the reaction

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products of β-O-4 dimer were acquired in F1 (13C) with 256 increments of 8 scans, and the total

181

acquisition time was about 37 min. The central DMSO solvent peak δppm (39.5, 2.49) was used for

182

calibration of correlation peaks. Volume integration of contours in HSQC spectrum used Bruker’s

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Topspin 4.0.3 software.

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3. RESULTS AND DISCUSSION

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3.1 Identification of phenylglycerol structures in ALs by 2D HSQC 8


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The 2D HSQC spectrum of AL from eucalyptus wood showed three strong signals (δC/δH

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74.4/4.43, 75.8/3.56 and 63.4/3.21-3.85, Figure 1A) close to those of C-H correlations from

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sidechain of phenylglycerol structures based on the reported data.34 In order to verify such structures,

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two types of model compounds, 3-methoxy 4-benzyloxy aryl glycerol (G-gly) and 3,5-dimethoxy-

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4-benzyloxy aryl glycerol (S-gly), representing the potential guaiacylglycerol and syringylglycerol

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structures in alkaline lignins, were synthesized and characterized with 2D HSQC NMR. The C-H

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correlations of G/S-glycerol sidechains are summarized in Table 1. When comparing HSQC spectra

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of the authenticated phenylglycerol compounds G-gly and S-gly with that of AL (Figure 1A, B and

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C), it was obviously that the three broad signals at δC/δH 73.2-75.2/4.34-4.54, 74.7-76.9/3.43-3.65

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and 61.2-65.6/3.19-3.89 for AL match well with those correlations in spectra of G-gly and S-gly

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corresponding to Cα-Hα, Cβ-Hβ and Cγ-Hγ correlations (Figure 1B and 1C). The broader

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correlations found in the spectrum of AL than those obtained from model compounds (single isomer)

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could be contributed to the isomeric/diverse structures and polymeric features of AL. The structures

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were further verified by comparing the HSQC spectra of acetylated reference compounds and AL

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(Figure 1D). The correlations associated with glycerol sidechain changed to δC/δH 72.6/5.86,

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71.3/5.34 and 61.5/3.91- 4.13 after acetylation. The acetylated AL also showed strong correlation

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signals at this area, and the signals of the lignin polymer completely overlapped with peaks of

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compounds. Both assignments on the native and acetylated ALs provided strong evidences that the

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three strong signals in the AL belong to phenylglycerol sidechain moiety.

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Table 1. 13C and 1H chemical shifts (ppm) of side chains of 3-methoxy 4-benzyloxy aryl glycerol (G-gly) and 3,5-dimethoxy-4-benzyloxy aryl glycerol (S-gly) in DMSO-d6.

Model

G-gly

S-gly

α

72.4/4.45

73.0/4.47 9



β γ

75.4/3.47 62.3/3.16 and 3.34

75.6/3.49 62.5/3.19 and 3.36

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209 210 211 212 213 214

Figure 1. Partial 2D HSQC NMR spectra of non-acetylated/acetylated eucalyptus alkaline lignin (AL) and phenylglycerol compounds for peak assignment. The unknown peaks in (A) eucalyptus AL were compared with the peaks from (B) G-gly and (C) S-gly. The phenylglycerol structure in AL was also confirmed by matching the peaks from acetylated compounds and acetylated AL (D).

3.2 Structural comparison between ALs and KLs revealed by 2D HSQC

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As shown in Figure 1A, the content of phenylglycerol structure revealed by 2D HSQC NMR

216

in the eucalyptus AL was very significant (~ 10%). However, it is interesting that such structures or

217

the signals were ignored or not recognized in many technical lignins before.10, 18, 20-21, 23 Therefore

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efforts were made here to quantitatively estimate these structures in ALs and KLs.

219

In native lignin, the non-phenolic β-O-4 unit is the dominant linkages, accounting for over 50-

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60% of all linkages. Pulping process under alkaline conditions could generate phenolic units by

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cleaving β-O-4 ether bonds. Although the reaction pathways of phenolic/non-phenolic β-O-4 10


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222

models in alkaline treatment has been reported, the quantitative comparison of their products under

223

alkaline (NaOH-only) and kraft (NaOH + Na2S) conditions was lacking.6, 9-10, 25 In current study,

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quantitation of products from phenolic β-O-4 models (dimers) suggested that alkaline process

225

resulted in higher yield of styryl ether products (39%, including 15% Z-isomer and 24% E-isomer),

226

than the kraft process did. Therefore, sulfides apparently facilitated the cleavage of the β-aryl ether

227

linkage, leading to higher guaiacol yield (from 21% to. 33%) and decreased the yield of styryl ether

228

(from 38% to 23%) under the kraft conditions (Scheme 1). These results can be explained by the

229

nucleophilic attack of HS- to the quinone methide intermediate at α-position forming a sulfite that

230

facilitates the cleavage of the β-aryl ether via neighboring assistance mechanism,7, 35 reducing the

231

formation of styryl ether. As for the non-phenolic dimer concerned, the yields of phenylglycerol

232

obtained in soda pulping were different from that obtained in kraft pulping, being 40% (based on

233

the reacted starting dimer), much higher than that (< 5%) from kraft pulping (Scheme 1). This

234

implied that the strong nucleophilic HS-/S2- in kraft process accelerates cleavage of -ether leading

235

to formation of mercaptide or polysulfide species (Sx2-),7, 35 instead the production of phenylglycerol

236

structure that was major products in Soda pulping.

11



237 238 239

Scheme 1. Quantitative analysis of the products from β-O-4 compounds after alkaline and kraft

240 241 242

quantity of reacted dimer (Figure S14 and S15). Less syringyl monomer was detected compared to the

formation of phenylpropyl derivatives after alkaline treatment, potentially due to some condensation reactions involving syringyl monomers.

243

Based on the above discussion about the model studies, more detailed structural features of

244

ALs and KLs from both eucalyptus and spruce were then comparatively analyzed by 2D HSQC

245

NMR. Carbon dioxide (CO2) was used to adjust the pH (to 8.5) of pulping liquors to precipitate and

246

recover the alkaline and kraft lignins. It is a “green” and low-cost process (called Ligninboost

247

process, commercialized in Europe) compared to the use of inorganic acids, and has been tested to

248

be preeminent in pulp/paper mills from an engineering and economic point of view.36 Although a

249

further reduction of pH value of pulping liquor by using acids can recover more lignin components,

250

the portion recovered under acidic condition has very low molecular weight, and contains more

251

carbohydrates.37 This portion was not considered in our work. The cellulolytic enzymatic lignins,

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CELs, were used as references. The aliphatic oxygenated and aromatic/unsaturated regions (δC/δH

treatments. The yields of products were determined by external standard method based on the mole

12


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253

45-135/2.5-8.0 ppm) were mainly concerned. The relative abundance of interunit linkages or

254

functional groups was estimated by integrating the corresponding correlated contours in their HSQC

255

spectra (the aromatic G2 (and S2,6) signals were used as lignin reference).

256

As shown in Figure 2, the dominant interunit linkage in CELs was β-aryl ether linkage (β-O-

257

4, A, 55-60%), followed by phenylcoumaran and resinol. Phenylglycerol structures were not

258

observed in the HSQC spectra of CELs (Figure 2), although it could be produced by ball-milling

259

during lignin isolation process.38 Pulping process with alkaline media promotes biomass

260

delignification by cleaving aryl ether bonds and solubilizing lignin.39-40 In the spectrum of ALs, a

261

small amount of β-O-4 linkage (6.0%) and phenylcoumaran structure (5.2%) were observed in the

262

spruce AL whereas no β-O-4 and phenylcoumaran can be identified in the eucalyptus AL. This

263

observation agrees that the β-aryl ether linkage of G units was more stable than that between G and

264

S units or S and S units.41-42 The resinol (β-β) structures with the stubborn C-C linkage was

265

substantially reserved during alkaline and kraft treatments. The signal at δC/δH 53.0/3.73 belongs

266

to the Cα-Hα correlation of α-5 linkage (E), arisen from condensed reaction of lignin fragments.16,

267

41

268

120.2/7.23, corresponding to Cα-Hα and Cβ-Hβ signals, in the spectra of spruce AL and KL. This

269

structure was mainly produced from phenylcoumaran (β-5) unit after pulping treatment.15, 42

A little stilbene structure (H) can be identified at the correlations of δC/δH 128.0/7.08 and δC/δH

270

Alkaline treatment of free phenolic β-O-4 structure produced styryl ether with two isomers:

271

Ftrans and Fcis, and their corresponding cross-signals of Cα-Hα can be respectively observed at δC/δH

272

112/6.11 and δC/δH 109.2/5.53 from the spectra of spruce AL and KL. The abundance of styryl ether

273

unit was 13.2% (7.5% trans and 5.7% cis) in spruce AL, higher than the value (6.2%, including

274

4.0% trans and 2.2% cis), in accordance with result of model reaction. Whereas, this structure was 13



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275

not detected in the eucalyptus AL. Presumably, in hardwood lignin most S unit are involved in

276

etherified β-O-4 structure that are cleaved leading to phenylglycerol structures.41-42

277 HO  HO  

O4

HO  

5

OMe

A (-O-4)

OMe

5 



 O

B (-5)



C ()

E (-5)

OH 



2

6 5

279

F (Styryl ether)

X (Phenylglycerol)

OH HO 

O

J

H (Stilbene)

2

6

OMe O

278

OMe OH

HO  HO  OH 

OMe

O 

O



O 

MeO





MeO

OMe O

G

S

Polysacharides, Unassigned signals

Figure 2. Partial 2D HSQC NMR spectra of enzyme lignin (CEL), alkaline lignin (AL) and kraft lignin 14


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280 281

(KL) of spruce and eucalyptus. The content of structural moieties was determined by integrating the

282

As shown in Figure 2, the phenylglycerol structure (X) can be readily recognized in the

283

aliphatic oxygenated side chain region of the spectra according to NMR data obtained from

284

reference compounds discussed. It was reported that the Cβ-Hβ corelation overlaps with the C4-H4

285

correlation signal of xylan.16, 43 Xylan was not observed in the lignin spectra of eucalyptus materials

286

while it could be identified from the spectra of spruce AL. Integrating Cβ-Hβ signal can result in an

287

overestimation of glycerol content in spruce AL. The relative contents of phenylglycerol estimated

288

by using Cα-Hα signal were 8.9% and 8.6%, respectively for spruce AL and eucalyptus AL.

289

Although small amounts of phenylglycerol were produced in Kraft pulping of β-aryl ether model

290

compound, the phenylglycerol structures were not detected in eucalyptus KL. However, in the

291

spectrum of spruce KL, signal accounting for 1.5% was observed. This signal could be also

292

responsible for Ar-CHOH-COOH (J) in KL, because the Cα-Hα signal of J overlaps with the Cα-

293

Hα signal of phenylglycerol.16,

294

15% in alkaline pulping process (16.8% and 14.3% for spruce eucalyptus respectively, produced

295

from the destruction of β-O-4 linkages). It should be noted that the cleavage of β-aryl ether bond in

296

lignin polymer produces phenolic β-O-4 units which could undergo a different reaction pathway

297

from non-phenolic units, explaining much less phenylglycerol structures resulted from lignin than

298

those from model compounds (Scheme 1).

contours (C-H correlation signals of G2 and/or S2,6 were used as references).

43

On the other hand, the yield of phenylglycerol units was about

299

The 2D HSQC analysis of acetylated lignins also showed that ALs had much more

300

phenylglycerol structures than KLs (Figure S16, Table 2), which was consistent with the results by

301

HSQC analysis of original samples. Moreover, the content of glycerol structure in acetylated lignins

302

determined by 2D HSQC was higher than the value obtained from underivatized lignins (Table 1). 15



303

The possible reason is that acetylation may have eliminated the hydrogen bonding associated with

304

glycerol sidechains, enhancing the intensity of corresponding correlation signals of NMR.

305

Nevertheless, it was still difficult to judge which way (acetylated or not) is better for quantifying

306

the phenylglycerol structure in lignins, because the intensity of C-H correlation signal in HSQC

307

spectrum is also affected by its chemical environments.

308 309

Table 2. Content of phenylglycerol end-groups in ALs and KLs determined by 2D HSQC NMR.

Types AL

KL

Eucalyptus

Spruce

Underivatized

8.6%

8.4%

Acetylated

13.4%

9.3%

Underivatized

nd

1.5%

Acetylated

nd

2.7%

310

Overall, the ALs recovered by CO2 neutralization have been showed by 2D HSQC to contain

311

much more phenylglycerol structure than KLs. The content of glycerol structure in ALs was

312

determined to be in the range of 8% - 14% dependent on their origins and evaluation methods (Table

313

2). The relatively severe pulping conditions cleaving almost all of β-aryl ether bonds in the lignin

314

might be responsible for a higher phenylglycerol content compared to some mild conditions (under

315

which some ether bonds are retained). Moreover, it was reported that the content of glycerol

316

structure was higher in lignin with higher molecular weight.12 Washing with dichloromethane

317

removed some low molecular weight fraction of hardwood (eucalyptus) lignins in addition to the

318

extractives (Table S2, S3), resulting a higher molecular weight fraction with a slight high content

319

of phenylglycerol structure. Therefore, complete breakdown of the abundant β-aryl ether bonds of

320

lignin followed by fractionation with dichloromethane extraction could produce technical lignins

321

with relatively simple structure enriched in glycerol and resinols (peaks of some structure might 16


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322

overlap with signal of methoxy group, Figure S1). For lignin modifications to produce the value-

323

added products, many methodologies including esterification, phenolation etc. aim to functionalize

324

hydroxyl groups.3 Therefore the ALs with high amount of phenylglycerol end-groups could be a

325

favorable feedstock for such chemical modifications. This type of AL polymer with unique

326

structural characteristic would have distinct properties useful for further material applications.

327 328

4. CONCLUSIONS

329

Herein, the phenylglycerol structures in ALs were unambiguously identified and confirmed by

330

2D HSQC NMR with use of authenticated model compounds. It is suggested that phenylglycerol

331

structure mainly generated from the non-phenolic β-aryl ether bonds under Soda pulping process

332

rather than the kraft pulping process. 2D HSQC NMR analysis of ALs from both hardwood and

333

softwood feedstocks showed that fractionated (purified) ALs with higher molecular weight had

334

much more glycerol end-groups (8%-14%) in comparison to the KLs. NMR analysis of acetylated

335

lignins revealed higher content of glycerol structure than that of underivatized lignin samples. These

336

new findings are valuable for better understanding of lignin reactions during chemical pulping

337

process, which will be beneficial to lignin processing and valorization.

338 339

ASSOCIATED CONTENT

340

Supporting Information

341

2D HSQC spectra of eucalyptus alkaline lignin and the products from non-phenolic β-aryl ether

342

compound (Figure S1, S3); Synthesis of non-acetylated/acetylated G/S-glycerol compounds and

343

styryl ether compounds; 1H NMR and 2D HSQC NMR of synthetized compounds (Figure S4-S13); 17



344

GC chromatograms of the products from reaction of model compounds (Figure S2, S14);

345

Calibration curves for quantification the products (Figure S15); 2D HSQC spectra of acetylated

346

alkaline and kraft lignins; Conditions for pulping processes (Table S1); Yield of recovered lignins

347

and their molecular weight distribution (Table S2, S3).

348 349

AUTHOR INFORMATION

350

Corresponding Author

351

✲ Corresponding

author: Fachuang Lu, E-mail: [email protected], Tel: +86 2-87113953

352 353

ORCID

354

Fachuang Lu: 0000-0002-6418-8992

355

Notes

356

The authors declare no competing financial interest.

357 358

ACKNOWLEDGEMENTS

359

The authors are grateful to the financial support for this work by the National Natural Science

360

Foundation of China (31770621), State Key Laboratory of Pulp and Paper Engineering (No.

361

2016TS03, 2018TS07, 201836), Guangdong Province Science Foundation for Cultivating National

362

Engineering Research Center for Efficient Utilization of Plant Fibers (2017B090903003) and the

363

Fundamental Research Funds for the Central Universities of SCUT, China (2018MS52).

364 365

REFERENCES 18


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485 486 487 488

Abstract graphic:

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graphic abstract



Figure 1. Partial 2D HSQC NMR spectra of non-acetylated/acetylated eucalyptus alkaline lignin (AL) and phenylglycerol compounds for peak assignment. The unknown peaks in (A) eucalyptus AL were compared with the peaks from (B) G-gly and (C) S-gly. The phen¬ylglycerol structure in AL was also confirmed by matching the peaks from acetylated compounds and acetylated AL (D). 169x125mm (300 x 300 DPI)


Page 24 of 26

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Scheme 1. Quantitative analysis of the products from β-O-4 compounds after alkaline and kraft treatments. The yields of products were determined by external standard method based on the mole quantity of reacted dimer (Figure S14 and S15). Less syringyl monomer was detected compared to the formation of phenylpropyl derivatives after alkaline treatment, potentially due to some condensation reactions involving syringyl monomers. 123x88mm (300 x 300 DPI)



Figure 2. Partial 2D HSQC NMR spectra of enzyme lignin (CEL), alkaline lignin (AL) and kraft lignin (KL) of spruce and eucalyptus. The content of structural moieties was determined by integrating the contours (C-H correlation signals of G2 and/or S2,6 were used as references). 107x120mm (300 x 300 DPI)


Page 26 of 26

Revealing Structural Differences between Alkaline and Kraft Lignins

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