Polymer Degradation and Stability 110 (2014) 184e194

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Polymer Degradation and Stability

journal homepage: www.elsevier.com/locate/polydegstab

Review article

Physicochemical characterization of ethanol organosolv lignin (EOL)

from Eucalyptus globulus: Effect of extraction conditions on the
molecular structure

n
Mauricio Ya ~ez-S a, *, Betty Matsuhiro a, Carolina Nun
~ ez a, Shaobo Pan b,
Christopher A. Hubbell , Poulomi Sannigrahi , Arthur J. Ragauskas b
b b

a
Laboratorio de Hidratos de Carbono, Departamento de Química, Universidad de Santiago de Chile (USACH), Departamento de Ciencias del Ambiente,
Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Correo 33, Chile
b
Institute of Paper Science and Technology (IPST), School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, United States

a r t i c l e i n f o a b s t r a c t

Article history: The aim of the present study was to investigate the effect of extraction conditions, mainly severity factor
Received 7 July 2014 (H-factor ¼ 3980e14,500) on the molecular structure of ethanol organosolv lignins extracted from
Received in revised form Eucalyptus globulus. Isolated lignins were structurally characterized by 1H NMR, 31P NMR, UVeVis, FT-IR
24 August 2014
spectroscopy and gel permeation chromatography. The results showed that an increase in the severity of
Accepted 27 August 2014
Available online 11 September 2014
the pretreatment decreased the molecular weight of the lignins within a 36e56% range with respect to
the untreated lignin (MWL). Moreover, the increase in severity of the organosolv treatment was
accompanied by strong decrease in the content of aliphatic hydroxyl groups and by an increase of
Keywords:
Organosolv
syringyl phenolic units and condensed phenolic structures. The condensed phenolic structures quanti-
Lignin fied in the organosolv lignin correspond to resinol, phenylcoumaran, dibenzodioxocin, spirodienone and
Lignocellulosic b-10 linkages.
Eucalyptus © 2014 Elsevier Ltd. All rights reserved.

1. Introduction
Lignin is the second most abundant terrestrial organic polymer
on earth after cellulose and plays an essential role in the architec-
ture of vascular plant cell walls [1]. Lignin contributes to the
compression strength of woody stems and to the water proofing of
tracheids cells and vessel elements within the xylem for the con-
duction of water and minerals throughout the plant [2].
Lignin is a branched and amorphous polyphenolic macromole-
cule (Fig. 1a) biosynthesized in the cell wall through copolymeri-
zation of one to three precursors consisting of p-hydroxy-cinnamyl
alcohols (monolignols) called coniferyl alcohol (R1 ¼ OMe, R2 ¼ H),
sinapyl alcohol (R1 ¼ OMe, R2 ¼ OMe) and p-coumaryl alcohol
(R1 ¼ H, R2 ¼ H). The polymerization process begins with the
enzymatic dehydrogenation of the phenolic hydroxyl groups from
the monolignols, which produced resonance stabilized phenoxy
* Corresponding author. Avenida Libertador Bernardo O'Higgins No 3363,

n Central, Santiago, Chile. Tel.: þ56 (0)27181153.
Estacio
E-mail addresses: mauricio.yanez@usach.cl, mauricioqc@gmail.com (M. Y

an~ez-
S).
radicals that can randomly combine with each other [3]. This
combining of radicals generates different types of inter-unit link-
ages including b-O-aryl ether (b-O-4), 4-O-5 (biphenyl ether) and
carboncarbon linkages like 1,2-diaryl propane (b-1), resinol (5-5),
biphenyl (5-50 ), and phenylcoumaran (b-5/a-O-4) [4] (Fig. 1b).
Currently, lignin is mainly obtained from the black liquor of
pulping process kraft hardwood and softwood which represents
94% of worldwide production of cellulose pulp [5]. However, this
lignin is directly used as a boiler combustible for energy generation
which also allows to recover the process reagents and energy [6],
only a small amount of this lignin has been used as raw material for
the obtention of aromatic chemicals such as vanillin [7], phenols
and methoxyphenols [8] and polymeric materials such as epoxy
resins [9], phenolic resins [10], polyolefins [11] and polyurethane
foam [12].
Despite the major advances in the use of lignin, especially kraft
and lignosulphonates [13], there is new interest on the use of
sulfur-free lignins, having always in mind that the renewable
source should be relatively pure and easy to obtain. Some of the
lignins presenting these characteristics are obtained from ligno-
cellulosic biomass pretreatment technologies which are mainly
focused on the generation of reactive substrates for the ethanol

http://dx.doi.org/10.1016/j.polymdegradstab.2014.08.026
0141-3910/© 2014 Elsevier Ltd. All rights reserved.

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M. Ya ~ez-S et al. / Polymer Degradation and Stability 110 (2014) 184e194
Fig. 1. (a) Schematic representation of a hardwood lignin structure (b) Common linkages between phenylpropane units in hardwood Lignin.
production [14]. Among the lignocellulosic biomass pretreatments,
the organosolv processes stands out as one of the most efficient
methods for obtaining high purity lignin. This extraction systems
use mixtures of organic solvent-water and under certain reaction
conditions has the ability to progressively break down and modify
the lignin macromolecule until the resulting molecular fragments
become small enough to dissolve in the aqueous liquor [15].
Among the organosolv pretreatments, ethanol organosolv pre-
treatment seems to be the most promising because the main
product of the bioprocess (ethanol) could be recycled and reused in
the first step of pretreatment [16]. By using ethanol organosolv
pretreatment, lignins have been isolated and characterized from
several raw materials such as Miscanthus giganteus [17], Buddleja
davidii [18], wheat straw [19] and softwood as Loblolly Pine [20];
moreover under the conditions applied in such processes it has
been possible to obtain fragments of low molecular weight relative
to native lignin. These depolymerized lignins could be interesting
starting materials for the preparation of high-value chemicals and
fuels.
Despite a considerable number of works on the structural
characterization of organosolv lignin there are very few studies
related to the effect of extraction conditions on the molecular
structure of lignin. In the present study, ethanol organosolv
pretreatments were performed on Eucalyptus globulus wood
under a wide range of severity of the process (H-
factor ¼ 3980e14,500), in order to investigate the effect of
extraction conditions on the molecular structure of isolated lig-
nins and also to better understand the lignin breakdown mech-
anism in the organosolv process.
2. Materials and methods
2.1. Raw material
Wood chips from 10 to 12 year-old E. globulus trees provided by
the Empresas CMPC S.A. (Santiago, Chile) were screened to
185
approximately 2.0 cm  2.5 cm  0.5 cm. The wood chips were
dried in a mechanical convection oven at 50  C to 8% moisture and
stored in plastic bags at 4  C. All solvents used were analytical grade
from J.T. Baker. The chemicals and deuterated solvents were ob-
tained from SigmaeAldrich.
2.2. Organosolv pretreatments
E. globulus wood chips were ethanol-organosolv-pretreated
under several conditions. The pretreatment processes were car-
ried out in a 3.75 L 4551 pressure Parr reactor equipped with a 4843
PID temperature controller, thermocouple, sampling and safety
valves, and fitted with internal cooling coil (Parr Instrument
Company, Moline, IL. USA). The reactor was loaded with 200 g of
wood chips (dry basis) and the solvent (30e70% v/v) ethanol at a
liquor:wood ratio of 6:1 (v/w). The reactor was heated at an average
rate of 2.5  C/min, starting at 20  C, to reach the desired set point
temperature (190  C or 200  C), which was kept within a range of
±1  C; after a residence time (15e75 min) the reactor was rapidly
cooled. The reaction mixture was vacuum filtered and the solid
fraction (pretreated material) was washed with 1.2 L of the solvent
mixture at the same concentration as the liquor and then, with 1 L
of hot water. The pretreated material was homogenized for 5 min at
250 rpm in a laboratory disintegrator, and screened in a BH-6/12
REGMED fiber classifier with flat screen (0.2 mm  20 mm) to
remove the rejects (non-defiberized wood chips and knots). The
rejects were dried in an oven at 50  C for 2 days and then weighed.
The screened pretreated material was centrifuged, weighed, and its
moisture content determined according to T 210 cm-03 TAPPI
method. The pretreated material (pulp) samples were analyzed to
determine Klason lignin and carbohydrate content, according to
T222 om-88 and T249 cm-85 TAPPI standard methods, respectively.
The severity of the process was calculated using the H-factor
parameter [21], which relates time and temperature in a single
variable and was calculated according to Sundquist using numerical
integration methods (Eq (1)) Table 1.
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M. Ya ~ez-S et al. / Polymer Degradation and Stability 110 (2014) 184e194

Table 1
Lignin Lignin
Experimental conditions of the ethanol organosolv pretreatments on Eucalyptus
globulus.
OH-
Condition Temperature ( C) Ethanol Time (min) H-factora + H2 O
concentration
(%, v/v) H3CO OCH3 H3CO OCH3
A 200 50 45 9624 OH O-
B 200 50 50 11,759
C 200 50 75 13,659
Fig. 2. Ionization of phenolic hydroxyl groups in lignin.
D 200 70 75 14,508
E 190 50 45 3948
F 190 50 75 6311 2.5.2. UVeVisible spectroscopy
G 190 30 45 3980 The analysis by UVeVis spectroscopy were performed on a Shi-
Z
a
t madzu Spectrophotometer UV-1650 PC spectrophotometer. The
H-factor, H ¼ eð43:2  16117=TÞdt; T: Reaction temperature, t: Reaction
0 determination of phenolic hydroxyl groups in the organosolv lignin
time. isolated samples was made by Ultraviolet difference spectroscopy
and with some modifications [23]. This method is based on the dif-
Zt   ference in absorption at 292 and 370 nm between phenolic units in
16117 neutral and alkaline solutions. The absorbance of EOL solution pH 12
H¼ e 43:2  dt (1)
T (boric acid/sodium borate buffer) was measured against a solution of
0
the same concentration at pH ¼ 6 (KH2PO4/NaOH buffer). The sub-
where T ¼ Reaction temperature, and t ¼ Reaction time. traction of two spectra provided the amount of phenolic hydroxyl
groups present in the sample. The measurements considered a molar
absorption coefficient (Dε) of the hydroxyl group of
2.3. Ethanol organosolv lignin (EOL) 4100 L cm1 mol1, taken as the average of the maximum absorbance
of a set of model compounds of lignin [24] Fig. 2.
The lignin was precipitated from the black liquor at pH 2 (adjusted
with 0.015 M sulfuric acid) [22]. The precipitated lignin was filtered 2.5.3. FT-IR spectroscopy
through a sintered glass Büchner funnel and washed with water at The FT-IR spectra were recorded on a BRUKER, VECTOR 22 IR
70  C to remove residual sugars. The purification was performed by spectrophotometer in KBr pellet.
dissolving 9.00 g of lignin in 45.00 mL of acetone (dried over mo-
lecular sieve) and filtered to remove impurities using a membrane 2.5.4. Determination of carbonyl groups by modified oximation
filter of 0.22 mm. Acetone was evaporated under reduced pressure The carbonyl group content of the organosolv lignin samples
and pure lignin was dried over P2O5 in a vacuum desiccator. was determined by a modified oximation, according to the

½ðOven  dry precipitated ligninðgÞ  100

Lignin recoveryð%Þ ¼
½ðOven  dry untreated woodðgÞ  ðKL=100Þ

where KL is the Klason lignin content of untreated wood (g Klason
lignin per 100 g of wood on dry basis).
2.4. Acetylation of lignin
Pure lignin (0.50 g) was dissolved in 6 mL of pyridine-acetic
anhydride mixture (1:1, v/v) and kept in the dark at room tem-
perature for 72 h, then the solution was added dropwise to 120 mL
of cold water containing1 mL of hydrochloric acid, under constant
stirring. The acetylated lignin precipitate was filtered using a
membrane filter of 0.22 microns, this was washed with nanopure
water and dried over P2O5 in a vacuum desiccator.
method described by Zakis et al. [25]. Lignin (80 mg), 2.00 mL of
dimethylsulfoxide (DMSO) and the oximating mixture (5.00 mL)
were placed in a tube, and air was expelled using nitrogen. The
sealed tube was heated at 80  C (±2  C) for 2 h. The reaction
mixture was cooled and then it was quantitatively transferred
into a titration glass using a minimum volume of distilled water.
The excess of triethanolamine (TEA) was potentiometrically
titrated with 0.1 N HCl to pH 3.3. A blank experiment was per-
formed without lignin, under the same conditions as before
Fig. 3.

R1 R1
2.5. Chemical characterization of organosolv lignin
O + NH2OH*HCl NOH + H2O + HCl
R2 R2
2.5.1. Elemental analysis
Oxime
The elemental analysis of C, H, N of the purified samples was
R1 = alkyl, aryl.
performed on an Elemental Analyzer EA1108 FISONS Elemental
Analyzer Instruments, the column used was PORAPAKQ column R2 = alkyl or H
and used a thermal conductivity detector were used; the oxygen HCl + (CH2CH2OH)3N (CH2CH2OH)3N*HCl
content was determined by difference. The determination was
performed using a calibration curve using acetanilide as standard. Fig. 3. Determination of carbonyl groups in lignin by modified oximation.

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M. Ya ~ez-S et al. / Polymer Degradation and Stability 110 (2014) 184e194 187

Table 3
Physicochemical characterization of ethanol organosolv lignin (EOL) extracted from
O
CH2OH Eucalyptus globulus.
P O
HO Condition Lignin MWD Elemental composition
O Lignin O H2 C O
P O recovery (%, w/w)
O O Lignin yielda (%)
O Mw Mn D %C %H %O %N
CDCl3
+ Cl P (g/mol) (g/mol) (Mw/Mn)
R2 R1 O Pyridine
OH R2 A 46.9 3990 1280 3.11 63.79 5.04 30.95 0.22
R1
B 51.2 3490 1240 2.81 63.96 8.10 27.54 0.46
R1=OMe, R2=H O O
P C 63.4 3820 1250 3.06 63.73 7.36 28.45 0.46
R1=R2=OMe
O D 53.4 4150 1350 3.07 63.18 7.46 28.99 0.37
R1=R2=H
E 40.5 4230 1340 3.16 62.33 7.33 29.81 0.52
F 40.5 4270 1360 3.14 64.09 8.16 27.06 0.48
Fig. 4. Derivatization of hydroxyl groups on the lignin polymer with TMDP. G 3.92 2960 1170 2.53 62.12 6.65 30.66 0.56
MWL 6700 2600 2.6 60.80 6.40 31.32
a
% on starting lignin in wood. MWD, molecular-weight distribution, Mn,
2.5.5. Lignin molecular-weight determination molecular-weight distribution number average molecular weight; MW, weight
Lignin sample (20 mg) was placed in a small vial to which average molecular weight, D, polydispersity. MWL: Milled wood lignin.
anhydrous pyridine (0.5 mL) and acetic anhydride (0.5 mL) were
added sequentially. The vial was capped, vortexed and allowed to 210 ppm spectra width in F1 (13C) dimension with 256 data points
stand at RT for 24 h, then ethanol (5 mL) was added to quench the (6.1 ms acquisition time), 3 s pulse delay, 1JCeH of 145 Hz, and 32
reaction. The vial was maintained uncapped overnight in a fume scans. The central solvent (DMSO-d6) peak (dC, 39.5 ppm; dC,
hood and then, then placed in a vacuum oven at 40  C for 3 h to 2.5 ppm) was used for chemical shift calibration. For quantitative
remove any residual volatiles. The acetylated lignin sample was 31
P NMR analysis, organosolv lignin (~25 mg) was dissolved in a
purified by dissolving in chloroform (1 mL) and then precipitated solution of pyridine/CDCl3 (500 mL) and derivatized with 100 mL of
into ethyl ether (75 mL). The resulting solid was collected by 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP)
filtration and dried under vacuum at 40  C for 1 h. Prior to GPC (Fig. 4). Endo-N-hydroxy-5-norbornene-2,3-dicarboximide (NHND)
analysis the acetylated lignin sample was dissolved in tetrahydro- was used as internal standard and the chemical shift of phosphi-
furan (1 mg/mL), filtered through a 0.45 mm filter and placed in a tylated NHND occurs at 151.9 ppm. The 31P NMR spectra were ac-
2 mL auto-sampler vial. The molecular weight distributions of the quired using an inverse gated decoupling pulse sequence with a
acetylated lignin samples were analyzed on an Agilent GPC SECu- 25 s pulse delay and 128 scans. NMR data were processed using the
rity 1200 system equipped with four Waters Styragel columns TopSpin 2.1 software (BrukerBioSpin).
(HR0.5, HR2, HR4, HR6), Agilent refractive index (RI) detector and
Agilent UV detector (270 nm) using THF as the mobile phase
3. Results and discussion
(1.0 mL/min) with injection volumes of 20 mL. A calibration curve
was created by fitting a 3rd order polynomial equation to the
3.1. Structural characterization
elution volumes of 16 narrow polystyrene standards ranging in
molecular weight from 580 to 3.6  106 g/mol. Data collection and
E. globulus wood was ethanol-organosolv-pretreated under
processing were performed using Polymer Standards Service
seven conditions as mentioned in the experimental section.
WinGPC Unity software. Number-averaged and weight-averaged
E. globulus has an initial lignin content of ~27% (w/w) oven-dried
molecular weights, Mn and Mw respectively, were calculated by
basis. The chemical composition of organosolv pulp was previ-
the PSS WinGPC software relative to the universal polystyrene
ously reported by Yan ~ ez-S et al. [14], some data are briefly shown
calibration curve.
(see Table 2). As can be seen in Table 3, at 50% ethanol concentra-
tion the increase in severity of the organosolv treatment from 3948
2.5.6. Structural analysis of EOL using 2D NMR and 31P NMR to 13,659 resulted in an increase in the recovery of EOL, reaching a
All NMR experiments were performed on a Bruker Avance-400 maximum of 63% of the Klason lignin in untreated wood. When
spectrometer operating at a frequency of 400.133 MHz for 1H, comparing the conditions C and D a decrease in the recovery of
100.59 MHz for 13C and 161.976 MHz for 31P. Conditions for the 2D lignin was observed, this is because that increasing the ethanol
HSQC NMR analysis were as follows: 13 ppm spectra width in F2 concentration (70%) decreases the concentration of hydronium ion
(1H) dimension with 1024 data points (95.9 ms acquisition time), (H3Oþ) in the solution and thus lowers the depolymerization of
lignin and its solubility in liquor.
Table 2
Chemical composition of pulp and physicochemical characterization of ethanol 3.2. Elemental analysis of EOL
organosolv lignin (EOL) extracted from Eucalyptus globulus.

Condition Chemical composition (% on fibers) The elemental composition of the isolated EOLs of E. globulus
Glucana Xylanb Klason ligninc
from pretreatment 1e7 are summarized in Table 3. The elemental

A 85.3 ± 0.59 3.1 ± 0.04 7.7 ± 0.33

B 80.24 ± 0.25 0.0 ± 0.0 8.73 ± 0.3
C 80.3 ± 0.55 4.1 ± 0.11 6.8 ± 0.02
D 79.9 ± 0.51 10.4 ± 0.03 8.8 ± 0.30
E 80.3 ± 0.21 4.9 ± 0.20 10.4 ± 0.09
F 78.1 ± 0.36 1.0 ± 0.0 9.3 ± 0.16
G 78.1 ± 0.77 4.3 ± 0.38 17.3 ± 0.28
a
Glucan ¼ glucose * 0.9.
b
Xylan ¼ xylose * 0.88, Arabinan  0.03%. Fig. 5. Lignin condensation reaction during autocatalyzed organosolv pretreatment of
c
Klason lignin (includes both acid-soluble and acid-insoluble). lignocellulosic biomass.
188
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M. Ya ~ez-S et al. / Polymer Degradation and Stability 110 (2014) 184e194

Fig. 6. Variation of the molecular weight (in gmol L1) with the severity of the process (H-factor).

analysis results show percentages of carbon between 62 and 64%

and hydrogen between 5 and 8%. The nitrogen content is between
0.2 and 0.6% which indicates a low level of contamination by pro-
teins and other nitrogenous compounds (Table 3). By comparing
the elemental composition of milled wood lignin (MWL) with EOLs,
we can see that the carbon content increased, while the oxygen
content decreased slightly, indicating that under the organosolv
conditions examined the EOLs undergo condensation reactions.
Hardwood such as E. globulus under severe temperature and
pressure conditions generates acetic acid by hydrolysis of acetyl
groups of xylans, such as O-acetyl-4-O-methyl-glucuronoxylan
[14]. The acetic acid released protonates the hydroxyl group in the
carbon Ca of the side chain of lignin, which is released as a
resonance-stabilized benzyl carbocation. This reactive carbocation
can readily form a bond with an electron-rich carbon atom in the
aromatic ring of another lignin unit i.e., through the free C5 or C6
position [15] (see Fig. 5).

Fig. 7. FT-IR spectrum of ethanol organosolv lignin isolated from E. globulus. Condition 3.3. GPC analysis of EOL
A (H-Factor: 9624).

The number-average molecular weight (Mn), weight-average

molecular weigth (Mw) and polydispersity (Mw/Mn) of milled
wood lignin and organosolv lignin from E. globulus were computed
from their chromatograms. The results are shown in Table 3. Under
the conditions of organosolv pretreatment applied (1e7), the mo-
lecular weight of the lignins decreased within the 36e56% range
with respect to the untreated lignin (MWL, Mw ¼ 6700 g/mol [26]),
indicating that the pretreatments degrade the structure of the
macromolecule to an appreciable degree. A similar effect in EOL
extracted of Miscanthus was reported by Brosse et al. [27], who
observed decreases in molecular weight of 43.5 and 48.5% g/mol for

Table 4
Results of CO group determination obtained by modified
oximation.

Condition H-factor CO (%, w/w)

A 9624 4.95
B 11,759 4.94
C 13,659 ND
D 14,508 3.42
E 3948 3.80
F 6311 3.04
G 3980 3.81
Fig. 8. Ionization difference spectra of organosolv lignins of E. globulus.

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M. Ya ~ez-S et al. / Polymer Degradation and Stability 110 (2014) 184e194 189

Table 5
Determination of non-conjugated and conjugated phenolic hydroxyl groups (%, w/w) by Ultraviolet difference spectroscopy.

Condition Difference ArOH (1) (mmol/g) ArOH (1) (%, w/w) Difference ArOH) (2) (mmol/g) ArOH(2) (%, w/w)
absortivity (DD) l ¼ 293 nm l ¼ 293 nm absortivity (DD) l ¼ 372 nm l ¼ 372 nm
l ¼ 293 nm (Dε ¼ 4100) l ¼ 372 nm (Dε ¼ 37,250)

A 0.101 1.19 2.02 0.126 0.163 0.27

C 0.103 1.07 1.82 0.120 0.137 0.23
D 0.094 1.09 1.84 0.120 0.153 0.26
E 0.09 0.83 1.41 0.113 0.114 0.195
F 0.143 1.12 1.90 0.2047 0.176 0.30
G 0.09 0.75 1.28 0.113 0.103 0.176

ArOH (1) ¼ Non-conjugated phenolic structures.

ArOH (2) ¼ Conjugated phenolic structures.

Table 6
31
Organosolv lignin structural characteristics calculated from P NMR data.

Chemical shift Assignment Organosolv lignin from E. globulus (mmoles/g of lignin)

range d31P NMR
A B C D E F G

145.5e150.0 Aliphatic (OH) 2.355 1.985 1.915 2.275 2.800 2.577 2.536
151.9 NHND (IS)
140.3e144.7 Condensed phenolic (OH) þ 2.875 2.981 3.002 2.707 2.538 2.756 2.836
Syringyl OH
139.0e140.3 Guaiacyl phenolic (OH) 0.589 0.600 0.579 0.590 0.615 0.581 0.552
138.2e139.0 Catechol (OH) 0.127 0.135 0.122 0.116 0.106 0.107 0.090
137.3e138.2 P-hydroxyphenyl (OH) 0.070 0.072 0.064 0.063 0.048 0.051 0.043
133.6e136.6 Carboxylic acids (OH) 0.243 0.256 0.230 0.184 0.146 0.198 0.175

Mw and Mn respectively. In the same way, studies conducted by
Hallac et al. [18], determined that molecular weight (Mw) of milled
wood lignin of Buddleja davidii decreases by ~85% after ethanol-
water pretreatment.
Under the studied conditions D, E and F, lignin samples were
obtained with a low degree of depolymerization in relation to
native lignin (MWL). In the case of condition D, this could be due to
a high concentration of ethanol in the liquor (70% (v/v)), which
generates lower hydrogen ion concentration (higher pH value),
decreasing the likelihood that this catalyzes the cleavage of ether
linkages. Such behavior is explained better as compared with lig-
nins extracted under conditions A to C, which used a lower ethanol
concentration (50% (v/v)) and thus generating a higher concen-
tration of hydronium ion in liquor (lower pH value), which largely
facilitates the breakdown of a and b-ether bonds in lignin and the
formation of molecular fragments of lower molecular weight.
Under the reaction condition G were obtained lignin with the
lowest molecular weight (2960 g/mol), this can be partly explained
due to the low concentration of ethanol (30%, v/v), which generates
a high concentration of hydronium ion in the liquor. However, the

31
Fig. 9. Quantitative P NMR spectra and signal assignment of organosolv lignin A from Eucalyptus globulus.
190
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M. Ya ~ez-S et al. / Polymer Degradation and Stability 110 (2014) 184e194

3,5

2,5

mmol.g -1of lignin 2 Aliphatic OH

Syringyl OH+ Condensed OH
Guaiacyl OH
1,5
Catechol OH
p-Hydroxyphenyl OH
1 Carboxylic acid

0,5

0
3000 5000 7000 9000 11000 13000

H-factor

Fig. 10. Variation of the hydroxyl group concentrations (in mmol g1 of lignin) with the severity of the process (H-factor).

low concentration of ethanol in the liquor decreases the degree of
delignification and hence the recovery of lignin in the liquor (3.9%).
Similar effect was reported by Sadler et al. during catalyzed ethanol
pulping of hybrid poplar wood [28]. Fig. 5 shows the effect of the
severity of the process on the molecular weight (Mn and Mw) and
polydispersity of the organosolv ethanol lignins. At a 50% ethanol
concentration, increasing the severity (H-factor) of the process
from 6300 to 11,760 decreases the molecular weight of lignin
by~18%. However, further increase in severity up to 13,659 resulted
in an increase of molecular weight of 330 g/mol, which indicates
that in this range of severity take place repolymerization reactions
of lignin. In this range of severity, the average polydispersity for the
lignins was 3.1, indicating a broad molecular weight distribution
(MWL: Mw/Mn ¼ 2.6) Fig. 6.
3.4. FT-IR spectroscopy
The IR absorption spectra of the seven EOL studied were
recorded in the 400e4000 cm1 region. The spectral profiles and
the relative intensities of the absorption bands of vibration are
similar in all EOL isolated, which indicates that the “core” of the
structure of lignin did not change significantly by changing the
reaction conditions. The absorption bands of vibration in the FT-IR
spectrum of lignin were assigned based on previously reported data
[29]. The FT-IR spectra for sample of ethanol organosolv lignin,
condition A is shown in Fig. 7.
The infrared spectrum of organosolv lignin shows at 3441 cm1
a broad intense OeH stretching absorption due to the phenolic
hydroxyl groups and aliphatic hydroxyl groups. The two bands at
2938 and 2840 are assigned to CeH stretching vibrations. The band
at 1705 cm1 is caused by stretching of unconjugated ketones,
conjugated aldehydes and carboxylic acids aromatic ring, and the
bands at 1605 and 1515 cm1 are caused by aromatic skeletal vi-
brations, while the band at 1461 cm1 corresponds to asymmetrical
CeH deformations in methyl (eCH3) and methylene (eCH2e)
groups [29]. The band at 1424 cm1 also corresponds to aromatic
skeletal vibrations (C]C) (guaiacyl-syringyl) combined with CeH
in-plane deformation. The band at 1328 cm1 corresponds to CeO
stretching (syringyl). The bands at 1217 and 1031 cm1 corresponds
to phenolic hydroxyl groups PheO(H) and PheOeC ether in

Fig. 11. Solvolytic cleavage of b-aryl ether linkage by a mechanism that involves the elimination of formaldehyde.

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M. Ya ~ez-S et al. / Polymer Degradation and Stability 110 (2014) 184e194 191

Table 7 3.5. Carbonyl groups

Molecular formulas C9 for the organosolv lignins studied.

Condition Formula C9 Molecular Carbonyl groups for all EOL lignins were determined quantita-
weight (g mol1) tively by the modified Oximating method [25]. In all the samples,
A C9H4.88O1.61(CH3O)1.38(OHAr)0.42 195.45 the content of CO is very close to the average of carbonyl in Alcell
(OHAlk)0.27(COOH)0.047 lignin of hardwoods (~5.0 w/w) [25]. However, there is not clear
C C9H8.92O1.13(CH3O)1.69(OHAr)0.44 201.07 relationship between increasing severity of the process and the
(OHAlk)0.23(COOH)0.046
increase of carbonyl groups in the molecular structure of the EOL
D C9H9.98O1.55(CH3O)1.25(OHAr)0.40 194.59
(OHAlk)0.26(COOH)0.036 (Table 4). These carbonyl groups are mainly present in the subunits
E C9H9.44O1.48(CH3O)1.52(OHAr)0.39 202.00 cinnamaldehyde ends and oxidized syringyl units, which were
(OHAlk)0.33(COOH)0.029 determined by 2D 13Ce1H HSQC NMR experiment (see section 3.8).
G C9H7.63O1.40(CH3O)1.78(OHAr)0.43 207.63
(OHAlk)0.31(COOH)0.036

In conditions B and F was not quantified the methoxyl groups. 3.6. UVeVis spectroscopy

syringyl and guaiacyl units, asymmetric and symmetric stretching
vibration respectively. Aliphatic ethers give one strong asymmetric
stretch at 1117 cm1, and a very weak symmetric stretch around
834 cm1. Finally the band at 912 cm1 corresponds to CeH aro-
matic (G þ S) out of plane deformation.
The phenolic hydroxyl groups of all EOL samples were deter-
mined by ultraviolet difference spectroscopy. The alkaline-neutral
difference spectra of organosolv lignin present three absorption
bands at wavelengths of 254, 293 and 372 nm (see Fig. 7). For the
determination of phenolic hydroxyl groups was used the difference

Fig. 12. A and B. HSQC spectrum of organosolv lignin from Eucalyptus globulus, condition A (a) Expanded side-chain region (dC/dH 50e95/2.5e6.0 ppm) and (b) Expanded aromatic
region (dC/dH 95e160/5.5e8.5 ppm).
192
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M. Ya ~ez-S et al. / Polymer Degradation and Stability 110 (2014) 184e194

absorptivity (DD) at 293 and 370 nm. These bands indicate the inter-unit linkages (substructures) present in the molecular
presence of non-conjugated and conjugated phenolic hydroxyl structure of lignins obtained. The substructures present in orga-
groups, respectively. As can be seen from Table 4, increasing the nosolv lignin samples were determined based on the presence of
process severity between 3900 and 9600, the number of hydroxyl cross-signals of 13Ce1H correlation. This signal assignment was
groups unconjugated increases and then decreases slightly in the performed based on work previously reported in other hardwoods
range between 9600 and 13,600 Fig. 8(Table 5). as Paulownia fortunei [31] and Eucalyptus grandis [32]. The HSQC
spectrum of organosolv lignin condition A is shown in Fig. 12. The
31
3.7. Quantitative P NMR analysis spectrum shows two regions corresponding to side-chain (dC/dH
50e95/2.5e6.0 ppm) and aromatic 13Ce1H (dC/dH 95e160/
The seven organosolv lignin samples were phosphitylated with 5.5e8.5 ppm) correlations. The main substructures present
2-chloro-4,4,5,5-tetramethyl-1,2,3-dioxaphospholane in presence assigned in the HSQC spectrum are listed in Table 8 and are
of Endo-N-hydroxy-5-norbornene-2,3-dicarboximide (NHND) as depicted in Fig. 13.
an internal standard agent and analyzed with quantitative 31P NMR The presence of strong cross-signals of syringyl units (S) and of
according to a method described by Granata and Argyropoulos [30]. the substructures b-O-40 aryl ether linked to a G and S unit (Aa,b(G)/
The concentrations of the hydroxyl groups were calculated on the Aa,b(S)), confirm the results obtained by 31P NMR, i.e., the greater
basis of the internal standard (i.e., NHND) and the respective in- amount of substructures in the molecular structure of EOL of
tegrated peak area. The signals attributed to functional groups in E. globulus correspond to syringyl phenolic structures. Moreover,
the lignin samples and the analysis of alkyl/aryl substituted hy- condensed phenolic structures quantified by 31P NMR correspond
droxyl groups was determined and summarized in Table 6. to resinol (B), phenylcoumaran (C), dibenzodioxocin (F), spi-
0
The quantitative 31P NMR spectrum of organosolv lignin (con- rodienone (D) and b-1 -linkages (E). However, the cross-signals
dition A) is also shown in Fig. 9. In all samples of organosolv lignin CbeHb and CgeHg for the substructure dibenzodioxocin (F) were
(EOL) examined, it was observed that the phenolic structures that not observed in the spectrum, indicating that these structures are
are present in greater amount corresponding to condensed found in low amounts in hardwoods [31]. Similarly, for the spi-
phenolic moieties and syringyl phenolic units, accounting for about rodienone substructure (D) were not observed the correlations
77e80% of the total phenolic hydroxyl (eOH) content and 41e51% CbeHb and Cb0 eHb0 . The CaeHa and CbeHb correlations for the
of the total hydroxyl content present in the molecule. Moreover, the cinnamyl alcohol end-groups (I) were also not observed. The
aliphatic hydroxyl groups representing about 34e46% of the total
hydroxyl content in the structure of lignins studied.
Fig. 10 shows the variations of the phenolic hydroxyl concen- Table 8
trations (in mmol g1 of lignin) with the H-factor of the organosolv Assignment of 13Ce1H correlation signals in the HSQC spectrum of organosolv lignin
pretreatment. As can be seen, the increase in severity of the orga- of E. globulus condition A.
nosolv pretreatment was accompanied by an increase of syringyl Labels Substructure Assignment dC/dH(ppm)
phenolic units and condensed phenolic structures and a strong
Cb (Phenylcoumaran) (C) Cbe0Hb 53.0/3.49
decrease in the content of aliphatic hydroxyl groups. The decrease Bb (Resinol) (B) CbeHb 53.4/3.07
of aliphatic hydroxyl can be partly explained due to the solvolytic Eb b-10 (E) CbeHb 53.6/2.87
cleavage of b-aryl ether linkage by a mechanism that involves the eOMe Methoxyls CeH 55.7/3.77
loss of the g-methylol group with elimination of formaldehyde. The Ag b-O-40 (A) CgeHg 59.5/3.75 and
3.45
enol ether formed is then hydrolyzed, causing the ether bond Db Spirodienone (D) CbeHb Not observed
cleavage to give guaiacol and 2-(4-hydroxy-3-methoxyphenyl) Ig Cinnamyl alcohol CgeHg 59.5/4.06
acetaldehyde (homovanillin). The guaiacol could be demethylated end-groups (I)
to give catechol (see Fig. 11). The homolytic cleavage of b-O-4 via (sinapyl/coniferyl)
Cy Phenylcoumaran (C) CgeHg 62.76/3.75
quinone methide intermediate causes the formation of b-1 sub-
Aa(G) b-O-40 linked to a G unit (A) CaeHa 71.4/4.80
structures [18]. The b-10 substructures can be degraded in acid Bg Resinol (B) CgeHg 70.9/4.20
medium to give stilbenes. These structures were determined at low Aa(S) b-O-40 linked to a S unit (A) CaeHa 72.1/4.90
concentrations using UVeVis spectroscopy (see section 3.5). Db ′ Spirodienone (D) Cb0 eHb0 Not observed
Hydroxyl groups of units as catechol and p-hydroxyphenyl fol- Da Spirodienone (D) Ca-Ha 77.2/5.13
Fa 5-50 (dibenzodioxocin) (F) Ca-Ha 81.3/4.78
lowed the same trend and remain almost unchanged under all Ab(G) b-O-40 linked to a G unit (A) CbeHb 85.74/4.17
experimental conditions applied. The amounts of carboxylic acid Ba Resinol (B) CaeHa 85.12/4.65
increased from 0.14 to 0.25 (mmol g1 of lignin) by increasing the Fb 5-50 (dibenzodioxocin)(F) CbeHb Not observed
severity of the process in a range between 3900 and 12,000. This Da ’ Spirodienone (D) Ca0 eHa’ 84.9/4.80
Ab(S) b-O-40 linked to a S unit (A) CbeHb 86.61/4.08
increase might be due to the oxidation of aldehydes groups and
Ca Phenylcoumaran (C) CaeHa 87.0/5.47
hydrolysis of ester bonds [27]. S2,6 Etherified syringyl units (S) C2eH2/C6eH6 104.3/6.73
J2,6(S) Sinapaldehyde end-groups (J) C2eH2/C6eH6 106.5/7.06
3.8. Molecular formulas of organosolv lignin S′2,6 Oxidized (Ca]O) phenolic C2eH2/C6eH6 106.7/7.35 and
syringyl units (S′) 7.32
G2 Guaiacyl units (G) C2eH2 110.3/6.94
The molecular formula of the lignin samples was obtained from G5 Guaiacyl units (G) C5eH5 115.3/6.78 and
elemental analysis and the quantitative analysis of hydroxyl groups 6.97
by 31P NMR. Methoxyl content was determined by 1H NMR (Table 7). G6 Guaiacyl units (G) C6eH6 118.8/6.78
Jb Cinnamyl aldehyde CbeHb 126.1/7.00
end-groups (J)
3.9. Structural analysis of E. globulus EOL using qualitative 2D
1 Ib Cinnamyl alcohol CbeHb Not observed
He13C correlation NMR end-groups (I)
Ia Cinnamyl alcohol CaeHa Not observed
All organosolv lignin samples were structurally characterized end-groups (I)
by 2D 1He13C HSQC NMR (heteronuclear single quantum corre- Ja Cinnamyl aldehyde CaeHa 153.5/7.6
end-groups (J)
lation). This technique provided information of the structure of

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M. Ya ~ez-S et al. / Polymer Degradation and Stability 110 (2014) 184e194
Fig. 13. Main substructures identified in ethanol organosolv lignin from Eucalyptus globulus.
aliphatic hydroxyl groups, quantified by 31P NMR belongs to the
substructures (A), (C), (E) and (G). Finally the HSQC spectra for all
EOLs analyzed present the same signals, so no changes were
observed in the molecular structure depending on the extraction
conditions applied.
4. Conclusions
In this work, we evaluated the effect of process severity on the
molecular structure of E. globulus ethanol organosolv lignin by
spectroscopic and cromatographic techniques. It can be concluded
from the structural analysis by 31P NMR that increasing the severity
factor in the range of 3980e13,600 the phenolic hydroxyl of
syringyl units increases and also increases the content of
condensed phenolic units. On the contrary, the content of aliphatic
hydroxyl content decreases. This can be explained by solvolytic
cleavage b-aryl ether linkage by a mechanism that involves the loss
of the g-methylol group with elimination of formaldehyde. HSQC
and FT-IR showed that the severity of process did not significantly
change the core of the organosolv lignin.
193
Acknowledgment
The authors gratefully thank FONDECYT for financial support

n de Capital Humano Avanzado en la academia
through Insercio
Project N 79100010 and DICYT Iniciacio

n (USACH) Project codigo
021241YS.
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