Water Solvent To Decrease Lignin

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Using green #-valerolactone/water solvent to decrease lignin heterogeneity by gradient precipitation Guanhua Wang, Xiaoqian Liu, Bo Yang, Chuanling Si, Ashak Mahmud Parvez, Jinmyung Jang, and Yonghao Ni ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b01641 • Publication Date (Web): 11 May 2019 Downloaded from http://pubs.acs.org on May 12, 2019

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ACS Sustainable Chemistry & Engineering

Using green γ-valerolactone/water solvent to decrease lignin heterogeneity by gradient precipitation Guanhua Wang †,‡*, Xiaoqian Liu †, Bo Yang ‡, Chuanling Si †, Ashak Mahmud Parvez §, Jinmyung Jang ‡,¶ and Yonghao Ni †,‡* †

Tianjin Key Laboratory of Pulp and Paper, College of Paper Making Science and Technology, Tianjin University of Science and Technology, Tianjin 300457, China ‡

Limerick Pulp & Paper Centre & Department of Chemical Engineering, University of New Brunswick, Fredericton, NB, E3B 5A3, Canada

§ Department

of Mechanical Engineering, University of New Brunswick, Fredericton, NB, E3B 5A3, Canada

¶

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto ON, M5B 0B5, Canada * Corresponding author. Tel.: +86 02260601313

Address: No.29 at 13th Avenue, Tianjin Economic Development Area (TEDA), Tianjin 300457, China E-mail address: [email protected] (Guanhua Wang)

[email protected] (Yonghao Ni)

1

ACS Paragon Plus Environment

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ABSTRACT Lignin heterogeneity, involving complex structure and high polydispersity, is a key challenge that restricts its value-added applications. Fractionation of heterogeneous lignin into several homogeneous subdivisions is an attractive and practical strategy to overcome this limitation. In this work, γ-valerolactone (GVL), a sugar-derived product, was used as a green solvent for lignin fractionation when mixed with water. The enzymatic hydrolysis lignin (EHL) was subdivided into three different fractions (F1, F2, and F3) by dissolving completely in 60% aqueous GVL and then following gradient precipitation in 40%, 30%, and 5% aqueous GVL solutions, sequentially. Detailed characterization techniques were conducted to provide a comprehensive evaluation of the three obtained lignin fractions. Moreover, the proposed fractionation mechanism was further investigated based on the Kamlet-Taft parameters. The gel permeation chromatography (GPC) analyses showed that the three fractions presented lower polydispersity than the parent EHL, furthermore, a gradual decreasing molecular weight due to the different solubility of various molecular weight lignins in aqueous GVL solvents. The structural analyses revealed that with the decrease of molecular weight, the guaiacyl unit content in lignin fractions decreased, with significant increases of functional groups (i.e. aromatic/aliphatic hydroxyl and carboxyl groups). The solvent recycling study showed that the aqueous GVL had a high recovery and the recycled GVL had the same lignin fractionation performance as fresh GVL. Overall, compared with traditional fractionation using multiple organic solvents, the present work provides a green and efficient route to fractionate lignin and therefore, significantly decreases its molecular weight polydispersity and structural heterogeneity. Keywords: Lignin heterogeneity, Lignin fractionation, γ-valerolactone, Lignin 2


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characterization, Molecular weight, Kamlet-Taft parameters INTRODUCTION Lignin, as the primary non-carbohydrate component of the terricolous plant cell wall, is the most abundant aromatic biopolymer in nature. Structurally, lignin is an amorphous phenolic polymer arising from copolymerization of three typical phenylpropanoid monomers through ether and C-C bonds. Lignin is usually considered as a low-value residue in current cellulose-based industries, such as pulping and bio-ethanol production. However, in order to promote the integrated biorefinery concept, there is an increasing interest in exploring more valuable applications of this vastly underused resource.1-2 Lignin is a typically heterogeneous biopolymer that has a complicated chemical structure with a wide molecular weight distribution.3-5 The lignin heterogeneity originates from the random radical polymerization of three different monomers in the biosynthesis process

6-7

and further aggravates due to the fragmentation/condensation

reactions during the pretreatment/extraction processes.8 Therefore, even from the same source and produced from the same process under the same conditions, the resultant lignin presents a broad molecular weight distribution. Generally, the molecular weight of lignin has a significant impact on its structure and reactivity and, to an even greater extent, on its performance for subsequent applications.3-5 For example, high molecular weight lignin has high carbon content and high viscosity, thus, is more suitable for carbon fiber preparation,9 whereas low molecular weight lignin has high biological activities (e.g. antioxidant, anti-tyrosinase and antimicrobial performances) due to its high reactive functional group content.10-12 Therefore, to achieve an efficient and high-value utilization of heterogeneous lignin, a lignin upgrading process by fractionation is required, which leads to the production of 3



specific lignin fractions with controllable molecular weight distributions and more homogeneous chemical characteristics.3, 5, 13 Until now, numerous processes have been developed to fractionate lignin, such as step-wise ultrafiltration with different cut-offs,14-15 gradient acid precipitation 4, 16 and successive solvent extraction.3,

13,

17

Among them, successive extraction of

heterogeneous lignin using various organic solvents is a commonly used fractionation method due to its high efficiency and applicability.3,

18

Thring et al. fractionated

Alcell® lignin into three parts by sequential extraction using diethyl ether and methanol, and found that the obtained fractions presented gradual increased molecular weight with decreased polydispersity.19 In another study, Li et al. classified bamboo lignin into five fractions by successive extraction with five different organic solvents and obtained similar results.17 It is important to note that lignin fractionation process by multiple organic solvents is a time-consuming and error-prone approach.20-21 Moreover, this process often relies on the use of large amounts of toxic organic solvents, which is not desirable in either laboratory or industrial environments. Therefore, the development of a simple and green lignin fractionation process is of fundamental and practical interest. γ-valerolactone (GVL), derived from sugars by dehydration and hydrogenation, is an important platform chemical, which is widely used for the synthesis of food and fuel additives, and polymer monomers.22 The high boiling point, low vapor pressure, excellent stability at ambient temperature, hypotoxicity and sustainability of GVL make it an excellent candidate as a green solvent.23-24 GVL has been used as a recyclable solvent in biomass pretreatment,25 non-enzymatic saccharification of biomass,26 and dehydration of hemicellulose to furfural.27 Li et al. developed a mild biomass pretreatment using a mixture of 80% GVL, and 20% water and the results 4


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indicated that the mixture was effective to remove lignin, mostly due to the excellent solubility of lignin in GVL.25 Recently, Xue et al. developed binary solvent systems consisting of GVL and one co-solvent (e.g. water, ionic liquid, DMSO, and DMF) to dissolve various types of lignin efficiently.28 Until now no attempt has been made to use GVL for lignin fractionation purpose. Considering the many benefits of GVL (e.g. stable, harmfulless, biodegradable and recyclable), this work is aimed at developing a novel and simple process using GVL/water solvent (GWS), instead of toxic solvents, to fractionate heterogeneous lignin. With the industrial-scale development of cellulosic ethanol, large number of enzymatic hydrolysis lignin (EHL) is discharged and consequently, further processing and valorization of this vastly underused resource is of increasing interest.10, 29 Thus, in this work, EHL was used as the parent lignin for the GVL-based fractionation. The three lignin fractions obtained were characterized by GPC, 2D-HSQC, 31P NMR, and Py-GC/MS to investigate their differences in terms of molecular weight and structural properties. Moreover, the solvatochromic parameters of GWS were further studied to verify the fractionation mechanism for the heterogeneous lignin. Finally, the solvent regeneration and subsequent reuse for lignin fractionation were further investigated. EXPERIMENT SECTION Experimental Materials Enzymatic hydrolysis lignin (EHL) was isolated from enzymatic hydrolysis residue (EHR) by alkaline extraction and acid precipitation according to the process described by Liu et al.30 EHR was obtained using corn stalk as raw material followed by steam explosion pretreatment (1.5 MPa steam pressure and 5 min retention time) and enzymatic hydrolysis (30 U/g cellulase loading, 50 °C temperature and 48 h reaction time). The EHR contained around 56.86% acid-insoluble lignin (w/w), 2.92% 5



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acid-soluble lignin (w/w), 27.92% carbohydrates (w/w), and 7.90% ash (w/w). GVL was purchased from Macklin Co., Ltd (Shanghai, China). Deuterated chloroform and cyclohexanol were obtained from Meryer Chemical Co. (Shanghai, China). Pyridine was purchased from Aladdin Co., Ltd and Chromium (III) acetylacetonate was purchased

from

Adamas

Reagent

Co.,

Ltd.

2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP) and Reichardt’s dye were purchased from purchased from Sigma Aldrich. They were analytically pure and were used without further purification. Lignin Fractionation The isolated EHL was processed according to the procedure shown in Figure 1 to obtain its three fractions. At first, 1.0 g dry EHL was completely dissolved in 20 ml GVL water solution (GWS) with a ratio of 60:40 (GVL/water, v/v) by stirring at ambient temperature. The solution was then diluted with deionized water to a GVL/water ratio (40:60, v/v) and after sufficient stirring (200 rpm, 5 min), the precipitated lignin (F1) was obtained by centrifugation at 8000 rpm for 5 min. Afterwards, the supernatant solution (GVL/water ratio 40:60, v/v) was further diluted with deionized water to a GVL/water ratio (30:70, v/v) and the second lignin fraction (F2) was precipitated and collected by centrifugation. Finally, the supernatant solution after centrifugation was diluted with deionized water to a GVL/water ratio (5:95, v/v) and the last lignin fraction (F3) was precipitated and recovered through centrifugation. All three lignin fractions were dried in a vacuum oven at 50 oC for 14 h and stored in a desiccator for further analysis. After fractionation, the 5% GVL water solution (5% GWS) was regenerated by rotary evaporation to remove water to 60% GVL water solution (60% GWS) and reused for the next run.

6


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Lignin Characterization The purity of lignin factions were measured using the procedure described by Wang and Chen,20 which was on the basis of the National Renewable Energy Laboratory (NREL) analytic standard. The molecular weight distribution of lignin samples was determined via gel permeation chromatography (GPC, Agilent 1200, USA) using a hydrophilic gel column (TSK G3000PWxl column) according to the literature.4 2D NMR spectra (HSQC) were acquired using a Bruker Avance 400 MHz at 25 oC. 0.1 g lignin was dissolved sufficiently in 0.5 ml DMSO-d6 for determination and Bruker standard pulse sequence ‘hsqcedetgpsisp 2.2’ was used.12 The quantitative analysis of lignin functional groups was performed by a nondestructive literature

31-32

31P

NMR technique following the method described in the previous

with minor modifications. Here, 0.02 g dry lignin was successively

treated with pyridine/deuterated chloroform solvent (1.6:1, v/v), cyclohexanol solution (10.85 mg/ml, the internal standard solution) and chromium (III) acetylacetonate solution. Finally, the lignin was phosphitylated with 100 μl 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP) and submitted to a quantitative

31P

NMR analysis. The spectra were processed using the MestReNova

software and the quantitative calculation results were acquired according to the method described elsewhere.33 The Py-GC/MS determination was conducted by a commercial pyrolyzer (Py-2020iS, Japan) coupled to a GC-MS apparatus (Agilent Techs. Inc. 7890A GC /5975C MS), using a fused silica capillary column (HP-5MS, 30m×0.25mm×0.25μm) from Agilent Technology.30 Approximately, 100 μg of lignin sample was pyrolyzed at 600 °C for 12 s with a heating rate of 2 oC/ms under helium. The obtained mass spectra were compared to the National Institute of Standards Library (NIST) and the 7



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total molar peak areas for each lignin degradation product were normalized. Each experiment was conducted at least two times to minimize the experimental error. Kamlet-Taft Parameter Measurement 20 μl 4-Nitroaninline (4NA) and N,N-diethyl-4-nitroaniline (DENA), and 200 μl Reichardt’s dye solutions were prepared in ethanol to a concentration of 1 mg/ml and the ethanol was evaporated under a stream of dry nitrogen. Then, the three dye solutions were obtained by adding 12.5 ml of GWSs with different GVL concentrations (i.e. 60% and 40%) and mixing on a rotary shaker at 200 rpm for 20 min. All dye/GWS solutions were detected using an UV spectrophotometer (350nm-700nm) and the maximum wavelength was recorded. The three key parameters, including solvent polarizability (π*), hydrogen bond donator (HBD) capacity (α) and hydrogen bond acceptor (HBA) capacity (β), can be expressed by the following correlation:

* 

 DENA max  27.52  3.183

(1)



ET 30  - 14.6 * -0.23 - 30.31 16.5

(2)



1.035   DENA max   4 NA max  2.64 2.8

(3)

ET 30   28591

(4)

 max

where, υ(DENA)max and υ(4NA)max are the maximum wavelengths of DENA and 4NA, respectively, in kilokeyser (kK, 10-3 cm-1). Here, λmax is the maximum wavelength of Reichardt’s dye in nm.34

8


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RESULTS AND DISCUSSION Lignin Fractionation by Gradient Precipitation in GWS Enzymatic hydrolysis lignin (EHL)

Recycled GVL

Dissolve in GVL-water (60:40, v/v)

Parent lignin liquor

Soluble lignin liquor


Add water to GVL/water (40:60, v/v) and centrifugation



Insoluble lignin fraction (F1)



GVL-water mixture

Figure 1. Schematic showing for the GVL-based gradient precipitation fractionation of enzymatic hydrolysis lignin (EHL).

In this work, a simple GVL-based fractionation process for heterogeneous lignin is proposed to take advantage of the unique properties and benefits of GVL, such as, excellent lignin solubility, low toxicity, and ease of recycling. The GVL-based fractionation process of enzymatic hydrolysis lignin (EHL) is illustrated in Figure 1. First the EHL was dissolved in 60% aqueous GVL solution and then distilled water was added into the solution to reach a GVL/water ratio (40:60, v/v). 41.13±0.15% of total lignin was precipitated, which was denoted as the first fraction (F1). The supernatant was further diluted by adding distilled water to reach a GVL/water ratio of 30:70 (v/v), and another part of lignin was precipitated (29.13±0.76% of total lignin), denoted as the second lignin fraction, F2. The last fraction (F3, 24.37±0.73% of total lignin) was precipitated, then recovered from the remaining lignin solution by adding more water to reach a GVL/water ratio of 5:95 (v/v). The three collected lignin fractions were dissolved in 1% NaOH aqueous solutions (with the same lignin 9



concentration of 1 mg/mL), and the brown color of these lignin solutions became lighter and lighter from F1 to F3 (shown in Figure 1), with the absorptivity at 280 nm of 0.283, 0.256, and 0.2423, respectively (UV spectra shown in Figure S1). Further detailed characterizations of the three lignin fractions were carried out and the results are presented in the following sections. Characterization of Lignin Fractions Chemical Composition The isolated EHL has a lignin content of 94.83% (Klason lignin and acid-soluble lignin). Accordingly, the three lignin fractions collected from the GVL-based fractionation process presented a very high purity. The impurities (carbohydrate and inorganic substance, although small quantities) decreased from F1 to F3 (Figure 2a). These results can be explained by the high solubility of carbohydrates and inorganic substances in water. Thus, lignin fractions precipitated in more water GWS (e.g. F2 and F3) showed low contents of carbohydrates and inorganic substances. Due to the decrease in sugar and ash contents, the lignin content increased from 94.30% (F1) to 95.81% (F2) and 96.36% (F3). Thus, the GVL-based gradient precipitation fractionation process can also be considered as a lignin purification process.

10


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a Klason lignin

Acid soluble lignin

Glucosan

Xylan+arbinan

Ash 100

100

F3

80

96 94

60

92

F2

90 40

88 86

F1

20

Fractions compositions (%)

Fractions yield (%)

98

5 0

0

Yield

EHL

F1

F2

F3

b

Detector response (280nm)

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


EHL

10

F1 F2 F3

12

14

16

18

Elution time (min)

Figure 2. Chemical compositions (a) and GPC results (b) of three lignin fractions obtained by the GVL-based gradient precipitation fractionation process, as well as those of the original EHL.

GPC A comparison of molecular weight distribution among the three lignin fractions, as well as the EHL is presented in Figure 2b. The chromatogram elution time of three lignin fractions increased gradually from 12.52 min (F1) to 12.98 min (F2), and

11



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finally reached to 13.42 min (F3). The weight-average molecular weight ( M w), number-average molecular weight ( M n) and polydispersity are listed in Table 1. The results indicated that F1 (precipitated in 40% GWS) had the highest molecular weight ( M w, 11,135 g/mol), followed by F2 (precipitated in 30% GWS) while F3 (precipitated in 5% GWS) showed the lowest molecular weight ( M w, 4,235 g/mol). Moreover, the molecular weight polydispersity of the three fractions notably decreased compared with that of the original EHL (Table 1). These results support the notion that the gradient precipitation process can effectively fractionate lignin and obtain different molecular weight fractions with relatively low polydispersity. In order to confirm the molecular weight-dependent fractionation using the GVL-based process, the solubility of different molecular weight lignins in the GWSs were determined. The three lignin samples with different molecular weight were obtained from a previously established ethanol-water fractionation process.20 The high molecular weight lignin was precipitated at high GVL content GWS (50-40%) while low molecular weight lignin was precipitated only at low GVL content GWS (20-5%) (Figure S2). Thus, the results support the conclusion that the GVL-based gradient precipitation method can realize the effective separation of lignins with different molecular weight from a heterogeneous lignin sample. Compared with other lignin fractionation methods using multiple organic solvents,17,

19

the proposed lignin

fractionation process is a simplified and sustainable method since green GVL is the only organic solvent used.

12


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Table 1 Molecular weight and functional group contents of three lignin fractions obtained by GVL-based gradient precipitation fractionation process, as well as those of the original EHL. Functional group contents analyzed by 31P NMR

Molecular weight distribution

M

w

M

n

D ( M w/ M n)

Syringyl OH

Guaiacyl OH

p-hydroxylic

Total phenolic

Aliphatic OH

COOH

(mmol/g)

(mmol/g)

phenyl (mmol/g)

OH (mmol/g)

(mmol/g)

(mmol/g)

F1

11135

7131

1.56

0.46

0.82

0.38

1.66

1.43

1.20

F2

8310

4704

1.77

0.52

0.77

0.46

1.75

1.40

1.54

F3

4235

2277

1.86

0.58

0.72

0.52

1.82

1.57

1.78

EHL

8930

4420

2.02

0.51

0.89

0.59

1.99

1.48

1.86

13


ACS Sustainable Chemistry & Engineering 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

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a

b

c

d

e

f

g

h

O

HO  R





O

OMe



O

MeO

O

O

OMe OH

A:R=OH

B

O

OH

OH

D

2

6 5

OR

G

2

6

OMe

5

OR

O

OH 6

OMe

G'

MeO

6

2

OR

S

A': R=lignin unit 14


OMe

MeO

S'

2

6

OMe

OR

H

5

OR

F

OMe

O 8

7

8

7

2

6

2

OR

O

O

OH

2

6

3

5

OR

P

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Figure 3. Side-chain (a, b, c, and d) and aromatic (e, f, g, and h) regions of 2D-NMR (HSQC) spectra of three lignin fractions (F1: b and f; F2: c and g; F3: d and h) obtained by GVL-based gradient precipitation fractionation process, as well as those of the original EHL (a and e). Main substructures identified by 2D NMR: (A) β-O-4’ aryl ether linkage; (A’) α, β-diaryl ethers (β-O-4’/α-O-4’); (B) phenyl-coumaran structure formed by β-5’ and α-O-4’ linkages; (D) dibenzodioxocin structure; (G) guaiacyl unit; (G’) guaiacyl unit bearing a carbonyl at Cα; (S) syringyl units; (S’) syringyl unit bearing a carbonyl at Cα; (H) p-hydroxyphenyl unit; (F) ferulic acid unit; (P) p-coumaric acid unit.

2D-NMR Analysis The 2D-NMR analysis technique was used to examine the structural differences of the three lignin fractions, as well as the original EHL. The side-chain regions (δC/δH 50−90/3.0-5.8) and aromatic (δC/δH 100−150/6.0−8.3) regions of the HSQC NMR spectra are shown in Figure 3a–d and Figure 3e-h, respectively. The main lignin substructures identified, based on previous publications,35-38 are also presented in Figure 3. The detailed assignments of the correlated signals are listed in Table S1. In the side-chain regions (Figure 3a-d), all lignin samples exhibited an obvious signal of methoxyl (δC/δH, 56.10/3.75). Three typical inter-unit linkages including β-O-4’ structure (A and A’), pinoresinol structure (B), and dibenzodioxocin structure (D), were confirmed. Among them, the aryl ether bond (β-O-4’ substructures, A) was the most predominant. The intensity of signals for β-O-4’ structure (A and A’) decreased with decreasing molecular weight from F1 to F3. In this work, the EHL was taken from a corn stalk biorefinery using steam explosion as a pretreatment process. The weak acid and high temperature conditions during the steam explosion pretreatment resulted in the breakage of β-O-4’ structure and therefore decreased the lignin molecular weight.35, 39 Thus, the low molecular weight fraction (F3) had relatively low β-O-4’ linkage contents. 15



In the aromatic region (Figure 3e-h), the main cross-signals from syringyl (S), guaiacyl (G) and p-hydroxyphenyl (H) were evident in all lignin samples. The S unit exhibited a prominent signal originated from C2,6-H2,6 correlation at δC/δH 105.1/6.68 ppm and the signal at δC/δH 128.4/7.17 ppm was attributed to the C2,6-H2,6 aromatic of H unit. The G units showed three correlations, which were C2-H2 (δC/δH 111.5/6.96 ppm), C5-H5 (δC/δH 114.9/6.76 ppm) and C6-H6 (δC/δH 119.9/6.74 ppm), respectively. Besides, two phenolic acid structures, including p-coumaric acid and ferulic acid, were also identified in the HSQC spectra of the lignin samples. In previous studies,20, 30

the phenolic acids were present in the low molecular weight fraction. However, in

this work, no obvious differences of phenolic acid content among the three fractions were found from the 2D-NMR results. The signal of Cα oxidized (Cα=O) guaiacyl units (G’) enhanced from F1 to F3 and the Cα oxidized syringyl unit (S’) was detected only in F3 (δC/δH 106.3/7.31 ppm, C2,6-H2,6). The Cα oxidized structures were formed by the cleavage of β-O-4’ structure during the steam explosion process.35, 39 Thus, it is reasonable that the F3 with low molecular weight has a high content of Cα oxidized units.

16


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Py-GC/MS O

Guaiacyl Syringyl Phenolic compound Heterocyclic compound Benzene Aliphatics

OH

OH

OH

O

OH OH

O

O

OH OH

O OH

OH

O

O OH

0

10

20

30

40

50

60

70

80

90

100

HO

O

O OH 0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

5

EHL

F1

F2

F3

10

15

20

Retention time (min) Figure 4. Py-GC/MS of three lignin fractions obtained by GVL-based gradient precipitation fractionation process, as well as those of the original EHL. The identities and relative abundances of compounds released by Py-GC/MS are listed in bar charts.

Py-GC/MS technique is able to provide useful information concerning the structure of lignin components, assuming that pyrolysis products represent, to a greater or lesser degree, the structural units forming the macromolecule.40 Thus, the three lignin fractions and the EHL were further analyzed by Py-GC/MS and their chromatograms are shown in Figure 4. Included are also the main identities and relative abundances of the released compounds as well as structural formula. The main products from lignin pyrolysis were phenolic compounds, which were principally from the breakage of unit 17



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linkages and the further cracking of the side chain of the three units.30, 41 All the lignin samples including EHL and its three lignin fractions had rather similar compositions. Six categories of compounds, including guaiacol, syringol, phenol, benzene, heterocyclic and aliphatic compounds were identified (Table S2). The principal products from lignin pyrolysis were guaiacol, syringol, and phenolic compounds, demonstrating the GSH type lignin. Based on the relative content of guaiacol, syringol and phenolic types after fast pyrolysis, the ratios of three lignin fractions were obtained.41-42 For the three lignin fractions from F1 to F3, these values were G55.99S10.12H33.88, G51.98S12.17H35.85, and G50.25S14.30H35.46, respectively, which meant that the high molecular weight lignin had relatively higher G unit content and lower S unit content compared with the low molecular weight lignin. The results can be explained by the fact that the C-C5 bond formed by guaiacyl units is not broken during the pretreatment process due to its higher stability.39,

43

Thus, the high

molecular weight lignin is expected to contain high guaiacyl unit. Besides, the low molecule weight lignin (F2 and F3) contained more phenol, benzene, heterocyclic and aliphatic compounds, which suggests that the side chains of low molecule weight lignin are cleaved more easily during the pyrolysis process.44

18


Page 19 of 35 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


31P

NMR Internal Standard

p-hydroxylic phenol OH

Aliphatic OH

COOH

Syringyl OH Guaiacyl OH

EHL

F1

F2

F3 148

146

144

142

140

138

136

134

ppm Figure 5. 31P NMR spectra of three lignin fractions obtained by GVL-based gradient precipitation fractionation process, as well as those of the original EHL.

The

31P

NMR spectra of the three lignin fractions are shown in Figure 5, from

which, the major hydroxyl group contents are included in Table 1. The dominant hydroxyl groups (syringyl OH, guaiacyl OH and p-hydroxylic phenyl OH) were observed in all lignin samples, confirming that the EHL was a typical GSH lignin. However, the contents of syringyl OH and p-hydroxylic phenyl in these three lignin fractions increased with the decrease of lignin molecular weight, while a completely opposite trend was observed for the content of guaiacyl OH. Besides, the carboxyl group (COOH) content also increased with decreasing molecular weight (Table 1), which coincided with the results in the literature.4, 13 It is noted that the contents of total phenolic OH and COOH in the EHL sample were higher than those in the three fractions. The reduction of total phenolic OH and COOH contents after fractionation may be caused by the fact that a small part of lignin with high total phenolic OH and 19



COOH contents in the EHL was not recovered by the GVL-based fractionation process. The lignin with high hydrophilic group contents (e.g. phenolic OH and COOH) exhibits good solubility in neutral water-rich solutions. Thus, the dissolved lignin (about 5.37% of total EHL) in the 5% aqueous GVL solution showed higher hydrophilic group contents compared with the recovered lignin fractions. Solubility Parameter of GWS

3.3

1.4

0.25

3.2

1.3

0.20

3.1

1.2

α

π*

0.30

β

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

Page 20 of 35

0.15

3.0

1.1

0.10

2.9

1.0

0.05

2.8

0.9 5%

30%

40%

60%

GVL concentration (%)

Figure 6. Kamlet-Taft α, β and π* parameters of different GVL/water solvents (GWS) at ambient temperature.

The mechanism of lignin fractionation using gradient precipitation in tunable GWS was analyzed by Kamlet-Taft parameters, which provides a quantitative measurement of the solvent hydrogen bond donator (HBD) capacity (α parameter), hydrogen bond acceptor (HBA) capacity (β parameter), and solvent polarizability (π* parameter). In this study, the Kamlet-Taft parameters were determined spectrophotometrically using three dyes and their relationships in the different GWSs (60%, 40%, 30%, and 5%) are shown in Figure 6. Evidently, the water content in GWS had a significant impact on the solubility parameters. The β parameters decreased with the presence of more water in GWS. The decrease of β parameter is due to the water’s relatively poor HBA 20


Page 21 of 35 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


capacity.34 Doherty et al. also found that the addition of water to ionic liquids decreased the solvents’ HBA capacity when they examined the relationship between Kamlet-Taft parameters of different ionic liquids and the solubility of lignin.34 A high HBA capacity is required for solvent systems to effectively dissolve lignin.28, 34 Thus, the EHL could be totally dissolved in 60% GWS, which presented the highest HBA capacity among the four different GWSs. With the addition of more water, the HBA capacity decreased sharply and the EHL precipitated consequently. Moreover, the addition of water to GWSs increased the solvent polarizability (increasing the π* parameter). The

31P

NMR analyses showed that low molecular

weight lignin had higher polar group contents (total phenolic OH, aliphatic OH, and COOH, Table 1), resulting in the increased polarity of the lignin molecule and its solubility in polar solutions. Therefore, compared with the high molecular weight lignin, the low molecular weight lignin had better solubility in the high-water-content GWS that had higher π* parameter (solvent polarizability). Through the addition of water, the lignin fraction with higher molecular weight is easier to precipitate due to its lower solubility and thus the separation of lignin parts with different molecular weight is realized by a gradual addition of water into the GWS containing heterogeneous lignin. Solvent Reuse The recycling of GVL is critical for the sustainability of this method. In this work, the 5% GWS after F3 precipitation was collected. Generally, GVL can be easily separated from the aqueous GVL solution by an energy-saving liquid CO2 extraction.25 Here, due to the much higher boiling point of GVL compared with water, a frequently-used rotary evaporation was applied to remove water from 5% GWS to regenerate 60% GWS. The regenerated 60% GWS was reused for EHL fractionation 21



following the same process as used for the fresh 60% GWS (Figure 1). The recovery of GVL after water removal using rotary evaporation was more than 95%. Due to the presence of small amounts of dissolved lignin (about 5 % of total EHL, Section 3.1), the color of the regenerated 60% GWS was pale yellow (Figure 7a). The UV spectrum of the regenerated 60% GWS is shown in Figure 7a. The dissolved lignin showed a maximum absorption at 300-310 nm (different from the other lignin fractions, F1, F2, and F3 in Figure S1), which might be caused by the phenolic acids having a maximum absorption at 320 nm due to the conjugated phenolic structure.16 The above results agreed with the high content of phenolic OH and COOH groups in the residual lignin that was not recovered after F3 precipitation (Section 3.2.5). The molecular weight distributions of lignin fractions obtained by the regenerated GWS were also characterized (Figure 7b), which were essentially the same as those using the fresh GWS (Figure 2b).

a

b

1.4


1.2 1.0

OD

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

Page 22 of 35

0.8

A

B

0.6 0.4 0.2 220

240

260

280

300

320

EHL

10

F1 F2 F3

12

14

16

18

Elution time (min)

Wavelength (nm)

Figure 7. (a) UV absorption spectra of the regenerated GWS using the fresh GWS as the reference, inset: (A) fresh GWS, (B) regenerated GWS; (b) molecular weight distributions of EHL and lignin fractions obtained by the gradient precipitation process using regenerated GWS.

CONCLUSIONS A novel process for efficient fractionation of heterogeneous EHL was proposed based on the gradient precipitation method using GVL/water mixture as the green 22


Page 23 of 35 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


solvent: EHL was first dissolved in 60% aqueous GVL and then progressively precipitated in 40%, 30%, and 5% aqueous GVL solutions. Three lignin fractions, with gradually decreased molecular weight, were obtained using the proposed process. Furthermore, all the fractions showed lower polydispersity than the parent EHL. The structural characterization suggested that with the decrease of lignin molecular weight from F1 to F3, the contents of S and H units increased while the β-O-4’ linkage and G unit content decreased. The functional group contents, including aliphatic/phenolic OH and COOH, increased with the decrease of lignin molecular weight. The oxidized G and S units, which were mainly formed due to cleavages of aryl ether bond, were enriched in F3. Due to the increase of polar group contents, low molecular weight lignin exhibited high polarity and solubility in high water content GVL solutions. These results were consistent with the Kamlet-Taft parameters determined experimentally for these aqueous GVL solutions. Since GVL is the only organic solvent used in the proposed process, this study provides a simple and green route to improve the lignin homogeneity. ASSOCIATED CONTENTS Supporting Information: UV spectra of lignin samples (Figure S1). Precipitation of lignins with different molecular weights in different GVL/water solutions (Figure S2). Assignments of 13C-1H

correlation signals in the HSQC spectra (Table S1). Assignments of major

peaks of Py-GC-MS (Table S2). ACKNOWLEDGMENTS Financial support for this study was kindly provided by National Natural Science Foundation of China (31700515), Natural Science Foundation of Tianjin City (16JCQNJC05900), Tianjin Municipal Education Commission (2017KJ023), and the 23



Canada Research Chairs program of the Government of Canada. REFERENCES (1) Ragauskas, A. J.; Beckham, G. T.; Biddy, M. J.; Chandra, R.; Chen, F.; Davis, M. F.; Davison, B. H.; Dixon, R. A.; Gilna, P.; Keller, M. Lignin valorization: improving lignin processing in the biorefinery. Science 2014, 344 (6185), 1246843. (2) Rinaldi, R.; Jastrzebski, R.; Clough, M. T.; Ralph, J.; Kennema, M.; Bruijnincx, P. C.; Weckhuysen, B. M. Paving the way for lignin valorisation: recent advances in bioengineering, biorefining and catalysis. Angew. Chem. Int. Edit. 2016, 55 (29), 8164-8215. (3) Jiang, X.; Savithri, D.; Du, X.; Pawar, S.; Jameel, H.; Chang, H.; Zhou, X. Fractionation and characterization of kraft lignin by sequential precipitation with various organic solvents. ACS Sustainable Chem. Eng. 2016, 5 (1), 835-842. (4) Wang, G.; Chen, H. Fractionation of alkali-extracted lignin from steam-exploded stalk by gradient acid precipitation. Sep. Purif. Technol. 2013, 105, 98-105. (5) Cui, C.; Sun, R.; Argyropoulos, D. S. Fractional precipitation of softwood kraft lignin: isolation of narrow fractions common to a variety of lignins. ACS Sustainable Chem. Eng. 2014, 2 (4), 959-968. (6) Lin, J.; He, X.; Hu, Y.; Kuang, T.; Ceulemans, R. Lignification and lignin heterogeneity for various age classes of bamboo (Phyllostachys pubescens) stems. Physiol. Plantarum 2002, 114 (2), 296-302. (7) Terashima, N.; Fukushima, K. Heterogeneity in formation of lignin-XI: an autoradiographic study of the heterogeneous formation and structure of pine lignin. Wood Sci. Technol. 1988, 22 (3), 259-270. (8) Samuel, R.; Pu, Y.; Raman, B.; Ragauskas, A. J. Structural characterization and comparison of switchgrass ball-milled lignin before and after dilute acid pretreatment. Appl. Biochem. Biotech. 2010, 162 (1), 62-74. (9) Baker, D. A.; Rials, T. G. Recent advances in low ‐ cost carbon fiber manufacture from lignin. J. Appl. Polym. Sci 2013, 130 (2), 713-728. (10) An, L.; Wang, G.; Jia, H.; Liu, C.; Sui, W.; Si, C. Fractionation of enzymatic hydrolysis lignin by sequential extraction for enhancing antioxidant performance. Int. J. Biol. Macromol. 2017, 99, 674-681. (11) Wang, R.; Wang, G.; Xia, Y.; Sui, W.; Si, C. Functionality study of lignin as a tyrosinase inhibitor: Influence of lignin heterogeneity on anti-tyrosinase activity. Int. J. Biol. Macromol. 2019, 128, 107-113. (12) Wang, G.; Xia, Y.; Liang, B.; Sui, W.; Si, C. Successive ethanol–water fractionation of enzymatic hydrolysis lignin to concentrate its antimicrobial activity. J. Chem. Technol. Biot. 2018, 93, 2977-2987. (13) Jääskeläinen, A. S.; Liitiä, T.; Mikkelson, A.; Tamminen, T. Aqueous organic solvent fractionation as means to improve lignin homogeneity and purity. Ind. Crop. Prod. 2017, 103, 51-58. (14) Huang, C.; He, J.; Narron, R.; Wang, Y.; Yong, Q. Characterization of Kraft Lignin Fractions Obtained by Sequential Ultrafiltration and Their Potential Application as a Biobased Component in Blends with Polyethylene. ACS Sustainable Chem. Eng. 2017, 5 (12), 11770-11779. (15) Toledano, A.; García, A.; Mondragon, I.; Labidi, J. Lignin separation and fractionation by ultrafiltration. Sep. Purif. Technol. 2010, 71 (1), 38-43. (16) Santos, P. S. B. d.; Erdocia, X.; Gatto, D. A.; Labidi, J. Characterisation of Kraft lignin separated by gradient acid precipitation. Ind. Crop. Prod. 2014, 55, 149-154. (17) Li, M.; Sun, S.; Xu, F.; Sun, R. Sequential solvent fractionation of heterogeneous bamboo organosolv lignin for value-added application. Sep. Purif. Technol. 2012, 101, 18-25. (18) Allegretti, C.; Fontanay, S.; Krauke, Y.; Luebbert, M.; Strini, A.; Troquet, J.; Turri, S.; Griffini, G.; D’Arrigo, P. Fractionation of Soda Pulp Lignin in Aqueous Solvent through Membrane-Assisted Ultrafiltration. ACS Sustainable Chem. Eng. 2018, 6 (7), 9056-9064. (19) Thring, R.; Vanderlaan, M.; Griffin, S. Fractionation of Alcell® lignin by sequential solvent extraction. J. Wood Chem. Technol. 1996, 16 (2), 139-154. (20) Wang, G.; Chen, H. Fractionation and characterization of lignin from steam-exploded corn stalk by sequential dissolution in ethanol–water solvent. Sep. Purif. Technol. 2013, 120, 402-409. (21) Saito, T.; Perkins, J. H.; Vautard, F.; Meyer, H. M.; Messman, J. M.; Tolnai, B.; Naskar, A. K. Methanol fractionation of softwood kraft lignin: Impact on the lignin properties. ChemSusChem 2014, 7 (1), 221-228. (22) Alonso, D. M.; Wettstein, S. G.; Dumesic, J. A. Gamma-valerolactone, a sustainable platform 24


Page 24 of 35

Page 25 of 35 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


molecule derived from lignocellulosic biomass. Green Chem. 2013, 15 (3), 584-595. (23) Horvath, I. T. Solvents from nature. Green Chem. 2008, 10 (10), 1024-1028. (24) Soh, L.; Eckelman, M. J. Green solvents in biomass processing. ACS Sustainable Chem. Eng. 2016, 4(11), 5821-5837. (25) Shuai, L.; Questell-Santiago, Y. M.; Luterbacher, J. S. A mild biomass pretreatment using γ-valerolactone for concentrated sugar production. Green Chem. 2016, 18 (4), 937-943. (26) Luterbacher, J. S.; Rand, J. M.; Alonso, D. M.; Han, J.; Youngquist, J. T.; Maravelias, C. T.; Pfleger, B. F.; Dumesic, J. A. Nonenzymatic sugar production from biomass using biomass-derived γ-valerolactone. Science 2014, 343 (6168), 277-280. (27) Gürbüz, E. I.; Gallo, J. M. R.; Alonso, D. M.; Wettstein, S. G.; Lim, W. Y.; Dumesic, J. A. Conversion of hemicellulose into furfural using solid acid catalysts in γ-valerolactone. Angew. Chem. Int. Edit. 2013, 52 (4), 1270-1274. (28) Xue, Z.; Zhao, X.; Sun, R.; Mu, T. Biomass-derived γ-valerolactone-based solvent systems for highly efficient dissolution of various lignins: Dissolution behavior and mechanism study. ACS Sustainable Chem. Eng. 2016, 4 (7), 3864-3870. (29) Guo, N.; Li, M.; Sun, X.; Wang, F.; Yang, R. Enzymatic hydrolysis lignin derived hierarchical porous carbon for supercapacitors in ionic liquids with high power and energy densities. Green Chem. 2017, 19 (11), 2595-2602. (30) Liu, C.; Si, C.; Wang, G.; Jia, H.; Ma, L. A novel and efficient process for lignin fractionation in biomass-derived glycerol-ethanol solvent system. Ind. Crop. Prod. 2018, 111, 201-211. (31) Granata, A.; Argyropoulos, D. S. 2-Chloro-4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaphospholane, a reagent for the accurate determination of the uncondensed and condensed phenolic moieties in lignins. J. Agr. Food Chem. 1995, 43 (6), 1538-1544. (32) Sadeghifar, H.; Well, T.; Le, R. K.; Sadeghifar, F.; Yuan, J. S.; Ragauskas, A. J. Fractionation of organosolv lignin using acetone: water and properties of the obtained fractions. ACS Sustainable Chem. Eng. 2016, 5(1), 580-587. (33) Sun, S.; Wen, J.; Ma, M.; Sun, R.; Jones, G. L. Structural features and antioxidant activities of degraded lignins from steam exploded bamboo stem. Ind. Crop. Prod. 2014, 56, 128-136. (34) Doherty, T. V.; Mora-Pale, M.; Foley, S. E.; Linhardt, R. J.; Dordick, J. S. Ionic liquid solvent properties as predictors of lignocellulose pretreatment efficacy. Green Chem. 2010, 12 (11), 1967-1975. (35) Wang, G.; Chen, H. Enhanced lignin extraction process from steam exploded corn stalk. Sep. Purif. Technol. 2016, 157, 93-101. (36) Yuan, T.; Sun, S.; Xu, F.; Sun, R., Characterization of lignin structures and lignin–carbohydrate complex (LCC) linkages by quantitative 13C and 2D HSQC NMR spectroscopy. J. Agr. Food Chem. 2011, 59 (19), 10604-10614. (37) Huang, C.; He, J.; Du, L.; Min, D.; Yong, Q. Structural characterization of the lignins from the green and yellow bamboo of bamboo culm (Phyllostachys pubescens). J. Wood Chem. Technol. 2016, 36 (3), 157-172. (38) Huang, C.; Su, Y.; Shi, J.; Yuan, C.; Zhai, S.; Yong, Q. Revealing the effects of centuries of ageing on the chemical structural features of lignin in archaeological fir woods. New J. Chem. 2019, 43 (8), 3520-3528. (39) Li, J.; Henriksson, G.; Gellerstedt, G. Lignin depolymerization/repolymerization and its critical role for delignification of aspen wood by steam explosion. Bioresour. Technol. 2007, 98 (16), 3061-3068. (40) Sequeiros, A.; Labidi, J. Characterization and determination of the S/G ratio via Py-GC/MS of agricultural and industrial residues. Ind. Crop. Prod. 2017, 97, 469-476. (41) Moghaddam, L.; Rencoret, J.; Maliger, V. R.; Rackemann, D. W.; Harrison, M. D.; Gutiérrez, A.; del Río, J. C.; Doherty, W. O. Structural characteristics of bagasse furfural residue and its lignin component. An NMR, Py-GC/MS, and FTIR study. ACS Sustainable Chem. Eng. 2017, 5 (6), 4846-4855. (42) Del Río, J. C.; Rencoret, J.; Prinsen, P.; Martínez, A. n. T.; Ralph, J.; Gutiérrez, A. Structural characterization of wheat straw lignin as revealed by analytical pyrolysis, 2D-NMR, and reductive cleavage methods. J. Agr. Food Chem. 2012, 60 (23), 5922-5935. (43) García, A.; Toledano, A.; Serrano, L.; Egüés, I.; González, M.; Marín, F.; Labidi, J. Characterization of lignins obtained by selective precipitation. Sep. Purif. Technol. 2009, 68 (2), 193-198. (44) An, L.; Si, C.; Wang, G.; Sui, W.; Tao, Z. Enhancing the solubility and antioxidant activity of high-molecular-weight lignin by moderate depolymerization via in situ ethanol/acid catalysis. Ind. Crop. Prod. 2019, 128, 177-185. 25



For Table of Contents Use Only Abstract Graphic

Synopsis: A gradient precipitation process using green γ-valerolactone (GVL)/water solvents is proposed to reduce the heterogeneity of sustainable lignin for subsequent value-added applications.

26


Page 26 of 35

Page 27 of 35 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


Abstract Graphic

Synopsis: A gradient precipitation process using green γ-valerolactone (GVL)/water solvents is proposed to reduce the heterogeneity of sustainable lignin for subsequent value-added applications.



Page 28 of 35

Enzymatic hydrolysis lignin (EHL)

Recycled GVL

Dissolve in GVL-water (60:40, v/v)

Parent lignin liquor









GVL-water mixture

Figure 1. Schematic showing for the GVL-based gradient precipitation fractionation of enzymatic hydrolysis lignin (EHL).


Page 29 of 35

a Klason lignin

Acid soluble lignin

Glucosan

Xylan+arbinan

Ash 100

100

F3

80

96 94

60

92

F2

90 40

88 86

20

F1

Fractions compositions (%)

Fractions yield (%)

98

5 0

0

Yield

EHL

F1

F2

F3

b


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


EHL

10

F1 F2 F3

12

14

16

18

Elution time (min)

Figure 2. Chemical compositions (a) and GPC results (b) of three lignin fractions obtained by the GVL-based gradient precipitation fractionation process, as well as those of the original EHL.



Page 30 of 35

a

b

c

d

e

f

g

h

O

HO  R





O

OMe



O

MeO

O

O

OMe

B

D

2

6 5

OMe

2

6 5

OMe

6

6

2

MeO

OMe

8

OR

OR

OR

OR

G'

S

S'

2

6

OMe

G

O 8

7

2

6

2

MeO

O

O

OH

O

OH

7

OH

A:R=OH

O

OH

OH

OR

H

5

OMe

6

2

5

3

OR

OR

F

P

A': R=lignin unit

Figure 3. Side-chain (a, b, c, and d) and aromatic (e, f, g, and h) regions of 2D-NMR (HSQC) spectra of three lignin fractions (F1: b and f; F2: c and g; F3: d and h) obtained by GVL-based gradient precipitation fractionation process, as well as those of the original EHL (a and e). Main substructures identified by 2D NMR: (A) β-O-4’ aryl ether linkage; (A’) α, β-diaryl ethers (β-O4’/α-O-4’); (B) phenyl-coumaran structure formed by β-5’ and α-O-4’ linkages; (D) dibenzodioxocin structure; (G) guaiacyl unit; (G’) guaiacyl unit bearing a carbonyl at Cα; (S) syringyl units; (S’) syringyl unit bearing a carbonyl at Cα; (H) p-hydroxyphenyl unit; (F) ferulic acid unit; (P) p-coumaric acid unit.


Page 31 of 35 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


O

Guaiacyl Syringyl Phenolic compound Heterocyclic compound Benzene Aliphatics

OH

O OH

OH

O OH OH

O

OH OH

O OH

OH

O

O OH

0

10

20

30

40

50

60

70

80

90

100

HO

EHL

O

O OH 0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

5

10

F1

F2

F3 15

20

Retention time (min)

Figure 4. Py-GC/MS of three lignin fractions obtained by GVL-based gradient precipitation fractionation process, as well as those of the original EHL. The identities and relative abundances of compounds released by Py-GC/MS are listed in bar charts.



Page 32 of 35

Internal Standard p-hydroxylic phenol OH

Aliphatic OH

COOH

Syringyl OH Guaiacyl OH

EHL

F1

F2

F3 148

146

144

142

140

138

136

134

ppm

Figure 5. 31P NMR spectra of three lignin fractions obtained by GVL-based gradient precipitation fractionation process, as well as those of the original EHL.


Page 33 of 35

3.3

1.4

0.25

3.2

1.3

0.20

3.1

1.2

α

π*

0.30

β

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


0.15

3.0

1.1

0.10

2.9

1.0

0.05

2.8

0.9 5%

30%

40%

60%

GVL concentration (%)

Figure 6. Kamlet-Taft α, β and π* parameters of different GVL/water solvents (GWS) at ambient temperature.



a

b

1.4


1.2 1.0

OD

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

0.8

A

B

0.6 0.4 0.2 220

Page 34 of 35

240

260

280

300

320

EHL

10

Wavelength (nm)

F1 F2 F3

12

14

16

18

Elution time (min)

Figure 7. (a) UV absorption spectra of the regenerated GWS using the fresh GWS as the reference, inset: (A) fresh GWS, (B) regenerated GWS; (b) molecular weight distributions of EHL and lignin fractions obtained by the gradient precipitation process using regenerated GWS.


Page 35 of 35 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


Table 1 Molecular weight and functional group contents of three lignin fractions obtained by GVL-based gradient precipitation fractionation process, as well as those of the original EHL.

Functional group contents analyzed by 31P NMR

Molecular weight distribution

M

w

M

n

Syringyl OH

Guaiacyl OH

p-hydroxylic

Total phenolic

Aliphatic OH

COOH

(mmol/g)

(mmol/g)

phenyl (mmol/g)

OH (mmol/g)

(mmol/g)

(mmol/g)

D ( M w/ M n)

F1

11135

7131

1.56

0.46

0.82

0.38

1.66

1.43

1.20

F2

8310

4704

1.77

0.52

0.77

0.46

1.75

1.40

1.54

F3

4235

2277

1.86

0.58

0.72

0.52

1.82

1.57

1.78

EHL

8930

4420

2.02

0.51

0.89

0.59

1.99

1.48

1.86


Water Solvent To Decrease Lignin

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