Biomass and Bioenergy xxx (2016) 1e14

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Biomass and Bioenergy

journal homepage: http://www.elsevier.com/locate/biombioe

Research paper

Using 2D NMR to characterize the structure of the low and high

molecular weight fractions of bio-oil obtained from LignoBoost™ kraft
lignin depolymerized in subcritical water
Cecilia Mattsson a, *, Sven-Ingvar Andersson b, Tallal Belkheiri b, Lars-Erik Åmand b,
Lars Olausson c, Lennart Vamling b, Hans Theliander a, d
a
Chalmers University of Technology, Department of Chemistry and Chemical Engineering, SE-412 96 Gothenburg, Sweden
b
Chalmers University of Technology, Department of Energy and Environment, SE-412 96 Gothenburg, Sweden
c
Valmet AB, Box 8734, SE-402 75 Gothenburg, Sweden
d
Wallenberg Wood Science Center, The Royal Institute of Technology, Chalmers University of Technology, SE-100 44 Stockholm, Sweden

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

Article history: In this work a multilevel analysis approach have been used for characterization of LignoBoost™ kraft
Received 30 January 2016 lignin and bio-oil produced from LignoBoost™ kraft lignin using a process based on subcritical water
Received in revised form (350
C, 25 MPa). LignoBoost™ kraft lignin and the different fractions of the bio-oil (light oil, heavy oil
31 August 2016
and suspended solids) was characterized with high field NMR (18.8 T, 2D13C, 1H-HSQC NMR and 13C-
Accepted 6 September 2016
Available online xxx
NMR), GPC, GC-MS and elemental composition to improve understanding of the subcritical process. By
using high resolution 2D HSQC NMR it was possible determine the chemical structures both on low and
high molecular weight fractions of the bio-oil. It was confirmed that the signals from the aliphatic lignin
Keywords:
Subcritical water
inter-unit linkages, i.e. b-O-40 , b-b0 , b-10 and b-50 , had disappeared from all of the bio-oil fractions studied.
Base-catalyzed depolymerization (BCD) This means that both the aliphatic carbon-oxygen (CeO) and to some extent carbon-carbon (CeC) bonds
Phenol in LignoBoost™ kraft lignin have been cleaved and an effective depolymerization has occurred. However,
Bio-oil re-polymerization into higher molecular weight (Mw) fractions takes place simultaneously. These higher
Char heteronuclear single quantum Mw fractions (heavy oil and suspended solids) were found to be re-polymerized macromolecules, with
coherence (HSQC) new structural networks based on guaiacol/disubstituted aromatic ethers and polyaromatic hydrocarbon
structures bound tightly together.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction heterogeneous, random, aromatic macromolecule, which means

that its exact structure is unknown. However, a vast amount of
Lignin is one of the three major components that form the work has already been carried out with the aim of defining its
structure of wood: approximately 20e30% of wood is composed of various building elements and type of bonds. The structure of
lignin. Lignin is a heterogeneous, random, aromatic macromolecule native lignin has still to be clarified; the closest structure is
that is cross-linked by carbon-oxygen (CeO) and carbon-carbon currently regarded as being milled wood lignin (MWL) [2,3]. The
(CeC) bond networks [1]. Although lignin structures vary, the MWL lignin from softwood has been found to be comprised of
main types are softwood, hardwood and annual plant lignin. Whilst different phenyl-propane units, i.e. coniferyl alcohol (>95%) and
the general characteristics of lignins are similar, there are variations para-coumaryl alcohol (<5%, Fig. 1) connected by aliphatic/aromatic
in the frequencies of the different bonds and side groups. This pa- CeC and CeO bonds (ether bonds, Fig. 1). The aliphatic inter-unit
per deals with softwood lignin. Lignin, as mentioned above, is a linkages found in softwood lignin are (Fig. 1) b-O-40 (b-ether,
45e50%), a-O-40 (a-ether, 6e8%) and b-b0 (pinoresinol, secoisolar-
iciresinol, 3%) and the aliphatic-aromatic are: b-10 (diphenyl
* Corresponding author. ethane, 7e10%) and b-50 (phenylcoumaran, 9e12%), whilst the ar-
E-mail addresses: [email protected] (C. Mattsson), sianders@ omatic connecting linkages are 4-O-50 (diphenyl ether, 4e8%) and
chalmers.se (S.-I. Andersson), [email protected] (T. Belkheiri), lars-erik.
5-50 [biphenyl and dibenzodioxocine (5-50 -a, b-O-40 ), 18e25%]
[email protected] (L.-E. Åmand), [email protected] (L. Olausson),
[email protected] (L. Vamling), [email protected] (H. Theliander).
[2,4]. During the kraft process lignin is solubilized by the cleavage

http://dx.doi.org/10.1016/j.biombioe.2016.09.004
0961-9534/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: C. Mattsson, et al., Using 2D NMR to characterize the structure of the low and high molecular weight fractions
of bio-oil obtained from LignoBoost™ kraft lignin depolymerized in subcritical water, Biomass and Bioenergy (2016), http://dx.doi.org/10.1016/
j.biombioe.2016.09.004
2 C. Mattsson et al. / Biomass and Bioenergy xxx (2016) 1e14

Fig. 1. Lignin linkages found in MWL softwood lignin and other common structural motifs. Bold face linkages indicate the carbon-carbon and carbon-oxygen bonds known to exist
in lignin [1].

of ether bonds, yielding an introduction of phenolic groups. The oxidation, catalysis and hydrolysis under sub and supercritical
most important structural change that occurs is the formation of conditions have been studied. All these thermochemical process
phenolic hydroxyl groups, which enhance the water solubility of struggle with understanding the complex depolymerization
lignin in an aqueous solution. It is known that bonds such as b-O-40 , mechanism, low yield and complex product mixtures of small ar-
a-O-4'and b-10 are hydrolyzed during the kraft process, and that 4- omatics as well as lignin repolymerization reactions to higher
O-50 , 5-50 , b-b0 and b-50 structures are usually more resistant to the molecular weight (Mw) byproducts (i.e. residual lignin, char and
alkaline environment of the kraft process and are therefore often coke) [12e15].
found in kraft lignin (Fig. 1) [4e6]. The rather harsh conditions also One promising technique for the depolymerization of lignin
cause other typical structural changes to the kraft lignin, such as involves procedures based on subcritical water to generate bio-oil
new CeC bonds from condensation reactions, unsaturation (i.e. [12,14e18]. Water gains unique properties when it is close to its
stilbene and vinyl ether structures) and a reduction in the lignin critical point (372.9
C, 22 MPa): it resembles an organic solvent
side chains of aliphatic structures [6e9]. The lignin obtained after a and can therefore be used in the solubilization of organic structures
kraft cook has a rather high molecular weight (Mw, 3e17 kDa) and, such as the phenolic lignin network. Water at these temperatures
due to the formation of CeC bonds, it is rather stable [10]. acts as reactant, catalyst and enhances solubility of both organic
Today, large quantities of lignin are dissolved from wood in the structures, inorganic ions and gases which yields exceptional op-
production of paper pulp. This lignin is mostly combusted at pre- portunities for promoting lignin depolymerization with an envi-
sent, and the heat used to produce steam. However, lignin is a ronmental friendly solvent [19e22]. Another advantage is that in
natural source of aromatic rings, i.e. phenols, guaiacols and syrin- the reactor it is subcritical conditions and the reactions proceeds in
gols, making it a potential renewable source for generating fuel and “one phase”, but after the reactor, when the pressure and temper-
chemicals that could replace a part of the current demand for fossil ature has been lowered (<350
C, <25 MPa) the system separates
fuels. A new, green, process technology for the separation and into two phase system i.e. bio-oil and water phase. By this means
purification of kraft lignin from the black liquor generated from the byproducts like: water, inorganic salts and more hydrophilic and
kraft pulping process currently exists, and is known as the Ligno- reactive intermediates (carboxylic acids, aldehydes and alcohols)
Boost™ process [11]. LignoBoost™ kraft lignin is precipitated from transports to the water phase This phase separation have been
alkaline black liquor with carbon dioxide, thereby generating a shown to be very beneficial for the bio-oil storage stability [23].
solid LignoBoost™ kraft lignin. This may be used in future bio- This opens up for the sustainable depolymerization of lignin by
refineries to develop new, high value, products from lignin. How- means of “green chemistry”.
ever, additional processes need to be developed if the kraft lignin is Several studies of subcritical water have shown that the reaction
to be transformed into new, high value, products. rates for breaking the various bonds in lignin are very high: that of
Recent years a huge effort for find a new thermochemical pro- aryl-alkyl ethers (i.e. b-O-4 and a-O-4) is very high at 270e290
C
cess technology for depolymerization of lignin to small aromatic already [24,25]. Catalysts and/or higher temperatures are necessary
structures i.e. pyrolysis, gasification, hydrogenolysis, chemical for bonds with a bond strength, such as CeC bonds [21,26].

Please cite this article in press as: C. Mattsson, et al., Using 2D NMR to characterize the structure of the low and high molecular weight fractions
of bio-oil obtained from LignoBoost™ kraft lignin depolymerized in subcritical water, Biomass and Bioenergy (2016), http://dx.doi.org/10.1016/
j.biombioe.2016.09.004
 C. Mattsson et al. / Biomass and Bioenergy xxx (2016) 1e14 3

Numerous studies of the sub/supercritical water depolymerization kraft lignin was performed in a continuous, high-pressure, bench-
of lignin have been carried out at various temperatures and pres- scale catalytic reactor filled with ZrO2 pellets and yielded bio-oil,
sures [12,14,15,17,18,27,28]. The reaction pathway is thought to start water soluble organics and char (deposits on the ZrO2 catalyst,
with lignin being depolymerized by hydrolysis and dealkylation Fig. 2) [49,60,61]. In addition, it is known from GC-MS analysis of
reactions to form aromatic monomers (i.e. phenol, guaiacol, cate- the bio-oil that the major phenolic monomeric/dimeric structures
chol and cresol), larger oligomeric structures and smaller aliphatic formed are: alkylphenol (methyl and
reactive intermediates (i.e. aldehydes and alcohols). However, these ethyl) > catechol > anisole > guaiacol > phenolic dimer (Are-
reactive functional groups may also be located on aromatic CH2eAr, AreCH2eCH2eAr). However, for a better understanding of
monomers as well as on the larger oligomeric structures. The ex- the depolymerization process in subcritical water, an additional
istence of many reactive groups/fragments mean that unfavorable structural analysis of the various bio-oil fractions is necessary.
repolymerization reactions accompany the depolymerization pro- This work is therefore focused on elucidating the structural
cess, causing cross-linking to fragments of higher Mw (e.g. residual composition of the different fractions (Fig. 2), i.e. light oil (DEE
lignin, heavy oil, char, solids, residual solids, phenolic char and soluble fraction), heavy oil (THF soluble fraction) and suspended
polyaromatic char) [29e32]. Recent investigations into continuous solids (insoluble THF residue) that are obtained from LignoBoost™
reactors have shown that depolymerization and repolymerization kraft lignin BCD in subcritical water (350
C, 25 MPa). In this work
are fast competing reactions that start immediately in sub/super- we have used the unique ability of the high resolution 2D HSQC
critical water [27,28]. Depolymerization is often promoted by using NMR (18.8 T), combined with GC-MS, along with Mw distribution
a base catalyst, i.e. BCD [18,25,33,34]. Other common additives measurements made by GPC and the determination of the
include capping agents/cosolvents, i.e. phenol, para-cresol elemental composition of the bio-oil fractions. By using a multilevel
[31,35e43], ethanol, methanol, 2-propanol and 1-butanol [43e50], analysis approach it's possible to gain new insight in both low and
to diminish the extent of the repolymerization reactions and thus high Mw structural composition of the bio-oil fractions (light oil,
enhance the yield of monomers. heavy oil and suspended solids).
The Bio-oil formed from depolymerization of lignin is a complex
mixture of low and high Mw structures. Today the most common 2. Experimental
analysis of the bio-oil is by extraction with organic solvents (DEE,
acetone, methanol and ethyl acetate) and analyzed with Gas 2.1. Materials
Chromatography-Mass Spectrometry (GC-MS), however, this yields
only structural characterization of volatile organic compounds in All reagents used in this study were purchased from Sigma-
the bio-oil (Mw 40e400 Da) which represents approximately 1/3 of eAldrich (Stockholm, Sweden) and used as received. The Ligno-
the total composition of the bio-oil. Typically, the low Mw fraction Boost™ kraft lignin used for subcritical water BCD to bio-oil
of bio-oil is composed of phenolic compounds like: guaiacol, originates from softwood and was produced at the Ba€ckhammar
catechol, phenol, alkyl phenols and vanillin [51e53]. Mill (Sweden).
The remaining part of the bio-oil the higher Mw fractions could
be unreacted lignin or repolymerized structures formed during the 2.2. Reaction conditions for subcritical based catalyzed water
process or, indeed, a combination of both [54]. It is currently depolymerization
believed that the high Mw fractions (i.e. char) developed in sub/
supercritical water are composed of low levels of phenolic or The bio-oil and solids used in this study were produced in BCD
alcoholic groups, aliphatic groups and aromatic structures as well subcritical water (350
C, 25 MPa) in a continuous, high-pressure,
as large clusters formed from the recombination of aromatic rings bench-scale catalytic reactor (500 cm3) filled with ZrO2 pellets.
(polycyclic aromatic hydrocarbons, PAH) [32,55e58]. A few studies The conditions used were: LignoBoost™ kraft lignin (5.5%), phenol
reported from various subcritical conditions point towards the fact (4.1%), K2CO3 (1.6%), KOH (0.2%), 350
C and 25 MPa. Calculations
that higher Mw fractions are composed of structural motifs other based on phenol-free products (based on TOC and elemental
than lignin. They show, for example: a higher carbon and lower analysis, yield calculations see supplementary data and Belkheiri
oxygen content than those present in lignin; a simplified mono- et al. [62] on a dry lignin basis yielded: 37.3% bio-oil, 18.7% accu-
meric compound pattern with pyrolysis-GC-MS and a residue mulated bio-oil (collected under the cleaning period), 25.8% water
(solids) that could not be used for the formation of new bio-oil [25] soluble organics (WSO) and 18.1% char (solid deposits on the
[57] [58]. catalyst, Fig. 2). The bench-scale BCD process used here has been
Today only a few publications can be found that cover both reported recently by Nguyen et al. [60,61] and Belkheiri et al. [62].
structural analysis of low and high Mw compounds from depoly-
merization of lignin in subcritical water with a multi-technique 2.3. Fractionation of bio-oil into light oil, heavy oil and suspended
analytical approach i.e. GC-MS, Gel Permeation Chromatography solids
(GPC), 13C solid-state NMR (cross-polarization/magic angle spin-
ning nuclear magnetic resonance, 13C CP/MAS NMR) and Fourier The exit stream from the continuous reactor was separated by
transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) centrifugation to remove the water phase (WSO); the organic phase
[57,59]. However, by utilizing one dimensional solid state NMR collected was named “bio-oil”. This bio-oil was fractionated further
together with high resolution FT-ICR/MS is it not possible to resolve using DEE and THF. A sample of 5.20 g Bio-oil was added to 200 g
any organic structural network in the high Mw structures. Knowl- DEE, stirred for 1 h and filtered through Duran glass filters P4
edge about the chemical structural composition of the higher Mw (nominal maximum pore size 10e16 mm). The filter was washed
fractions from subcritical liquefaction processes is crucial and this with DEE until the filtrate was clear; it was then evaporated to yield
can be obtained by use of the high resolution two-dimensional the DEE-soluble fraction of the bio-oil (in this case 3.93 g of light
13 1
C, H-correlated Heteronuclear Single Quantum Coherence NMR oil). The remaining filter cake was dried for 24 h before THF (40:1
technique (2D HSQC NMR). mass ratio) was added; it was stirred for 1 h before being filtered
Our group has previously performed BCD on LignoBoost™ kraft through Duran glass filters P4. The filter was washed with THF until
lignin in subcritical water (K2CO3, 350
C, 25 MPa) with the addition the filtrate was clear; it was then evaporated to yield the THF-
of a capping agent i.e. phenol. The depolymerization reaction of soluble fraction of the bio-oil (in this case 0.99 g of heavy oil).

Please cite this article in press as: C. Mattsson, et al., Using 2D NMR to characterize the structure of the low and high molecular weight fractions
of bio-oil obtained from LignoBoost™ kraft lignin depolymerized in subcritical water, Biomass and Bioenergy (2016), http://dx.doi.org/10.1016/
j.biombioe.2016.09.004
4 C. Mattsson et al. / Biomass and Bioenergy xxx (2016) 1e14

Fig. 2. Flow diagram of the subcritical water-based catalyzed depolymerization process and the resulting products. WSO: water soluble organics; DEE: diethyl ether; THF:
tetrahydrofuran.

The remaining solids (filter cake) were dried at 105
C for 1 h and 90
pulse, 250 ms acquisition time, 3 s pulse delay, 1JCeH of 145 Hz,
yielded 0.76 g of suspended solids (SS). The total yield of all frac- 16 scans and the acquisition of 8192 data points (for 1H) and 256
tions is calculated to 5.68 g and differ from initial bio-oil weight increments (for 13C). The pulse widths of 1H and 13C were
(5.2 g), this discrepancy have to be attributed to residuals of solvent p1 ¼ 6.9 ms and p3 ¼ 12.9 ms, respectively. The spectral widths of 1H
(H2O, THF and diethyl ether) and insufficient drying of the fractions. and 13C were 20 ppm and 240.00 ppm, respectively (16,025.6 and
48,275 Hz). HSQC data processing and plots were carried out using
2.4. Elemental analysis the MestReNova v10.0.0 software default processing template, with
automatic phase and baseline correction.
The elemental composition of the solvent free lignin bio-oil
fractions were analyzed at SP (The Technical Research Institute of 2.7. GC-MS spectroscopic analysis
Sweden) in Borås, Sweden. The contents of carbon, hydrogen and
nitrogen were measured according to the SS-EN 15,104 method The volatile compounds in the light oil (1 g, dissolved/sus-
using a Leco CHN 628 instrument (Leco Corporation, St. Joseph, pended in DEE with S/F of 43 and mixed with a known amount of
Michigan, USA), whereas the sulphur content was measured ac- internal standard 2,6-dimethoxyphenol) were determined by a gas
cording to the SS-EN 15,289 method with an ELTRA CS-580A Helios chromatograph (Agilent 7890A, Stockholm, Sweden) connected to
instrument (Eltra GmbH, Haan, Germany). a mass spectrometer (Agilent 5975C, Stockholm, Sweden) oper-
ating in an electron ionization mode. The analytes are separated in
2.5. Determination of molecular weight a chromatographic column HP-5MS (length: 30 m; internal diam-
eter: 0.25 mm; thickness of stationary phase: 0.25 mm) by injecting
GPC was run with a PL-GPC 50 plus integrated system connected 1 mm3 of sample via an autosampler (Agilent 7693A), using helium
with RI and UV detectors. (280 nm, Polymer Laboratories, Varian at 1 cm3 min1 as the carrier gas. The injector temperature is set at
Inc.) Two series coupled PolarGel-M column and a guard column 300
C and the temperature programme of the GC oven is 45
C for
(300  7.5 mm and 50  7.5 mm, 8 mm) with DMSO/LiBr 2.25 min, 2
C min1 up to 300
C, 300
C for 10 min. The MS source
(0.01 mol dm3) as mobile phase (500 mm3 min1) was used. A 10- and quadrupole temperatures are set at 250
C and 150
C,
point calibration curve with Pullulan standards was used to respectively. Semi-quantification was performed, using 2,6-
determine the Mw and polydispersity index (PD) (708,000, dimethoxyphenol as the internal standard, according to the
375,000, 200,000, 107,000, 47,100, 21,100, 11,100, 5900, 667 and method published previously by Nguyen et al. [60,61]. Spectral
180 Da, Polysaccharide Calibrations Kit, PL2090-0100, Varian). Data interpretation was carried out using the NIST MS Search program
analysis was performed using Cirrus GPC software Version 3.2. (Version 2.0) operating on the NIST/EPA/NIH Mass Spectral Data-
Samples (250 mgL1) were dissolved in mobile phase DMSO/LiBr base 2011 (NIST 11).
(0.01 mol dm3), and filtered through a syringe filter (GHP, Acro-
disc, d ¼ 13 mm, 0.2 mm GHP membrane). 3. Results and discussion

3.1. Elemental analysis of the bio-oil fractions and LignoBoost™

2.6. NMR spectroscopic analysis
kraft lignin
NMR analysis was performed on bio-oil (100 mg), light oil
Investigation of the elemental composition of the different bio-
(100 mg), heavy oil (70 mg), LignoBoost™ kraft lignin (50 mg) and
oil fractions allowed the H/C atomic ratio to be estimated (Table 1).
suspended solids (20 mg), with each sample being dissolved in
The order of the H/C atomic ratio in the different fractions was
200 mm3 dimethyl sulphoxide-d6 (DMSO-d6). The 1H and, 13C NMR
found to be: light oil (1.21) > heavy oil (0.98) > suspended solids
and HSQC spectra were recorded at 25
C on a Bruker Avance III HD
(0.84). This indicates that the carbon component increases within
18.8 T NMR spectrometer (Rheinstetten, Germany) equipped with a
3 mm TCI Cryoprobe (cold 1H and 13C channels) operating at a
frequency of 800 MHz for 1H and 201 MHz to detect the presence of Table 1
13
C. The 1H spectra were recorded at a 30
pulse angle, 5 s pulse Elemental analysis of the various bio-oil fractions and LignoBoost™ kraft lignin.
delay, 1024 scans (for LignoBoost™ kraft lignin, heavy oil and SS),
C (%) H (%) N (%) S (%) O (%) H/C O/C
256 scans (for light oil) and 2.56 s acquisition time. The 13C spectra
Light oil (DEE soluble) 72.1 7.3 <0.10 0.31 1.21
were recorded with an inverse-gated decoupling sequence, 90
e e
Heavy oil (THF soluble) 72.9 6.0 0.17 0.72 e 0.98 e
pulse angle, 12 s pulse delay, 3200 scans (for LignoBoost™ kraft Suspended solids 65.3 4.6 0.19 0.37 e 0.84 e
lignin, heavy oil and SS) and 640 scans (for light oil), and 1.36 s Bio-oil 75.3 6.4 0.11 0.40 15b 1.01 0.15
acquisition time [63]. The central solvent peak was used for cali- LignoBoost™ kraft lignina 65.6 5.7 0.09 1.85 26b 1.04 0.30
bration of the chemical shift. The HSQC spectra were recorded us- a
Published previously by Nguyen et al. [60,61].
ing a standard Bruker pulse sequence “hsqcedetgpsisp2.3” with a b
Oxygen is calculated by difference.

Please cite this article in press as: C. Mattsson, et al., Using 2D NMR to characterize the structure of the low and high molecular weight fractions
of bio-oil obtained from LignoBoost™ kraft lignin depolymerized in subcritical water, Biomass and Bioenergy (2016), http://dx.doi.org/10.1016/
j.biombioe.2016.09.004
 C. Mattsson et al. / Biomass and Bioenergy xxx (2016) 1e14 5

the series, and more aromatic and condensed structures are 3.3. Characterization of 13C NMR in the bio-oil fractions and
therefore to be expected in the fractions with lower values i.e. LignoBoost™ kraft lignin
suspended solids [64,65].
3.3.1. Integration of the 13C NMR in the aromatic region of the bio-
oil fractions and LignoBoost™ kraft lignin
The 13C NMR aromatic region dC 106e162.5 ppm was integrated
3.2. Determination of the molecular weight of the bio-oil fractions with the lignin chemical shift integration regions used in the
and LignoBoost™ kraft lignin using GPC literature (106e154 ppm, Table 2S) [66] with the addition of a
mono phenolic/aryl ether region (dC 154e162.5 ppm, estimated
When subcritical water is used, the BCD of LignoBoost™ kraft from the lignin database published by Ralph et al. [67]). This
lignin yields a bio-oil with a new pattern of peaks in the distribu- analysis was made to provide a rough estimation of the aromatic
tion of Mw in the GPC (red line, Fig. 3) compared with LignoBoost™ carbon-carbon (AreC), aromatic carbon-hydrogen (AreH) and ar-
kraft lignin (blue line, Fig. 3). The first two peaks (Peaks A and B, omatic carbon-oxygen (AreO) bonds in the bio-oil fractions as well
Fig. 3) in the bio-oil correspond to depolymerized monomeric/ as in the LignoBoost™ kraft lignin, as well as to calculate the ratio of
dimeric compounds with an Mw distribution of approx. phenolic vs. guiacolic-like aromatic rings in the various fractions
60e240 Da. However, a biphasic peak of larger Mw fragments are (Table 2S). The aromatic 13C NMR ratios calculated for AreH/AreC
also found in the bio-oil with an Mw distribution of 6.5 kDa (PD are plotted against the AreO/AreC ratios in a Van Krevelen plot
1.97, Peak D, Fig. 3). Although this displaces an Mw distribution that (Fig. 4). This clearly shows that the heavy oil and suspended solids
is lower than the starting material used in the process, i.e. Ligno- are based on a more substituted and/or aromatic-like structure
Boost™ kraft lignin, 16.7 kDa, PD 3.17, it is not possible to deter- when compared with LignoBoost™ kraft lignin [i.e. heavy oil and
mine, using GPC measurements alone, whether this is unreacted suspended solids have a lower AreH/AreC ratio (0.6 and 0.5, resp.)
lignin or new, repolymerized structures [66]. Fractionation of the than LignoBoost™ kraft lignin (0.9), Fig. 4] [68].
bio-oil makes it possible to observe that the light oil corresponds to
the depolymerized Peaks A and B found in the bio-oil, i.e. the 3.3.2. Interpretation of the 13C NMR of the methoxyl group region of
monomeric/dimeric fraction (green line, Fig. 3). Moreover, heavy oil the bio-oil fractions and LignoBoost™ kraft lignin (dC 55e57 ppm)
(purple line, Fig. 3) and suspended solids (orange line, Fig. 3) were Overlaying the methoxyl region of the different bio-oil fractions
found to consist of fragments of higher Mw: 5.4 and 19.5 kDa, and LignoBoost™ kraft lignin in 13C NMR meant that the differences
respectively. It was, however, discovered that the suspended solids in the chemical environment/substitution pattern could be
had a higher distribution of Mw (19.5 kDa, orange line, Fig. 3) than revealed, i.e. LignoBoost™ kraft lignin, heavy oil and suspended
LignoBoost™ kraft lignin (16.7 kDa, blue line, Fig. 3). solids have methoxyl groups in the same range (dC 55.5e56.7 ppm,

Fig. 3. Molecular weight (Mw) distribution of the bio-oil fractions and LignoBoost™ kraft lignin measured using Gel Permeation Chromatography: SS: suspended solids (For values
of Mw see Table 1S.).

Please cite this article in press as: C. Mattsson, et al., Using 2D NMR to characterize the structure of the low and high molecular weight fractions
of bio-oil obtained from LignoBoost™ kraft lignin depolymerized in subcritical water, Biomass and Bioenergy (2016), http://dx.doi.org/10.1016/
j.biombioe.2016.09.004
6 C. Mattsson et al. / Biomass and Bioenergy xxx (2016) 1e14

1.0 oxygenated aliphatic region (dC/dH 52.5e90.0/2.8e5.7 ppm, Fig. 7)

Light oil Kraft
0.9 and an aromatic region (dC/dH 90.0e155/4.0e9.2 ppm, Fig. 8 and
Bio-oil Lignin
0.8 Fig. 1S). Additionally, a few low intensity aldehydes were detected
0.7 in the region dC/dH 190.0e204/9.5e10.5 ppm (Fig. 2S). Assignments
of 13Ce1H correlation signals in the 2D NMR spectra of Ligno-
Ar-H/Ar-C

0.6
Heavy oil Boost™ kraft lignin and the bio-oil fractions were identified ac-
0.5
Suspended solids cording to the literature (i.e. the lignin model compounds data base
0.4
of Ralph et al. [67]), kraft lignin from softwood [7,8,70,71], bio-oils
0.3
from various sources [30,72e74] and the estimation of chemical
0.2
shifts obtained using ChemBioDraw Ultra (13.0.2.3021, Perkin
0.1
Elmer, Cambridge Soft, UK). The chemical shifts of the different
0.0 regions are reported in Figs. 1Se5S.
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
Ar-O/Ar-C
3.4.1. Aliphatic region 2D NMR (dC/dH 8.0e52.5/0.5e4.5 ppm)
Fig. 4. A Van Krevelen plot of the ratios for AreH/AreC and AreO/AreC obtained from The aliphatic 2D NMR region (dC/dH 8.0e52.5/0.5e4.5 ppm)
the integration of 13C NMR in the aromatic region (dC 106e162.5 ppm) of the shown in Fig. 6 and Fig. 3S was divided into methyl (CH3), methy-
subcritical oil fractions and LignoBoost™ kraft lignin. Ar: aromatic. Tabulated data and
lene (CH2) and methine (CH) groups, combined with the distance of
integration regions are reported in Table 2S. When bio-oil is measured that contains
both monomeric and polymeric carbons in the same sample, the majority of signals
the alkyl group from the aromatic nucleus: a, b and g (Table 2) [73]
come from the former: this is not a representative 13C NMR ratio for describing the [52,75]. Lignin is generally based on phenyl-propane units and
composition of the bio-oil. therefore a majority of the peaks can be found in the a, b and g-
positions from the aromatic ring. Guaiacol rings form 95% of the
major aromatic composition of softwood (G, Table 2, Fig. 6).
Although other compounds such as extractives, i.e. fatty acids and
Fig. 5). This leads to the conclusion that the methoxyl groups found
resins/tall oil, are also found in kraft lignin, their concentration is
in these two fractions are “lignin-like” (i.e. based on a guaiacol-like
expected to be relative low [7,8,73,76]. Most of the cross-peaks in
pattern). In contrast, the light oil (red line, Fig. 5) displaces the
the bio-oil fractions were, however, found to be connected in the a-
mono-substituted methoxyl signal emitted mainly from anisole (dC
position to an aromatic ring, which indicates a shortening of the
55.17 ppm [67]) and the guaiacol methoxyl group (dC 55.87 ppm)
aliphatic chains in the bio-oil when compared to the LignoBoost™
located more downfield [53]. In addition, this 13C NMR analysis
kraft lignin.
corresponds well with the phenolic monomers/dimers found in
Obvious differences could be detected when the cross-peak
light oil using GC-MS analysis (Table 3S).
patterns in the aliphatic region of the bio-oil fractions were
compared with those of LignoBoost™ kraft lignin. Here, the most
3.4. 2D NMR characterization of the bio-oil fractions and characteristic cross-peaks of lignin are found in the region of the
LignoBoost™ kraft lignin CH2 groups between dC/dH 25e37/1.2e3.0 ppm (Fig. 6, black rings).
They correspond to the main aliphatic network present in lignin G-
2D NMR was run on the three bio-oil fractions and compared CH2-CH2-CH(R)-OH/R located at the b (dC/dH 33e37/1.4e1.8 ppm)
with the crude batch of (unpurified) LignoBoost™ kraft lignin and a (dC/dH 30e37/2.1e2.9 ppm) positions, respectively, to a
[10,69]. The spectra were interpreted qualitatively and divided into guaiacol ring. These typical cross-peaks in the lignin network can
three spectral regions: an aliphatic region (dC/dH 8.0e52.5/ be seen, to some extent, in heavy oil. Light oil and suspended solids,
0.5e4.5 ppm, Fig. 6), an inter-unit aliphatic lignin region/ on the other hand, are devoid of these aliphatic structures: these
have instead been replaced with other aliphatic linkages, indicated
with red circles in Fig. 6, i.e. ortho- and meta/para-methyl and ethyl-
substituted phenolic/guaiacol-like ring structures, aromatic rings
connected by ethylene (eCH2eCH2e) or methylene (eCH2e)
bridges, substituting bridge head methine carbons a to the aro-
matic ring and branched b-CH3 groups.

3.4.2. The inter-unit aliphatic lignin region/oxygenated aliphatic

region dC/dH 52.5e90.0/2.8e5.7 ppm in 2D NMR
This is the region where carbons next to alcohol, ether and Ar-
methoxyl groups as well as carbohydrate sugars resonate in 2D
NMR (dC/dH 52.5e90.0/2.8e5.7 ppm, Fig. 7). All bio-oil fractions
have a methoxyl signal between (dC/dH 55.20e56.09/
3.64e3.81 ppm, Fig. 7); that of LignoBoost™ kraft lignin is dC/dH
56.04/3.74 ppm, see discussion in Section 2.3.2 regarding the sub-
stitution position on the aromatic ring. The known aliphatic inter-
unit linkages b-O-40 (b-ether), b-b0 (pinoresinol), b-b0 (secoisolar-
iciresinol), b-10 (diphenyl ethane, week signal) and b-50 (phenyl-
Fig. 5. The 13C NMR methoxyl region (dC 55e57 ppm) of the subcritical oil fractions: coumaran) could be found for the LignoBoost™ kraft lignin (Fig. 1).
light oil (red line), heavy oil (blue line), suspended solids (light blue line) and Ligno- However, the a-O-40 and dibenzodioxocine (5-50 -a, b-O-40 ) struc-
Boost™ kraft lignin (green line). Light oil [dC 55.17 ppm (anisole), dC 55.23 ppm tures were absent, as they were probably hydrolyzed during the
(alkylated anisole derivatives), dC 55.87 ppm (guaiacol), dC 56.05 and 56.13 ppm kraft process (Figs. 1 and 7) [5,77]. The weak cross-peaks of the b-10
(alkylated guaiacol derivatives)], heavy oil (dC 56.22 ppm), suspended solids (dC
56.11 ppm) and LignoBoost™ kraft lignin (dC 56.07 ppm). (For interpretation of the
structures are due to the degradation of these structures in the kraft
references to colour in this figure legend, the reader is referred to the web version of process (i.e. stilbenes) [5,6]; this composition is according to pre-
this article.) vious studies of kraft lignin [7,8,76]. Nevertheless, it is not clear

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of bio-oil obtained from LignoBoost™ kraft lignin depolymerized in subcritical water, Biomass and Bioenergy (2016), http://dx.doi.org/10.1016/
j.biombioe.2016.09.004
 C. Mattsson et al. / Biomass and Bioenergy xxx (2016) 1e14 7

Fig. 6. The aliphatic region dC/dH 8.0e52.5/0.5e4.5 ppm of 2D NMR spectra obtained from the subcritical oil fractions light oil, heavy oil, suspended solids and LignoBoost™ kraft
lignin. Cross-peaks: CH/CH3 (green-yellow-orange-red) and CH2 (blue). Black rings: lignin structural motifs. Red rings: new cross-peaks found in the bio-oil fractions. The chemical
shifts are shown in Fig. 3S. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

whether b-b0 (secoisolariciresinol, Fig. 1) forms during the kraft 6.6e6.9 ppm) and G6 (dC/dH 119.3/6.7e6.8 ppm) [7,8]. Also, cross-
€nnols et al. disclosed a
process or exists in native lignin. Recently, Ro peaks corresponding to oxidized guaiacol units (e.g. a-carbonyl
structural analysis of LignoBoost™ kraft lignin (Mw cut-off at groups) were found to have cross-peaks of lower intensity (G0 , G0 2
15 kDa) using 2D NMR that supports our findings [76]. Further- and G0 6, Fig. 8). Another region, located downfield from the main
more, assignment of the different cross-peaks in LignoBoost™ kraft guaiacol cross-peaks, has peaks of lower intensity dC/dH 122e130/
lignin is possible; more detailed information is given in Fig. 4S. 6.9e7.8 ppm that correspond to stilbene structures (dC/dH 127e129/
It is clear from visual inspection of the 2D NMR spectra in this 7.0e7.4 ppm, Fig. 8). Moreover, it cannot be excluded that PAH
region of the different bio-oil fractions that all inter-unit linkages structures (dC/dH 125e135/7.2e8.2 ppm, Fig. 8) may exist in
(i.e. CeO linkages) from LignoBoost™ kraft lignin have disappeared LignoBoost™ kraft lignin.
(Fig. 7). This means that aliphatic CeO has been cleaved under One simple way of analyzing the aromatic 2D NMR region
subcritical conditions, i.e. in the known lignin inter-unit structures (Fig. 8) is by highlighting the most pronounced regions in Ligno-
b-O-40 , b-b0 , b-10 and b-50 . This is an important finding: it proves Boost™ kraft lignin: guaiacyl G2, G5 and G6, oxidized guaiacyl G0
that all of the lignin is reacted under subcritical water conditions and G0 6 and the stilbene structures (Ar-CH]CH-Ar). A clear trend
(350
C, 25 MPa) in this continuous reactor system. It also dem- can be seen in the intensity of the cross-peak regions G2, G5 and G6,
onstrates that the amount of oxygen connected to the aliphatic i.e. LignoBoost™ kraft lignin > heavy oil > suspended solid (Fig. 8).
carbons in bio-oil is very low. The content of atomic oxygen This indicates either that the aromatic G units for heavy oil and
detected in the bio-oil by means of elemental analysis (Table 1) suspended solids are substituted with more new CeC bonds
comes, to a large extent, almost exclusively from oxygen-containing compared with lignin, or that the guaiacyl units are transformed
groups (-OH and eOMe/R) connected to an aromatic ring, i.e. into other aromatic structures. Light oil, on the other hand, has a
phenols, guaiacols and other aromatic ethers. This is discussed pattern of cross-peaks different to that of LignoBoost™ kraft lignin:
more thoroughly in Sections 3.5.1 and 3.5.2. one of the main reasons for this is that all monomeric/dimeric ar-
omatics derived from lignin and phenol (i.e. added and formed
derivatives) end up in this fraction. It is known, from GC-MS
3.4.3. The aromatic region (dC/dH 90.0e155/4.0e9.2 ppm) in 2D analysis, that phenol and phenol derivatives dominate this frac-
NMR tion (Fig. 9, Table 3S) [60,61]; interpretation of the various mono-
The main CH cross-signals of LignoBoost™ kraft lignin in the meric phenol derivatives present in light oil can be found in
aromatic region correspond to the guaiacol ring (G) and the known Table 5S and Figs. 1S and 5S.
substitution pattern of softwood kraft lignin (Fig. 8): G2 (dC/dH
110.7e110.8/6.9, 112.2e112.4/6.8e6.9 ppm), G5 (dC/dH 115.5e115.9/

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of bio-oil obtained from LignoBoost™ kraft lignin depolymerized in subcritical water, Biomass and Bioenergy (2016), http://dx.doi.org/10.1016/
j.biombioe.2016.09.004
8 C. Mattsson et al. / Biomass and Bioenergy xxx (2016) 1e14

Fig. 7. The inter-unit aliphatic lignin region/oxygenated aliphatic region dC/dH 52.5e90.0/2.8e5.7 ppm of 2D NMR spectra of the subcritical oil fractions light oil, heavy oil, sus-
pended solids and LignoBoost™ kraft lignin. Cross-peaks: CH/CH3 (green-yellow-orange-red) and CH2 (blue). Black rings: lignin structural motifs. Red rings: new cross-peaks found
in the bio-oil fractions. The chemical shifts are reported in Fig. 4S. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)

3.5. Summary of the elucidation of the structure

3.5.1. Proof of the depolymerization concept: LignoBoost™ kraft

lignin
Table 2 None of the different fractions of bio-oil produced from BCD of
General aliphatic chemical shift regions of the 2D NMR spectruma. LignoBoost™ kraft lignin subcritical water depolymerization, i.e.
light oil, heavy oil and suspended solids, were found to have any
signs of the lignin inter-unit oxygen connections in the 2D NMR (dC/
dH 52.5e90.0/2.8e5.7 ppm, Fig. 7). This indicates that the same type
of deoxygenation reaction proceeded in them all. It leads to the
assumption that lignin is initially solubilized/hydrolyzed in the
subcritical phase during our conditions (350
C, 25 MPa), and that
the subsequent depolymerization and repolymerization reactions
Chemical shift region 1H (ppm) Description that occur yield monomeric/dimeric molecules and repolymerized
0e1 CH3 g or further to Ar
macromolecules, respectively. It also proves that the higher Mw
1e1.4 CH3, CH2 b to Ar fractions (i.e. heavy oil and suspended solids) are not comprised of
CH g to Ar unreacted LignoBoost™ kraft lignin (i.e. residual lignin). The
1.4e1.9 CH2, CH b to Ar breakage of lignin inter-unit linkages has also been reported in
1.9e3.5 CH3, CH2, CH a to Ar
similar continuous flow reactor systems (350
C, 20.7 MPa and
3.5e4.5 CH2, CH a to two Ar
AreCH2eAr, Ar-CH(R)-Ar 315
C, 13 MPa), which supports our finding [74,78].
Chemical shift region13C (ppm) Description

10e16 CH3 3.5.2. Carbon-oxygen connections in the bio-oil fractions

16e23 CH2, CH3 Regarding the location of the CeO bonds in the heavy oil and
23e30 CH, CH2, CH3
suspended solids, all of the NMR analyses indicate that the main
30e38 CH2, CH2 a and CH2 b in
GeCH2eCH2eCH2eOH oxygen content (CeO) of these fractions are connected to aromatic
38e44 CH, CH2, CH3 with EWG rings: (a) 2D NMR shows that almost no aliphatic hydroxyl or ether
44e52.5 Restricted alkyl CH and CH2 bonds remain (Fig. 7); (b) 13C NMR integration reveals that the CeO
a
Abbreviations: G: guaiacol ring; Ar: aromatic ring; EWG: electron withdrawing bond is located almost exclusively in the disubstituted aromatic
group; R: alkyl or aryl-group. ether/guaiacol chemical shift region (Table 2S); (c) The methoxyl

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of bio-oil obtained from LignoBoost™ kraft lignin depolymerized in subcritical water, Biomass and Bioenergy (2016), http://dx.doi.org/10.1016/
j.biombioe.2016.09.004
 C. Mattsson et al. / Biomass and Bioenergy xxx (2016) 1e14 9

Fig. 8. The aromatic region dC/dH 105e155/6.0e9.2 ppm of the 2D NMR spectra of the subcritical oil fractions light oil, heavy oil, suspended solids and LignoBoost™ kraft lignin.
Cross-peaks: CH (green-yellow-orange-red) and CH2 (blue). Black rings: lignin structural motifs. Red rings: new cross-peaks found in bio-oil fractions. The full aromatic region dC/dH
90.0e155/4.0e9.2 ppm is shown in Fig. 1S. G: guaiacol unit; G0 : oxidized guaiacol units; PAH: polyaromatic hydrocarbon. The chemical shifts are reported in Fig. 1S. (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 9. The most pronounced aromatic monomers and dimers in the light oil estimated by GC-MS. Percentage and structural information from GC-MS for the different compounds
can be found in Table 3S.

group (-OCH3) in 13C NMR was found to be in a guaiacol-like light oil fraction was found to consist of a mixture of phenol and
environment (Fig. 5). The conclusion can be thus drawn that CeO guaiacol-like rings at a ratio of 2.4:1 (13C NMR, Table 2S). Together
bonds are found in the form of hydroxyl, methoxyl or aromatic with the knowledge that the main patterns of the oxygen aromatics
ether groups in guaiacol, catechol or other aromatic disubstituted in the other high Mw fractions were disubstituted aryl ethers al-
ether structures. However, 1H NMR (Table 4S) showed that the lows the assumption to be made that the majority of the phenolic
content of free phenolics was low in the suspended solids: this derivatives end up in the light oil fraction. One reason for this could
yields probably a higher fraction of aromatic ethers and methoxyl be that alkylated phenols are less reactive than substituted guaiacol
groups in the suspended solids than in the heavy oil. However, the structures, which has been postulated previously by other research

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of bio-oil obtained from LignoBoost™ kraft lignin depolymerized in subcritical water, Biomass and Bioenergy (2016), http://dx.doi.org/10.1016/
j.biombioe.2016.09.004
10 C. Mattsson et al. / Biomass and Bioenergy xxx (2016) 1e14

groups, and thereby leads to an enrichment of phenol structures in aromatic-CH (18), vinylic (Ar-CH]CH-R, 19) and allylic (Ar-C-CH]
the light oil [30,56,79]. Also, our group has previously reported a CH-R, 20) connected aromatic substituents, stilbenes (Ar-CH]CH-
blank run with subcritical water, phenol and base (K2CO3, 350
C, Ar, 11) and condensed PAH-like structures (21), as well as the
25 MPa) in which the phenol showed no tendency at all to react in aliphatic carbon bridge heads methylene (a-CH2, 22) and methine
the presence of base [60]. Placed in context, guaiacol model com- (a-CH, 23) substituted directly onto aromatic rings. It should be
pounds have been found to be reactive in sub/supercritical condi- pointed out here that none of these connections are to an oxygen
tions [80e83], which is in agreement with earlier work where the atom. One new linkage with methylene oxygen (Ar-O-CH2-R, 24)
guaiacol ring was presupposed as being responsible for the poly- was found both in heavy oil and suspended solids, and a fraction of
merization reactions leading to structures with a higher Mw methylene-g-hydroxyls (Ar-C-C-CH2-OH/R, 7) was found in the
[29,83,84]. heavy oil. The new methylene oxygen linkage (24) may be located
in a tetrahydrofuran-like ring structure due to it resembling the
3.5.3. Structural features of heavy oil and suspended solids vs. shift with b-b0 and dihydrobenzofuran (fused to an aromatic ring)
LignoBoost™ kraft lignin structures [72]. In addition, heavy oil seems to be built up to a great
Summarizing the different analyses, it seems that heavy oil and extent of more flexible bond structures, with two carbon bridges
suspended solids are comprised of new repolymerized macro- (eCH2eCH2e, 9), ethyl (25) and propyl groups (8, Fig. 10). These
molecules because: (a) no lignin inter-unit oxygen connections structures cannot be found in suspended solids, and are instead
were found in the 2D NMR; (b) the aliphatic a- and b-CH2 cross- substituted on one carbon bridge head (i.e. Ar-CH-, 23) and have
peak networks typical of lignin are, to a large extent, not present linkages with unsaturated bonds (i.e. vinylic 19, allylic 20 and stil-
in heavy oil and suspended solids; (c) the cross-peak patterns of bene 11, Fig. 10). These structural findings agree with previous main
aromatic guaiacyl G2, G5 and G6 in heavy oil and suspended solids conclusions drawn from the formation of char in a sub/supercritical
are not the same as for those in LignoBoost™ kraft lignin, thus phase, i.e. there is a loss of phenolic or alcoholic groups and an
indicating another pattern of aromatic substitution; (d) the Mw increase of aliphatic groups and aromatic structures, as well as a
distribution of the bio-oil (Peak D) is approx. 10 Da lower than in recombination of aromatic rings [32,54e56,68].
LignoBoost™ kraft lignin; (e) plotting the H/C and O/C ratios from
the aromatic region of 13C NMR in a van Krevelen plot (Fig. 4) yields 3.5.4. Structural features of light oil
a chemical structure that clearly has a lower content of oxygen and GC-MS analysis of the light oil fraction showed the main con-
a higher content of carbon than LignoBoost™ kraft lignin; (f) the H/ stituents as being phenol, anisole and guaiacol units substituted
C atomic ratios acquired from the elemental analysis also indicate a with small alkyl groups, i.e. methyl, ethyl and propyl, or linked by
higher content of total carbon for the heavy oil and suspended one and two carbon bridges (eCH2e, eCH2eCH2e, Fig. 9 and
solids (both aromatic and aliphatic carbons included) vs. Ligno- Table 3S). By using 2D NMR, the total composition of the light oil
Boost™ kraft lignin (Table 1). These structural changes could be could be investigated and a good agreement with the GC-MS results
caused by the loss of CeH or CeO bonds in favour of the formation was found (Fig. 11). An additional finding of NMR was that the light
of CeC bonds, thereby giving a more substituted and/or condensed oil, along with the other high Mw fractions, was devoid of aliphatic
structure. oxygen lignin inter-unit connections. However, two cross-peaks
A summary of the structural features found in the different bio- corresponding to methylene-g-hydroxyls (Are-
oil fractions and LignoBoost™ kraft lignin can be found in Fig. 10, CH2eCH2eCH2eOH/R, Fig. 7) were detected, indicating that a
which is a hypothetical macromolecular structural diagram for the fraction of aliphatic hydroxyl groups remains in the light oil. It is
visualization of the different chemical fragments/linkages found clear from the 2D NMR that a larger fraction of the light oil is
using 2D NMR. connected to diphenylmethane (AreCH2eAr) and diphenylethane
Whilst the structure of LignoBoost™ kraft lignin is modified (AreCH2eCH2eAr) structures that have not been detected previ-
compared with the MWL softwood structure proposed (Fig. 6S) ously by GC-MS, probably due to the fact that they are located on
[4,85], the known inter-unit linkages b-O-40 (1), 4-O-50 (2), 5-50 (3), dimers and trimeric aromatic structures. In addition, other aro-
b-b0 [pinoresinol (4) and secoisolariciresinol (5)] and b-50 (6) are matic substituents, such as aldehyde and methyl ketone groups,
still present in it (Fig. 10). It is interesting to note that many have been detected in smaller amounts by 2D NMR.
different aliphatic structural motifs can be found, indicating that a
reduction in the aliphatic oxygen region of the lignin proceeds 3.5.5. Structures of diphenylmethane and diphenylethane
during the kraft cook: dihydroconiferyl alcohol (Are- One intriguing finding of this study is that diphenylmethane
CH2eCH2eCH2eOH, 7, Fig. 10), propyl side chains (8, Fig. 10) as well structures could only be found in the light oil fraction. This struc-
as two carbon bridges (eCH2eCH2e, 9) and CH2-groups in a-po- ture class has been investigated intensely because of the suspicion
sition (10) to the aromatic ring are likely to be found [1]. In addition, that it is a constituent of the condensed structures formed in lignin
other known kraft lignin structural motif can be found: stilbene during the kraft process. However, this assumption has been found
structures (11, Fig. 10), aliphatic a-hydroxyl group (12) and methine to be incorrect, as revealed by recent 2D NMR characterizations of
groups (a-CH, 13) connected to an aromatic ring (a-5 condensed kraft lignin [7,8]. It is also known that formaldehyde and phenol
structure). heated with a base catalyst will form phenol-formaldehyde resins
The bio-oil fractions heavy oil and suspended solids have none [35,88]. Thus one possible explanation here is that phenol/guaiacol
of the original lignin inter-unit CeO bondings left in b-O-40 , b-b0 fragments react with released formaldehyde to form diphenyl-
and b-5’. However the CeC skeleton of b-b0 , b-50 and b-10 may still methane structures during subcritical depolymerization [43].
be present in linkages and side chains, i.e. stilbene (11) and aliphatic Formaldehyde has been shown to be formed as a byproduct of the
linkages (9 and 14, Fig. 10). The aromatic carbon-carbon 5-50 (3) and cleavage of Cb-Cg bonds in the phenyl-propane structures of lignin
carbon-oxygen 4-O-50 (2) linkages of lignin have been reported to and to participate in repolymerization reactions by forming
be more resistant to subcritical depolymerization and so are most diphenylmethane structures. This leads to the common assumption
likely to be unaltered in the bio-oil fractions [19,30,86,87]. The being made that these compounds are a part of the structures of the
structural features present both in the heavy oil and suspended solids and the heavy oil [35e37,39]. However, our results indicate
solids are (Fig. 10): aromatic methyl groups (a-CH3, 15), branched b- that these structures go to the monomeric/dimeric light oil and are
CH3 groups [Ar-CH0-2-(CH3)1-3), tert-butyl (16) or similar (17)], not a part of the higher Mw structures (i.e. heavy oil and suspended

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of bio-oil obtained from LignoBoost™ kraft lignin depolymerized in subcritical water, Biomass and Bioenergy (2016), http://dx.doi.org/10.1016/
j.biombioe.2016.09.004
 C. Mattsson et al. / Biomass and Bioenergy xxx (2016) 1e14 11

Fig. 10. The chemical structures of the linkages found by 2D NMR linked together in a hypothetical macromolecule pattern, allowing the changes in the heavy oil and suspended
solid bio-oil fractions vs. LignoBoost™ kraft lignin to be visualized (N.B. not proportional to their natural occurrence in the macromolecule). Lignin linkages are in bold print: b-O-40
(1), 4-O-50 (2) and 5-50 (3), b-b0 [pinoresinol (4) and secoisolariciresinol (5)] and b-50 (6).

Fig. 11. Examples of structural fragments identified from 13C NMR and 2D NMR in the light oil fraction obtained from the subcritical depolymerization of LignoBoost™ kraft lignin.

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of bio-oil obtained from LignoBoost™ kraft lignin depolymerized in subcritical water, Biomass and Bioenergy (2016), http://dx.doi.org/10.1016/
j.biombioe.2016.09.004
12 C. Mattsson et al. / Biomass and Bioenergy xxx (2016) 1e14

Acknowledgements

This work is supported by grants from Chalmers Energy Initia-

tive e LignoFuel Project, Valmet AB and The Swedish Energy
Fig. 12. A possible reduction pathway for stilbene to form the diphenylethane struc- Agency. Our thanks go to Ms. Cecilia Persson for her technical
tures found in light oil and heavy oil. support with the NMR analysis at the Swedish NMR Centre atThe
University of Gothenburg, Mr. Tommy Friberg for his analytical
contributions to the experiments.
solids) (Fig. 6). Furthermore, these diphenylmethane structures
have been postulated as being produced primarily when phenol is Appendix A. Supplementary data
used as the capping agent [35e37,39]. The use of 2D NMR (Figs. 6
and 12) and GC-MS (Fig. 9) analysis of the light oil meant that, Supplementary data related to this article can be found at http://
based on aromatics connected to a two carbon bridge (eCH2e- dx.doi.org/10.1016/j.biombioe.2016.09.004.
CH2e), a new type of dimeric structure could be assigned, namely
diphenylethane. This structure class has been reported recently by
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C, 9 MPa) with
phenol additives [31]. Analysis of 2D NMR leads to a group of [1] C. Heitner, D. Dimmel, J.A. Schmidt, Lignin and Lignans: Advances in Chem-
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structural connection between the monomeric light oil and the 2008, p. 201.
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oligomer, Biomacromolecules 12 (11) (2011) 3928e3935.
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Please cite this article in press as: C. Mattsson, et al., Using 2D NMR to characterize the structure of the low and high molecular weight fractions
of bio-oil obtained from LignoBoost™ kraft lignin depolymerized in subcritical water, Biomass and Bioenergy (2016), http://dx.doi.org/10.1016/
j.biombioe.2016.09.004