Purification, structural characterization, and modification of organosolv wheat straw lignin

Article abstract of DOI:10.1021/acs.jafc.5b02071

Biolignin, a wheat straw lignin produced by acetic acid/formic acid/water hydrolysis, was characterized by ³¹P and ¹³C-¹H 2D NMR spectroscopy and by size-exclusion chromatography. Biolignin is a mixture of low molar mass compounds (M_n = 1660 g/mol) made up of S, G, and H units and of coumaric and ferulic acid units. β-5 and β-O-4 interunit linkages are partially acylated in the γ-position by acetate and p-coumarate groups. Deacylated samples with a low content of contaminants were obtained by combining alkaline hydrolysis and solvent extraction. The high phenolic OH content found by ³¹P NMR reflects the presence of condensed aromatic units, such as 5-5 units. Reaction of purified lignin with ethanol and ethane-1,2-diol yielded esterified lignins much more soluble than Biolignin in common organic solvents. During this reaction, the secondary OH of β-O-4 linkages was simultaneously etherified. Phenol hydroxyethylation by 2-chloroethanol yielded samples containing only aliphatic hydroxyl groups.

Full text of DOI:10.1021/acs.jafc.5b02071

Article
pubs.acs.org/JAFC
Purification, Structural Characterization, and Modification of
Organosolv Wheat Straw Lignin
Laurie Mbotchak, Clara Le Morvan, Khanh Linh Duong, Brigitte Rousseau, Martine Tessier,
and Alain Fradet*
Sorbonne Universites
Paris, France
́ ́ ́
, UPMC Univ Paris 06, Institut Parisien de Chimie Moleculaire UMR 8232, Chimie des Polymeres, F-75005
CNRS, IPCM UMR 8232, F-75005, Paris, France
S
* Supporting Information
ABSTRACT: Biolignin, a wheat straw lignin produced by acetic acid/formic acid/water hydrolysis, was characterized by 31P and
13C−1H 2D NMR spectroscopy and by size-exclusion chromatography. Biolignin is a mixture of low molar mass compounds (Mn
= 1660 g/mol) made up of S, G, and H units and of coumaric and ferulic acid units. β-5 and β-O-4 interunit linkages are partially
acylated in the γ-position by acetate and p-coumarate groups. Deacylated samples with a low content of contaminants were
obtained by combining alkaline hydrolysis and solvent extraction. The high phenolic OH content found by 31P NMR reflects the
presence of condensed aromatic units, such as 5−5 units. Reaction of purified lignin with ethanol and ethane-1,2-diol yielded
esterified lignins much more soluble than Biolignin in common organic solvents. During this reaction, the secondary OH of β-O-
4 linkages was simultaneously etherified. Phenol hydroxyethylation by 2-chloroethanol yielded samples containing only aliphatic
hydroxyl groups.
KEYWORDS: wheat straw lignin, NMR, esterification, etherification
late).25 However, several issues have to be addressed before
INTRODUCTION
■
lignins can find extensive applications in polymeric materials:
(1) Technical lignins are rarely recovered in pure form. While
the major portion of hemicelluloses is removed during the
fractionation step, the presence of residual carbohydrates in
crude lignin fractions (i.e., obtained directly after the
delignification process) cannot be excluded. Wax and sugar
content in a lignin sample are closely related to the process and to
the pre- or posttreatment steps used for lignin isolation. Their
presence has to be carefully considered as it may influence lignin
reactivity and, therefore, the properties of final materials.
Treatment of straw by apolar/polar solvents mixtures prior to
the fractionation process is used in some cases to produce
dewaxed straw.3−5 On the other hand, basic hydrolysis is an
efficient way to remove residual polysaccharides by cleavage of
ester linkages between hemicellulose and lignin, yielding almost
polysaccharide-free technical lignins.4,6,26,27
(2) As mentioned above, the structure of technical lignins
depends heavily on the conditions used in the delignification and
purification processes. In order to elaborate materials with
controlled properties and to ensure synthesis repeatability, an in-
depth knowledge of lignin structure and reactivity is required.
(3) With regard to polymer synthesis, the major drawbacks of
technical lignins are their low solubility in organic medium, their
functional group heterogeneity, and their relatively poor
hydroxyl group accessibility.
Conversion of lignin to value-added products is one of the
challenges for the expanding biobased products industry. In this
context, the utilization of lignin from grasses, and especially from
crop residues, has attracted increasing research attention.1,2
Wheat straw lignin is a major industrial crop residue and its
separation, purification, and structure have been extensively
studied.3−12 Like lignins from other grasses, wheat straw lignin is
a complex polymer formed of three types of aromatic units,
syringyl (S), guaiacyl (G), and 4-hydroxyphenyl (H) units,
linked to each other by short aliphatic “side chains” of various
types through C−C or C−O−C bonds. It presents a relatively
high content of acetate and p-hydroxycinnamate groups,
predominantly bound at the γ-position of side chains.13 In
plants, lignin is linked to polysaccharides, mainly hemicellulose,
by ether or ester groups, thus ensuring structural cohesion and
rigidity to cell walls. The delignification process, designed to
cleave lignin−saccharide bonds, leads to more or less extensive
modification of native lignin structure. Therefore, the
composition of the resulting technical lignins, for example, the
relative abundance of S/G/H units, the nature of side chains, the
presence of cinnamate and acetate groups and the content in
functional groups (alcohols, phenols, and carboxylic acids), is
highly dependent on the delignification process conditions.14
Technical lignins are highly functionalized aromatic com-
pounds and, as such, particularly attractive for use as
comonomers in polymeric materials.15 Lignins have thus been
used as rigid polyols in polyester,15−18 polyurethane,19−21 and
epoxy22−24 thermosetting resins and, after modification, for the
synthesis of starlike polystyrene and poly(methyl methacry-
Received: February 11, 2015
Revised: May 9, 2015
Accepted: May 10, 2015
Published: May 11, 2015
© 2015 American Chemical Society
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Journal of Agricultural and Food Chemistry
Article
overnight at 50 °C under vacuum to yield 0.75 g of ethanol-soluble
modified lignin (s-Lp-Et).
To overcome these issues, preliminary purification and
chemical modification of technical lignins have been described:
for example, esterification of hydroxyl groups by anhydrides,28
reaction with epichlorhydrin,22 or reaction with propylene
oxide.29−31 While the characterization of oxypropylated lignin
with modern methods has been reported,31,32 most modified
technical lignins, however, have subsequently been used without
detailed characterization.
Modification of Lignin with Ethane-1,2-diol. Lp (5 g), ethane-
1,2-diol (75 mL) and p-toluenesulfonic acid monohydrate as a catalyst
(530 mg, 0.6 wt %) were heated at 90 °C for 20 h. The homogeneous
mixture was poured into 500 mL of deionized water. The precipitate was
filtered on sintered glass filter (porosity 5), washed with deionized water
until neutral pH, and dried at 50 °C under vacuum for 24 h to give 4.95 g
of ethane-1,2-diol-modified lignin (Lp-EG).
The aim of this study was to evaluate the influence of
purification steps and of esterification and etherification chemical
modifications of wheat straw lignin on its chemical and structural
characteristics. This study was undertaken on wheat straw lignin
Modification of Lignin with 2-Chloroethanol. Lp (3.1 g) was
dissolved in 30 mL of 1 M aqueous NaOH at 90 °C. 2-Chloroethanol (6
mL, 84 mmol) was added dropwise and the mixture was heated at 90 °C
for 20 h. The mixture was poured into 300 mL of water and neutralized
with 2 M aqueous HCl. The precipitate was filtered on sintered glass
filter (porosity 5) and dried at 50 °C under vacuum for 24 h to give 2.80
g of 2-chloroethanol-modified lignin (Lp-CE).
Synthesis of 4-(2-Hydroxyethoxy)cinnamic Acids. 4-(2-
Hydroxyethoxy)cinnamic acids were synthesized by reacting the
corresponding 4-hydroxycinnamic acids with 2-chloroethanol in alkaline
medium according to a procedure described for hydroxyethoxylation of
4-hydroxybenzoic acid.35
(2E)-3-[4-(2-Hydroxyethoxy)phenyl]prop-2-enoic Acid, 20. A sol-
ution of p-coumaric acid (8.2 g, 50 mmol) in 40 mL of 3 M aqueous
NaOH was heated to 50 °C. 2-Chloroethanol (5 mL, 75 mmol) was
added slowly, and the mixture was heated at 90 °C for 6 h. After cooling,
the mixture was poured into 50 mL of deionized water and acidified with
1 M aqueous HCl. The residue was filtered and washed with acetone to
remove residual p-coumaric acid and ester byproducts. Product 20 was
recrystallized from ethanol: yield 6.2 g (60%). 1H NMR (DMSO-d6) δ,
ppm 7.61 (d, J = 8 Hz, H-2, H-6), 7.54 (d, J = 16 Hz, H-α), 6.96 (d, J = 8
Hz, H-3, H-5), 6.36 (d, J = 16 Hz, H-β), 4.02 (t, J = 6 Hz, −CH2−OAr),
3.72 (t, J = 6 Hz, −CH2−OH). 13C NMR (DMSO-d6) δ, ppm 167.7 (C-
γ), 160.3 (C-4), 143.6 (C-α), 129.8 (C-2, C-6), 126.6 (C-1), 116.4 (C-
β), 114.8 (C-3, C-5), 69.6 (−CH2−OAr), 59.4 (−CH2−OH).
(2E)-3-[4-(2-Hydroxyethoxy)-3-methoxyphenyl]prop-2-enoic
Acid, 21. Compound 21 was synthesized from ferulic acid following the
same procedure: yield 5.6 g (47%). 1H NMR (DMSO-d6) δ, ppm 7.51
(d, J = 16 Hz, H-α), 7.31 (d, J4 = 2 Hz, H-2), 7.17 (dd, J4 = 2 Hz, J3 = 8
Hz, H-6), 6.97 (d, J3 = 8 Hz, H-5), 6.44 (d, J = 16 Hz, H-β), 4.00 (t, J = 6
Hz, −CH2−OAr), 3.81 (s, −CH3-O−), 3.72 (t, J = 6 Hz, −CH2−OH).
13C NMR (DMSO-d6) δ, ppm 167.9 (C-γ), 150.1 (C-4), 149.0 (C-3),
produced by the CIMV (Compagnie Industrielle de la Matier
̀
e
Vegetale) acetic acid/formic acid/water fractionation proc-
́
́
ess,11,33 available under the trade name Biolignin. Lignin was
first purified by hydrolysis and solvent extraction and then
modified by esterification with ethanol or ethane-1,2-diol to
mask acid groups or by etherification with 2-chloroethanol in
alkaline conditions to convert phenol groups into more reactive
primary alcohols. The modification reactions were expected to
provide samples with enhanced solubility, homogenized
functionality in terms of function types, and improved reactivity
for future applications as comonomer in polymeric materials.
These samples were thoroughly characterized by means of 31P
and 13C−1H 2D NMR spectroscopy and size-exclusion
chromatography (SEC).
MATERIALS AND METHODS
■
Materials. Absolute ethanol, ethane-1,2-diol (99.8%), 2-chloroetha-
nol (99%), p-coumaric acid (98%), ferulic acid (99%), sinapic acid
(98%), p-toluenesulfonic acid monohydrate (98.5%), sulfuric acid
(99%), 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (95%),
anhydrous pyridine (99.8%), chromium(III) acetylacetonate (97%),
and acetic anhydride (≥99%) were obtained from Sigma−Aldrich
(Saint-Quentin-Fallavier, France). N-Hydroxy-6-norbornene-2,3-dicar-
boximide (>96%) was obtained from Fluka (Saint-Quentin-Fallavier,
France). All reagents were used without further purification.
Biolignin (Klason lignin content 89.8%)34 was obtained from CIMV
144.1 (C-α), 127.0 (C-1), 122.5 (C-6), 116.6 (C-β), 112.5 (C-5), 110.4
(C-2), 70.1 (−CH2−OAr), 59.4 (−CH2−OH), 55.5 (CH3O−).
(2E)-3-[4-(2-Hydroxyethoxy)-3,5-dimethoxyphenyl]prop-2-enoic
Acid, 22. A solution of sinapic acid (0.9 g, 4 mmol) in 5 mL of 3 M
aqueous NaOH was heated to 50 °C. 2-Chloroethanol (0.65 mL, 9.5
mmol) was added slowly, and the mixture was heated at 90 °C for 6 h.
After cooling, the mixture was poured into 20 mL of deionized water,
acidified with 1 M aqueous HCl, and extracted with ethyl acetate (3 × 20
mL). Organic phases were washed with brine and concentrated under
reduced pressure. Product 22 was recrystallized from ethyl acetate: yield
0.11 g (10%). 1H NMR (DMSO-d6) δ, ppm 7.52 (d, J = 16 Hz, H-α),
7.03 (d, J = 2 Hz, H-2, H-6), 6.53 (d, J = 16 Hz, H-β), 3.90 (t, J = 6 Hz,
̀ ́ ́
(Compagnie Industrielle de la Matiere Vegetale, Neuilly, France).
Wheat straw from Triticum aestivum L. subsp. aestivum, seeded in
November 2011, was harvested in Champagne region (France) in July
2012. Biolignin was extracted by the CIMV process with acetic acid/
formic acid/water 30:55:15 v/v/v) at 105 °C under atmospheric
pressure.12,33
Lignin Purification. Biolignin (20.19 g) was hydrolyzed by 1 M
aqueous NaOH (300 mL) at 80 °C for 4 h. The reaction mixture was
then acidified to pH 2 with 3 M aqueous HCl. The precipitate was
filtered and washed thoroughly with deionized water until neutral pH,
then dried at 50 °C under reduced pressure for 24 h to give 18.13 g of
hydrolyzed Biolignin (Lh; yield 90 wt %). The acidic filtrate was
concentrated under reduced pressure to afford a dried hydrolysis
residue, Rh.
Lh was further purified by repeated washing steps with CH2Cl2 (2×)
and ethyl acetate (3×) as follows: Lh (17 g) was poured into 100 mL of
solvent, heated to reflux for 1 h, and filtered. After drying under reduced
pressure, 13.33 g of purified Biolignin (Lp) was obtained (overall yield
from Biolignin 70.5 wt %). Organic phases were evaporated to yield the
purification residue, Rp.
Modification of Lignin with Ethanol. Lp (2 g), ethanol (30 mL),
and p-toluenesulfonic acid monohydrate as a catalyst (200 mg, 0.8 wt %)
were heated at 80 °C for 20 h. The insoluble fraction was filtered on
sintered glass filter (porosity 5), washed with deionized water until
neutral pH, and dried overnight at 50 °C under vacuum to give 1.25 g of
ethanol-insoluble modified lignin (i-Lp-Et). The alcohol phase was
concentrated under reduced pressure. The residue was precipitated in
200 mL of water, filtered on sintered glass filter (porosity 5), and dried
−CH2−OAr), 3.81 (s, CH3-O−), 3.61 (t, J = 6 Hz, −CH2−OH). 13
C
NMR (DMSO-d6) δ, ppm 167.6 (C-γ), 153.0 (C-3, C-5), 144.0 (C-α),
138.4 (C-4), 129.6 (C-1), 118.4 (C-β), 105.8 (C-2, C-6), 74.1 (−CH2−
OAr), 60.1 (−CH2−OH), 56.0 (CH3O−).
Ethyl (2E)-3-[4-(2-Hydroxyethoxy)-3-methoxyphenyl]prop-2-
enoate, 23. A mixture of 22 (4.02 g, 16.9 mmol), ethanol (60 mL),
and 18 M sulfuric acid as a catalyst (310 mg, 0.6 wt %) was refluxed for
20 h. After cooling, the solution was concentrated under vacuum to yield
an orange-colored viscous product. This product was dissolved in 100
mL of diethyl ether, and this solution was washed twice with 30 mL of
0.5 M aqueous solution of NaHCO3, twice with 30 mL of saturated
NaCl solution, then twice with 30 mL of deionized water. The organic
phase was dried on MgSO4, filtered, and evaporated to dryness under
vacuum. The resulting white powder was dried at 60 °C under vacuum
for 12 h: yield 3.25 g (72%). 1H NMR (DMSO-d6) δ, ppm 7.57 (d, J = 16
Hz, H-α), 7.35 (d, J4 = 2 Hz, H-2), 7.21 (dd, J4 = 2 Hz, J3 = 8 Hz, H-6),
6.98 (d, J3 = 8 Hz, H-5), 6.54 (d, J = 16 Hz, H-β), 4.86 (t, J = 5 Hz,
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−OH), 4.17 (q, J = 7 Hz, −CH2−OOC−), 4.01 (t, J = 5 Hz, −CH2−
OAr), 3.81 (s, CH3−O−), 3.72 (q, J = 5 Hz, −CH2−OH), 1.25 (t, J = 7
Hz, −CH3). 13C NMR (DMSO-d6) δ, ppm 166.4 (C-γ), 150.3 (C-4),
149.0 (C-3), 144.5 (C-α), 126.8 (C-1), 122.8 (C-6), 115.5 (C-β), 112.4
(C-5), 110.5 (C-2), 70.05 (−CH2−OAr), 59.7 (−CH2−OOC−), 59.35
(−CH2−OH), 55.5 (CH3O−), 14.1 (−CH3).
Spectroscopic Methods. One-dimensional (1D) 1H and 31P NMR
spectra and two dimensional (2D) 13C−1H NMR correlation spectra
were recorded on a Avance 500 spectrometer (Bruker, Karlsruhe,
Germany), at 500 MHz (1H NMR), 125 MHz (13C NMR), or 202 MHz
(31P NMR) with a 5 mm double-resonance broadband inverse probe
(BBI). The 1D 13C NMR spectra were recorded on the same
spectrometer at 125 MHz with a 10 mm double-resonance broadband
observe probe (BBO). Lignin samples were dissolved in deuterated
dimethyl sulfoxide, DMSO-d6 (1H, 13C, and 2D 13C−1H NMR), or
CDCl3/pyridine mixture (31P NMR).
The 2D 13C−1H correlation spectra were recorded through a phase-
sensitive gradient-enhanced 2D heteronuclear single quantum coher-
ence (HSQC) with echo−antiecho experiment (HSQCETGP
sequence). The 2D 13C−1H long-range correlation heteronuclear
multiple bond correlation (HMBC) spectra were recorded via
heteronuclear zero and double quantum coherence experiment
(HMBCGPLPNDQF sequence). Chemical shifts were referenced to
residual DMSO-d5 (δ = 2.50 ppm) for 1H NMR and to DMSO-d6 (δ =
39.43 ppm) for 13C NMR.
31P NMR experiments were carried out following a slightly modified
previously reported method:36 Lignin (30 mg) was dissolved in a
CDCl3/pyridine mixture (1:1.6 v/v, 0.5 mL). The phosphitylation
reagent, 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (150 μL),
and the internal standard, N-hydroxy-6-norbornene-2,3-dicarboximide
(100 μL of 0.1 M solution in 1:1.6 v/v CDCl3/pyridine mixture)37 were
added successively. Chromium(III) acetylacetonate (100 μL of 0.014 M
solution) in the same CDCl3/pyridine mixture was added to the solution
in order to homogenize and accelerate phosphorus relaxation. Spectra
were recorded with a 25 s relaxation time and an average number of
10 000 scans. The quantitative results shown are the average of duplicate
NMR experiments. Chemical shifts are relative to the signal of the
phospholane hydrolysis product at 132.2 ppm. The integral value of the
internal standard was used for calculations of the absolute amount of
each functional group.
Size-Exclusion Chromatography. SEC analyses were performed
on a 300 × 7.5 mm i.d., 5 μm Mixed-C (200−(2 × 106) Å) PLgel three-
column set (Agilent Technologies, Les Ulis, France), connected to a
VE5200 automatic injector (Malvern Instruments Ltd., Guyancourt,
France) and a model 515 pump and a model 410 differential
refractometer (Waters Corp., Guyancourt, France). Tetrahydrofuran
(THF) was used as eluent (1 mL/min). Acetylated lignin solution in
THF (100 μL, 5 g/L) was injected. Chromatograms were processed by
use of OmniSEC software. Polystyrene standards (Polymer Labo-
ratories) were used for the calibration.
Lignin Acetylation. Lignin (220 mg) was reacted with acetic
anhydride (5 mL) and pyridine (5 mL) at room temperature for 72 h.38
The resulting mixture was poured in water (100 mL). The precipitated
acetylated lignin was filtered and dissolved in CH2Cl2. The solution was
dried over MgSO4 and evaporated under reduced pressure to afford
acetylated lignin (210 mg, 95% yield).
RESULTS AND DISCUSSION
■
Figure 1. 2D 13C−1H HSQC NMR spectra of (A) Biolignin, (B) Lh,
and (C) Lp: expansion of the lignin side-chain domain. Structure
notations are given in Figure 3 and discussed in main text.
Purification and Characterization of Biolignin. Biolignin
was first hydrolyzed in 1 M aqueous NaOH solution for 4 h at 80
°C, leading to hydrolyzed Biolignin (Lh). This alkaline
hydrolysis leads to the cleavage of ester bonds, including those
involved in carbohydrate−lignin complexes, and allows the
elimination of residual polysaccharides and esters together with
some aromatic compounds.27 Moreover, traces of acetic and
formic acids present in the starting material and resulting from
the delignification process are also removed. In order to remove
water-insoluble impurities, such as low molar mass aromatic
compounds and waxes, Lh was then extracted with dichloro-
methane and ethyl acetate, yielding a final, purified Biolignin
(Lp) with highly reduced contaminant content. The purification
steps were followed by 1H NMR. Analysis of the hydrolysis and
purification residues confirmed the elimination of polysacchar-
ides and of fatty acids, p-coumaric acid, and ferulic acid,
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Figure 2. 2D 13C−1H HSQC NMR spectra of (A) Biolignin, (B) Lh, and (C) Lp: expansion of the aromatic domain. Structure notations are given in
Figure 4 and discussed in main text.
Figure 3. Structures, atom numbering, and HSQC correlations (δC/δH, ppm) of interunit linkages found in wheat straw lignin (R1−4 = H, CH3O; R′ = H,
CO).
respectively, together with various low molar mass aromatic
compounds.
in Biolignin, as shown by the correlations corresponding to their
β- and γ- positions, the latter being more intense (Figure 1A).
These two correlations are no longer present after hydrolysis in
Lh and Lp spectra (Figure 1B,C). α-Oxidized alkyl−aryl ether
structures, 3, are also detected by a correlation corresponding to
the β-position. Correlations corresponding to the α-, β-, and γ-
positions of β-5 (phenylcoumaran), 4, and β−β (resinol)
linkages, 5, are of much lower intensity as compared to those
2D 13C−1H HSQC NMR Analyses of Biolignin, Lh, and Lp. 2D
13C−1H HSQC NMR analyses were performed in DMSO-d6
(Figures 1 and 2). Correlations were assigned according to
literature (Figures 3 and 4).7,27,38−40 The β-O-4 linkages (alkyl−
aryl ether), 1, of H, G, and S units are easily detected by their α, β,
and γ resonances. γ-Esterified β-O-4 linkages, 2, are also present
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Figure 4. Structures, atom numbering, and 2D 13C−1H HSQC NMR correlations (δC/δH, ppm) of aromatic units found in wheat straw lignin.
of β-O-4, which, therefore, seem to be predominant. However, it
is difficult to conclude on their relative abundance, since the
NMR sequence used for these analyses does not allow cross-peak
quantitation.
In the aromatic domain (Figure 2A), the resonances of H, G,
and S units are observed together with moieties derived from p-
coumaric and ferulic acids. As previously reported in studies on
wheat straw lignin,41 sinapic acid derivatives are not present,
since their characteristic signal at 106.1/6.98 ppm (2,6-
Figure 5. Structure of model 4-(2-hydroxyethoxy)cinnamic derivatives.
positions) is not detected. In Biolignin, the presence of free p-
coumaric and ferulic acids 7 and 9, and of their esters 8 and 10, is
reflected by their 2,6-, α- and β-correlations. A cross-peak close to
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Table 2. Number- and Mass-Average Molar Masses and
a
Molar-Mass Dispersities of Acetylated Lignin Samples
sample
Biolignin
Lp
i-Lp-Et s-Lp-Et Lp-EG Lp-CE
Mw (g/mol)
Mn (g/mol)
ĐM
31890
1660
19.2
18400
1560
11.8
24270
2100
11.6
4160
1340
3.1
23520
1765
13.3
4633
1155
4.0
a
Determined by size-exclusion chromatography.
are not detected in Biolignin spectra. The acidic conditions of the
CIMV process probably lead to the elimination of this type of
compound.
31P NMR Spectroscopy. 31P NMR spectroscopy was used to
differentiate and quantitate the different types of free hydroxyl
groups (aliphatic, phenolic, and carboxylic) in Biolignin and Lp,
after phosphitylation by 2-chloro-4,4,5,5-tetramethyl-1,3,2-diox-
aphospholane, as described by Argyropoulos and co-workers.8,36
N-Hydroxy-5-norbornene-2,3-dicarboximide was used as inter-
nal standard instead of cyclohexanol,37 as its signal at 151.9 ppm
is well separated from those of lignin. It should be noted that the
un-, mono-, and disubstituted 4-hydroxyphenyl groups quanti-
tated by this method correspond only to a fraction of the H, G,
and S units present in lignin, that is, those bearing free phenol
groups. Moreover, the disubstituted 4-hydroxyphenyl structures
detected by this method comprise syringyl units with free phenol
groups, as well as condensed phenolic units.36
There are two main differences between the spectra of
Biolignin and Lp (Figure 6): (1) lower relative intensity of
unconjugated carboxylic acid resonances with respect to
conjugated ones (134.70 and 135.05 ppm,27 respectively) in
the spectrum of Lp, due to the elimination of fatty acids and
residual acetic and formic acids during the hydrolysis and
purification steps; and (2) disappearance of the sharp peaks
emerging from broad signals in the phenolic OH region of
Biolignin spectrum (un-, mono-, and disubstituted 4-hydrox-
yphenyl groups at 137−144 ppm). The well-resolved peaks at
137.80 and 139.40 ppm are assigned to p-coumaric and ferulic
acids, respectively.27
Aliphatic, phenolic, and acidic hydroxyl contents were
determined by integration of the corresponding peaks with
respect to the internal standard (Table 1). In Biolignin, phenolic
OH groups are preponderant, with the aliphatic/phenolic OH
ratio rOH being close to 0.8, and acidic OH groups represent
about 10% of total OH content. Monosubstituted 4-hydrox-
yphenyl OH unit content is slightly higher than that of
disubstituted units (mono/disubstituted = 1.2), while unsub-
stituted 4-hydroxyphenyl units represent ca. 15% of total
phenolic groups. Hydrolysis and purification lead to a slight
increase in total OH content (6.90 vs 5.61 mmol/g) without
Figure 6. 31P NMR spectra of (A) phosphitylated Biolignin and (B)
phosphitylated purified Biolignin Lp. (*) Impurity; (**) internal
standard; (a) disubstituted p-hydroxyphenyl structure as syringyl and
condensed phenolic units; (b) monosubstituted p-hydroxyphenyl
structure as guaiacyl units; (c) unsubstituted p-hydroxyphenyl structure.
the 6-correlation of 9 and 10 was assigned to p-etherified ferulic
acid 11, with the help of a model of these moieties, namely, (2E)-
3-[4-(2-hydroxyethoxy)-3-methoxyphenyl]prop-2-enoic acid 21
(Figure 5). Low molar mass aromatic derivatives are also
detected: syringic acid 14, vanillin 15, and syringaldehyde 16.
Correlations at 122.9/7.54, 112.8/6.78, and 149.1/8.10 ppm
were assigned to the CH groups of furaldehyde 18, a product of
polysaccharide degradation, by comparison with the spectrum of
an authentic sample. The CHO correlation of 18 was also
observed at 178.3/9.61 ppm (region not shown).
After hydrolysis (Figure 2B), p-coumarates and ferulates 8 and
10 are no longer detected, but free acids 7, 9, and 14 and
aldehydes 15 and 16 are still present, and a correlation
corresponding to acetosyringone 17 appears. These low molar
mass compounds are removed during the solvent extraction
steps, and the spectrum of Lp (Figure 2C) exhibits a very
simplified pattern consisting mainly of signals relative to H, G,
and S units and to p-etherified ferulic acid (11). As found for
other lignin types,13 p-etherified ferulates are an integral part of
the lignin backbone. These findings were confirmed by the 2D
NMR study of hydrolysis and solvent extraction residues.
Milled wood (MW) wheat straw lignin is also reported to
contain flavonoids, such as tricin [5,7-dihydroxy-2-(4-hydroxy-
3,5-dimethoxyphenyl)-4H-chromen-4-one], which presents
strong and well-resolved HSQC signals.7 However, these signals
a
Table 1. Hydroxyl Content of Biolignin, Lp, Lp-Et, Lp-EG, and Lp-CE Lignins
OH content (mmol/g)
OH content (% of total OH + COOH)
b
b
b
sample
phenolic
aliphatic
COOH
(a)
(b)
(c)
aliphatic
COOH
Biolignin
Lp
2.84
3.49
3.12
2.08
2.24
2.57
1.13
2.67
0.53
0.84
0.04
0.04
19.40
22.50
32.40
20.0
23.35
21.60
30.70
17.40
3.40
7.85
6.50
9.50
6.00
0.90
39.95
37.25
26.30
55.70
87.80
9.45
12.15
1.10
c
s-Lp-Et
Lp-EG
0.90
d
Lp-CE
3.30
4.60
a
b
Determined by quantitative 31P NMR after phosphitylation with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane. (a) Di-, (b) mono-, and (c)
c
d
unsubstituted 4-hydroxyphenyl OH, respectively. Ethanol-soluble fraction of Lp-Et. OH content (millimoles per gram) was not determined since
Lp-CE is not completely soluble in phosphitylation medium. OH content (percent) was calculated on the soluble fraction.
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Figure 7. 2D 13C−1H HSQC NMR spectra of (A) s-Lp-Et and (B) Lp-EG: expansion of the lignin side-chain domain. Structure notations are given in
Figure 8. EG, residual ethane-1,2-diol.
much affecting the aliphatic/phenolic ratio and, therefore, lignin
main structure. Although free aliphatic and cinnamic acids were
removed by the hydrolysis and purification steps, COOH
concentration increases, due to hydrolysis of the ester functions
of 4-O-etherified ferulates.
hydrolysis of p-coumarates present in lignin structure. A similar
p-coumarate hydrolysis during the acidic CIMV delignification
process could also be involved to explain the low unsubstituted 4-
hydroxyphenyl unit content of Biolignin.
Size-Exclusion Chromatography. The impact of the
hydrolysis and purification steps on average molar masses and
molar mass distributions was evaluated by SEC (Table 2). The
samples were acetylated, in order to improve their solubility in
THF and to avoid the formation of aggregates.46 Biolignin is a
mixture of oligomeric compounds (Mn = 1660 g/mol) exhibiting
a very large molar-mass dispersity, close to ĐM = 19. The
hydrolysis and purification steps do not affect molar mass since
the Mn of Lp lies in the same range. The molar-mass dispersity of
Lp is lower, but still quite high (ĐM = 11.8). The Mn values
previously reported for various technical wheat straw lignins
(1000−3780 g/mol)7,9,12,44 are close to those found for Biolignin
in this work, while literature ĐM values vary between 1.79 and
10.6.44 However, it is difficult to compare these SEC values, since
delignification and/or analysis conditions are different from each
other. In this work, molar mass values are compared before and
after chemical modification, in order to detect potential lignin
backbone changes.
Modification of Purified Biolignin. Modification of lignin
prior to its use as macromonomer presents many advantages.
Besides improving sample solubility in common organic solvents,
this could allow selective masking of some functions and
homogenizing of reactive group types. Lp was modified by three
methods: esterification by (i) ethanol or (ii) ethane-1,2-diol to
mask acid groups and (iii) etherification with 2-chloroethanol
under alkaline conditions to replace phenol groups by more
reactive aliphatic OH groups.
The high total phenolic group content indirectly reflects the
presence of condensed phenolic units, such as 5−5 units, 6
(Figure 3). Shoulders at ca. 143 and 142 ppm in the 31P NMR
spectra of phosphitylated lignins have been assigned to
condensed phenolic structures.36 However, it is difficult to
make any accurate quantitation in this region, even by
deconvolution, due to the unknown number and width of
signals that overlap in this region. Condensed phenolic units
might also be detected in the aromatic region of HSQC spectra.
On the basis of NMR literature data on model compounds,42,43
the 5−5 structure arising from coniferyl alcohol should give two
signals (2- and 6-positions), close to the 2- and 6-correlations of
G units. Unassigned correlations at 109.5/7.10 and 119.4/6.95
ppm are observed in the HSQC spectrum of Lp (Figure 2C).
They could reflect the presence of 5−5 units, but this has yet to
be confirmed.
Comparison of 31P NMR literature data on technical wheat
straw lignins highlights the importance of the fractionation
method on the chemical composition of resulting lignin samples.
When either bases, for example, soda wheat straw lignin
(GreenValue SA),32 or organic acids44 are used, lignins samples
exhibit aliphatic/phenolic OH ratio rOH ≤ 1, close to that found
for Biolignin in the present work. On the other hand, rOH is close
to 2.4−3 for MW wheat straw lignin8 and dioxane/HCl lignin45
samples. The un/mono/disubstituted 4-hydroxyphenyl unit
distribution is also process-dependent, with MW samples
exhibiting higher unsubstituted unit content as compared to
samples obtained by alkaline or acidic processes. Crestini and
Argyropoulos8 studied the effect of 2 M alkaline hydrolysis on
MW wheat straw lignin. They observed a strong decrease in
unsubstituted 4-hydroxyphenyl unit content, reaching values
close to that found above for Biolignin and Lp. They showed that
this was mainly due to release of p-coumaric acid by the basic
Lignin Modification by Esterification. Both esterification
reactions were catalyzed by p-toluenesulfonic acid. When
esterifications were carried out in ethanol, the reaction mixture
remained heterogeneous. After reaction, the insoluble fraction, i-
Lp-Et, was isolated from the soluble fraction, s-Lp-Et, by
filtration. Both samples were soluble in DMSO-d6 and were
1
analyzed by H and 2D NMR spectroscopy. Spectra with very
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Figure 8. Structures, atom numbering and HSQC correlations (δC/δH, ppm) of modified lignin units found in Lp-Et, Lp-EG, and Lp-CE (R1−4 = H,
CH3O; R′ = aliphatic chain).
similar profiles were obtained, so that only the spectra of s-Lp-Et
will be further discussed. On the other hand, esterification with
ethane-1,2-diol led very rapidly to a homogeneous reaction
mixture and afforded a sample, Lp-EG, that was soluble in
common organic solvents such as CH2Cl2, THF, and DMSO.
The aliphatic domains of s-Lp-Et and Lp-EG HSQC NMR
spectra are presented in Figure 7 with assignments in Figure 8.
The spectrum of s-Lp-Et exhibits two distinct signals at 59.3/4.04
and 59.4/4.16 ppm (Figure 7A), assigned to the methylenes of
ethyl ester groups of nonlignin aliphatic esters 25, and of p-
etherified ethyl ferulate moieties 26, respectively. This was
confirmed by long-range correlations between the corresponding
carbonyl carbons and the methylene protons of ethyl esters at
172.45/4.04 and 166.25/4.16 ppm, respectively, observed in the
HMBC 13C−1H NMR spectrum of s-Lp-Et. These assignments
were also confirmed by comparisons with the spectra of ethyl
(2E)-3-[4-(2-hydroxyethoxy)-3-methoxyphenyl]prop-2-enoate
23, a model of p-etherified ethyl ferulate groups (Figure 5). The
HSQC NMR spectrum also reveals that the secondary alcohols
of β-O-4 structures were simultaneously etherified: The α-CH
signal of β-O-4 linkages (1-α) is shifted to 79.70/4.48 ppm after
reaction (24-α), that is, in the secondary ether resonance region.
Moreover, new correlations are detected at 63.7/3.32 ppm
(Figure 7A) and 14.80/1.08 ppm (not shown), which
correspond, respectively, to methylene and methyl of the ethyl
ether group of 24. Etherification of the secondary alcohol of β-O-
4 linkages has already been reported for Miscanthus giganteus
lignin obtained by the ethanol−water−HCl process.47
Similar results were obtained when the reaction was carried
out with ethane-1,2-diol. The −COO−CH2−CH2−OH meth-
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unaffected by the chemical modification. A decrease in the
aliphatic/phenolic OH ratio is observed for s-Lp-Et (rOH = 0.36),
due to formation of ethyl ethers 24. In the case of Lp-EG, the
aliphatic/aromatic OH ratio increases (rOH = 1.28). In this case,
the formation of hydroxyethyl ethers 27 does not change total
aliphatic hydroxyl group content, while the hydroxyethylation of
11 leads to etherified hydroxyethyl ferulates 29 and to hydroxyl
group concentration increase.
SEC analysis shows that s-Lp-Et exhibits lower Mn (1340 g/
mol) and dispersity (ĐM = 3.1) than Lp, while the Mn of the
ethanol-insoluble fraction is slightly higher (2100 g/mol) and
dispersity is similar (ĐM = 11.6). Lp and Lp-EG present very
close Mn and molar-mass dispersity (Table 2). Cleavage of lignin
β-O-4 linkages and their rearrangement into lignin-based
polymeric materials was reported for beech MW lignin
liquefaction with ethane-1,2-diol.48 The experimental hydrox-
yethylation conditions used in the present work (0.6% p-
toluenesulfonic acid, 90 °C) are significantly milder than those
used for wood liquefaction (3% p-toluenesulfonic acid, 150 °C)
and appears to leave β-O-4 linkages unchanged, with the
exception of the α-hydroxyethoxylation reaction discussed
above.
Lignin Modification by Etherification with 2-Chloroetha-
nol. Etherification of Lp phenol groups with 2-chloroethanol
under alkaline conditions yields a modified lignin, Lp-CE, that is
insoluble in CH2Cl2 and only partially soluble in DMSO. Lp-CE
characterization was, therefore, carried out on the soluble
fraction. Analysis of the HSQC aliphatic domain (Figure 10A)
shows that phenol groups have reacted, since three different
signals are observed at 69−74/3.8−4.0 ppm, assigned to the Ar−
O−CH2−CH2−OH methylene of 2-hydroxyethoxy groups in
the 4-position of H, G, and S aromatic rings (Figure 8).
Assignments of the resulting etherified units H′, G′, and S′ were
made with the help of model compounds 20, 21, and 22 (Figure
5) obtained by reaction of coumaric, ferulic, and sinapic acids
with 2-chloroethanol in similar conditions. The −CH2−OH
methylenes of these groups and of β-O-4 linkages (1-γ) give
overlapping cross-peaks. During the reaction, carboxylic acids are
partly esterified into hydroxyethyl esters since signals corre-
sponding to ferulate (29-9 and 29-10) and aliphatic esters (28-9
and 28-10) are also detected. On the other hand, β-O-4 aliphatic
secondary hydroxyl groups remain unchanged, since no signal
corresponding to aliphatic ethers (24-α) is detected at 79.7/4.48
ppm. Etherification of phenol groups by 2-chloroethanol is
reflected by new correlations at 112.55/6.86 (G′-5) and 113.90/
6.84 ppm (H′-3,5) (Figure 10B).
Figure 9. 31P NMR spectra of phosphitylated lignin samples: (A) Lp,
(B) s-Lp-Et, (C) Lp-EG, and (D) Lp-CE. (*) Impurity; (**) internal
standard; (a) disubstituted p-hydroxyphenyl structures as syringyl and
condensed phenolic units; (b) monosubstituted p-hydroxyphenyl
structures as guaiacyl units; (c) unsubstituted p-hydroxyphenyl
structures.
Figure 10. 2D 13C−1H HSQC NMR spectra of Lp-CE: expansion of
lignin (A) aliphatic and (B) aromatic domains. Structure notations are
given in Figure 8. EG, residual ethane-1,2-diol.
Lp-CE was also analyzed by 31P NMR (Figure 9). Since the
sample was not fully soluble in phosphitylation medium,
quantitative determination of OH groups was not possible.
The relative aliphatic/aromatic OH content was nevertheless
determined on the soluble fraction, showing an almost 7-fold
decrease in phenol content, with unchanged ratio of un-, mono-,
and disubstituted 4-hydroxyphenyl units. Hydroxyethylation by
2-chloroethanol in basic medium, therefore, appears to be a very
efficient modification method of lignin phenol groups.
Since Lp-CE was only partially soluble in THF, even after
acetylation, the SEC analyses were performed on the soluble
fraction after filtration, leading to lower molar mass (Mn = 1155
g/mol) and dispersity (ĐM = 4.0) than Lp, as expected for a
fractionated polymer sample.
ylenes of non-lignin aliphatic esters 28 and ferulates 29 are clearly
identified at 65.20/4.01 and 65.25/4.14 ppm (Figure 7B). As
discussed above in the case of modification by ethanol, α-
etherified β-O-4 structures 27 are also observed. No noticeable
differences were observed in the aromatic domain, except a slight
shift of the α- and β-resonances of feruloyl moieties after
reaction, 29.
Comparison of Lp, s-Lp-Et, and Lp-EG 13C NMR spectra
confirmed the preceding conclusions, in particular with the 2
ppm upfield shift of −COO− resonances (esterification of acids)
and the detection of new peaks assigned to −CH2−O− ether
resonances of 24 and 27 at 63.7 and 70.1 ppm, respectively.
The 31P NMR spectra (Figure 9) show that, as expected,
carboxylic acid content strongly decreases, from 0.84 mmol/g in
Lp to 0.04 mmol/g in s-Lp-Et and in Lp-EG (Table 1). The ratio
of un-, mono-, and disubstituted 4-hydroxyphenyl units remains
Finally, we can conclude that alkaline hydrolysis (1 M NaOH
at 80 °C) and solvent extraction of Biolignin yields a purified,
deacylated lignin sample (Lp) with very low content of
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contaminants and that the purification does not significantly
affect starting lignin main structure and molar masses. Lp
contains primary and secondary alcohol groups, carboxylic acid
groups, and phenolic OH groups. The large phenolic OH
content reflects the existence of condensed aromatic structures,
such as units of 5−5 type, which bring two additional phenolic
OH groups per lignin molecule.
(2-hydroxyethoxy)-3,5-dimethoxyphenyl unit; MW, milled
wood
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ASSOCIATED CONTENT
* Supporting Information
■
S
1H NMR spectra of Biolignin, Lh, Lp, Rh, and Rp; 2D 13C−1H
HSQC NMR spectra of Rh and Rp side-chain and aromatic
domains; 2D 13C−1H HMBC NMR spectrum of Rp; 2D
13C−1H HMBC NMR spectrum of s-Lp-Et; and 13C NMR
spectra of Lp, s-Lp-Et, and Lp-EG. The Supporting Information
is available free of charge on the ACS Publications website at
DOI: 10.1021/acs.jafc.5b02071.
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AUTHOR INFORMATION
Corresponding Author
*Telephone +33-144273804; fax +33-144277089; e-mail alain.
fradet@upmc.fr.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. Bouchra Benjelloun-Mlayah and Professor Michel
Delmas (CIMV) for lignin samples and for helpful discussions.
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ABBREVIATIONS
■
Lh, hydrolyzed Biolignin; Lp, purified Biolignin after hydrolysis
and solvent extraction; Rh, hydrolysis residue; Rp, solvent
extraction residue; Lp-Et, reaction product of purified Biolignin
with ethanol; s-Lp-Et, ethanol-soluble fraction of Lp-Et; i-Lp-Et,
ethanol-insoluble fraction of Lp-Et; Lp-EG, reaction product of
purified Biolignin with ethane-1,2-diol; Lp-CE, reaction product
of purified Biolignin with 2-chloroethanol; G, guaiacyl unit; H, 4-
hydroxyphenyl unit; S, syringyl unit; G′, 4-(2-hydroxyethoxy)-3-
methoxyphenyl unit; H′, 4-(2-hydroxyethoxy)phenyl unit; S′, 4-
́
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DOI: 10.1021/acs.jafc.5b02071
J. Agric. Food Chem. 2015, 63, 5178−5188