Compositional analysis of organosolv poplar lignin by using high-performance liquid chromatography/high-resolution multi-stage tandem mass spectrometry

Article abstract of DOI:10.1039/d0gc03398g

Organosolv treatment is an efficient and environmentally friendly process to degrade lignin into small compounds. The capability of characterizing the individual compounds in the complex mixtures formed upon organosolv treatment is essential for the optimization of the further lignin conversion processes and for the rational genetic engineering of plants used to produce lignin in order to improve lignin properties. In this study, an organosolv poplar lignin sample was initially analyzed by high-resolution mass spectrometry coupled with negative-ion mode electrospray ionization ((?)ESI HRMS). Lignin monomers and dimers were found to constitute the majority of the compounds in the organosolv lignin sample. Larger lignin oligomers, such as trimers and tetramers, and some not lignin-related compounds, were also detected. A high-performance liquid chromatograph/linear quadrupole ion trap/orbitrap mass spectrometer capable of multi-stage high-resolution tandem mass spectrometry experiments (HRMSⁿ), equipped with an (?)ESI source (HPLC/(?)ESI HRMSⁿ), was employed to separate the unknown compounds in the organosolv mixture and to obtain structural information for the deprotonated compoundsviacollision-activated dissociation (CAD) HRMSⁿexperiments. To improve the understanding of the CAD behavior of deprotonated lignin-related compounds, 16 deprotonated model compounds with different functionalities and linkage types were examined. This approach enabled the assignment of likely structures for several lignin monomers, dimers, trimers, and tetramers, and some not lignin-related compounds, most likely fatty acids. Based on the proposed structures, compounds in the organosolv lignin sample contain β-O-4, 5-5, β-5, and possibly also 4-O-5 linkages. Most compounds contain G- and S-monomeric units although a small amount of H-units were also detected.

Full text of DOI:10.1039/d0gc03398g

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Compositional analysis of organosolv poplar
lignin by using high-performance liquid
chromatography/high-resolution multi-stage
tandem mass spectrometry†
Cite this: Green Chem., 2021, 23,
83
9
a
a
a
b
a
Jifa Zhang, Yuan Jiang, Leah F. Easterling, Anton Anstner, Wanru Li,
a
a
b
Kawthar Z. Alzarieni, Xueming Dong, Joseph Bozell
Hilkka I. Kenttämaa
and
a
*
Organosolv treatment is an efficient and environmentally friendly process to degrade lignin into small
compounds. The capability of characterizing the individual compounds in the complex mixtures formed
upon organosolv treatment is essential for the optimization of the further lignin conversion processes and
for the rational genetic engineering of plants used to produce lignin in order to improve lignin properties.
In this study, an organosolv poplar lignin sample was initially analyzed by high-resolution mass spec-
trometry coupled with negative-ion mode electrospray ionization ((−)ESI HRMS). Lignin monomers and
dimers were found to constitute the majority of the compounds in the organosolv lignin sample. Larger
lignin oligomers, such as trimers and tetramers, and some not lignin-related compounds, were also
detected. A high-performance liquid chromatograph/linear quadrupole ion trap/orbitrap mass spectro-
n
meter capable of multi-stage high-resolution tandem mass spectrometry experiments (HRMS ), equipped
n
with an (−)ESI source (HPLC/(−)ESI HRMS ), was employed to separate the unknown compounds in the
organosolv mixture and to obtain structural information for the deprotonated compounds via collision-
n
activated dissociation (CAD) HRMS experiments. To improve the understanding of the CAD behavior of
deprotonated lignin-related compounds, 16 deprotonated model compounds with different functional-
ities and linkage types were examined. This approach enabled the assignment of likely structures for
several lignin monomers, dimers, trimers, and tetramers, and some not lignin-related compounds, most
likely fatty acids. Based on the proposed structures, compounds in the organosolv lignin sample contain
β-O-4, 5–5, β-5, and possibly also 4-O-5 linkages. Most compounds contain G- and S-monomeric units
although a small amount of H-units were also detected.
Received 13th October 2020,
Accepted 6th January 2021
DOI: 10.1039/d0gc03398g
rsc.li/greenchem
2
,3
abundance, easy accessibility, and renewability. It is com-
Introduction
posed of three major components: cellulose, hemicellulose,
2
The depletion of fossil fuels, compounded by the deterioration and lignin. Lignin is the most abundant aromatic biopolymer
of the global environment caused by their production and in nature and therefore an attractive source for aromatic com-
extensive usage, has prompted the search for alternative renew- pounds. Lignin has an extremely complex, three-dimensional
1
–7
able sources for energy and materials.
Lignocellulosic structure as it is formed via radical coupling reactions of three
biomass holds great potential to fulfill this goal due to its high major monolignols, p-coumaryl alcohol, coniferyl alcohol, and
8
–16
sinapyl alcohol (Fig. 1).
These monolignols correspond to
p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units,
respectively, in lignin. These basic units are cross-linked
together by different inter-unit linkages, among which the
a
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette,
Indiana 47907, USA. E-mail: hilkka@purdue.edu
b
University of Tennessee, Center for Renewable Carbon, Knoxville, Tennessee, USA
1
5,16
†
Electronic supplementary information (ESI) available: Materials, chemicals β-O-4 is the most abundant.
Other linkage types include
n
15,16
used, instrumental conditions, experimental procedures, HRMS CAD spectra of
β-5, 5–5, 4-O-5, β-1 and β–β linkages (Fig. 1).
n
ionized lignin model compounds, CAD HRMS spectra of unknown deproto-
Many efforts have been dedicated to converting lignin into
nated compounds in the organosolv lignin sample, synthesis procedures, NMR
data for the synthesized compounds, and tables of Cartesian coordinates, elec-
tronic energies, zero-point vibrational energies, and 298 K thermal contributions
for all computed structures. See DOI: 10.1039/d0gc03398g
2
,4–6,17,18
biofuel or valuable chemicals.
Organosolv treatment
is an environmentally friendly and efficient process to isolate
1
9–22
lignin and degrade it into small compounds.
During the
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Fig. 1 A. The three most common monolignols of lignin. B. The three most common monomeric units in lignin. C. Some of the most common lin-
kages in lignin (highlighted in blue).
organosolv treatment, many of the labile β-O-4 bonds in lignin may have been changed during pyrolysis. Identification of the
are cleaved. The extent to which they are cleaved is determined individual compounds in the lignin degradation products,
6
by the exact conditions used. Further, various reactions can therefore, requires other methods.
take place at the aliphatic hydroxyl groups: they can be oxi-
HPLC/high-resolution multi-stage tandem mass spec-
n
dized to aldehyde, ketone, or carboxylic acid groups or con- trometry (HRMS ) based on collision-activated dissociation
2
3
verted to ether or ester functionalities. Repolymerization (CAD) is a powerful method for the separation and structural
reactions among the produced monomers can also occur to elucidation of organic compounds in complex mixtures.
2
4–26
produce bigger oligomers.
Therefore, a very complex Negative-ion mode electrospray ionization is widely used for
mixture of organic compounds is usually generated. The struc- the ionization of lignin-related compounds for mass spectro-
2
3,27,36
tures of most of the compounds in these mixtures are metric analysis
unknown.
as many lignin-related compounds have
2
3,27
This presents a problem as a prerequisite for a relatively acidic phenol group. Upon (−)ESI, the phenol
the optimization of further upgrading processes is the ability group often gets deprotonated to afford phenoxide anions.
to characterize the compounds in these complex mixtures. Quantum chemical calculations at the M06-2X/6-311++G(d,p)
Furthermore, this is also a prerequisite for rational genetic level of theory carried out on a deprotonated β-O-4 type lignin
3
7
engineering of plants in order to improve the plant raw dimer in the gas phase indicate that the energy required to
2
8
Modifications of lignin remove a proton from the phenol group is about 15 kcal mol−1
materials for industrial usage.
content and composition via genetic engineering could have less than removal from an aliphatic hydroxyl group (in spite of
2
8,29
great economic and environmental benefits.
stabilizing hydrogen-bonding interactions), suggesting that
Although many techniques are available for the analysis of deprotonation predominantly occurs on the phenol moiety in
mixtures generated upon degradation of lignin, including the gas phase.
n
nuclear magnetic resonance (NMR) spectroscopy, Fourier-
Previous HRMS studies based on (−)ESI and CAD experi-
transform infrared resonance (FT-IR) spectroscopy, and gel ments have provided some structural information for deproto-
2
3,27
permeation chromatography (GPC), most of these techniques nated unknown compounds in organosolv lignin samples.
only provide bulk information for the mixtures.
8
,19,21,30–33
The limitations of these studies mostly arise from insufficient
Analytical pyrolysis coupled to gas chromatography with mass knowledge of the CAD behavior of deprotonated lignin-related
2
3,27
n
spectrometric detection (py-GC/MS) can be used to attempt to model compounds.
In a (−)ESI HRMS study, the most
analyze some of the individual compounds in the lignin degra- important fragmentation pathways and likely mechanisms
3
4,35
dation mixtures.
However, only volatile lignin-related com- were elucidated for deprotonated oligomeric lignin
3
7
pounds can be examined by this method and their structures β-O-4 model compounds. Their fragmentation can be cate-
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gorized into three major pathways (for
a
dimer, see trimers or bigger oligomers does not fragment further upon
3
9
Scheme 1A): (1) elimination of the charge-remote unit(s) (B in CAD. These unique dissociation behaviors identified for
Scheme 1A) via the cleavage of the Cβ–O bond to form frag- model compounds should enable the determination of
ment ions containing unit A; (2) elimination of the charged whether β-O-4, β-5 or 5–5 linkages exist in unknown oligomeric
end unit A via the cleavage of a Cβ-O bond to form fragment lignin ions.
ions containing unit B; and (3) elimination of water and for-
During organosolv treatment, various reactions can take
maldehyde from the aliphatic hydroxyl groups in the side place on the aliphatic hydroxyl groups of lignin monomers
chain. The formation of the two fragment ions of pathways 1 and oligomers, such as oxidation to a keto, aldehyde or car-
and
Scheme 1A).
unknown oligomeric ion implies that the unknown analyte mentation of the ionized modified compounds different from
contains a β-O-4 linkage. that of their native forms. The characteristic fragmentation
2
involve the cleavage of the β-O-4 linkage boxylic acid functionality or conversion to an ether or ester
3
7–39
23,40
(
Observation of these fragment ions for an functionality.
These new functionalities can make the frag-
On the other hand, a previous fragmentation study of one patterns of some simple deprotonated lignin model com-
deprotonated β-5 type lignin dimer model compound by using pounds with an aldehyde, carboxylic acid, or ester functional-
n
the (−)APCI HRMS technique suggested that deprotonated ity and compounds with two functionalities in the side chain
n
23,36
β-5 type lignin dimers can lose their charged end unit A as a have been investigated using the (−)ESI HRMS technique.
quinone methide-like neutral molecule (Q; see Scheme 1B) However, the fragmentation patterns of several relevant,
3
8
upon CAD in all three major fragmentation pathways.
simple lignin-related model compounds (e.g., alcohols,
Pathway 1 involves the elimination of water followed by the ketones, and catechols), and some model compounds with
elimination of the neutral molecule Q while pathway 2 involves two functionalities in the side chain (e.g., β-keto esters), have
2
3,36
the elimination of formaldehyde followed by the elimination not been examined.
Further, while the majority of lignin is
3
8
of Q. Pathway 3 only involves the elimination of Q. Based on composed of the H-, G- and S-monomers discussed above,
the proposed pathways, the eliminated quinone methide-like other minor monomeric units can also be found, such as cate-
neutral molecule Q has the formula C
for the H-, G-, and S-monomeric units, respectively ated H- and G-monomers, respectively.
Scheme 1B). Thus, the elimination of the charged end unit A tion patterns of these deprotonated monomers have not been
as a neutral molecule Q with a formula of C O, C , or reported. Therefore, the fragmentation behavior of deproto-
may be indicative of the presence of a β-5 linkage in nated lignin model compounds with hydroxy, aldehyde, keto
an unknown ion. or catechol functionalities, compounds with two functional-
Examination of CAD of five deprotonated oligomeric lignin ities in the side chain, and some other compounds not pre-
7 6 8 8 2
H O, C H O , and chol and 5-hydroxyl G, which can be considered as hydroxyl-
2
8,41–43
C
9
H
10
O
3
The fragmenta-
(
7
H
6
8 8 2
H O
9 10 3
C H O
n
model compounds with a 5–5 linkage by using (−)ESI HRMS
demonstrated that the 5–5 linkage stays intact upon CAD.
viously studied were investigated here to be able to more accu-
rately interpret the fragmentation behavior observed for depro-
3
9
For deprotonated compounds with two or more linkages, the tonated unknown lignin-related compounds. Also, deproto-
characteristic dissociation behaviors of the β-O-4 and β-5 lin- nated 5–5 type lignin dimer model compounds, deprotonated
kages can be identified based on the dissociation patterns 2,2′-biphenol and deprotonated 5,5′-bisvanillin containing an
shown in Scheme 1. The dimeric 5–5 unit in deprotonated aldehyde functionality, were studied to better understand the
3
7–39
Scheme 1 Fragmentation pathways reported for deprotonated lignin model compounds containing a β-O-4 linkage
(left, part A) and for a
38
compound containing a β-5 linkage (right, part B). Based on our unpublished data, the structures indicated for the ionic products may be incorrect
for part B.
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dissociation of deprotonated compounds with 5–5 linkages rate of 100 μL min− . The resulting mixture was introduced
and different functional groups in the side chains. into the ESI source and the analytes were ionized using (−)
In this study, the (−)ESI HRMS technique was utilized ESI. A N sheath gas flow rate of 30 (arbitrary units), N auxili-
to obtain chemical composition information on a poplar ary gas flow rate of 10 (arbitrary units), spray voltage of 3.5
1
2
2
n
organosolv lignin sample. Further, a HPLC/(−)ESI HRMS
kV, ion transfer capillary temperature of 275 °C, and capillary
(wherein n refers to the number of ion isolation steps) voltage −10 V were utilized.
method based on CAD was employed to provide structural
For CAD studies, each of the 16 lignin model compounds
information for many of the unknown degradation products was prepared at a concentration of approximately 1 mM in LC/
in the organosolv lignin sample. The previously published MS grade methanol. Each solution was introduced into the
diagnostic fragmentation patterns determined for deproto- mass spectrometer via a syringe drive at a flow rate of 10 µL
3
7
38
−1
nated model compounds with β-O-4, β-5, and 5–5 lin- min . The analytes were ionized by (−)ESI and the analyte
3
9
kages, as well as the fragmentation patterns of the model ions were transferred into the linear quadrupole ion trap of
compound discussed above, were utilized to assign likely the mass spectrometer. An isolation width of 2 m/z units was
structures for the major unknown compounds in the organo- used to isolate the ions of interest for CAD. A normalized col-
solv lignin mixture.
lision energy of 35, a q value of 0.25, and an activation time of
0 ms were used to induce fragmentation of the isolated ions
3
in the ion trap. The most abundant fragment ions were iso-
3
lated and subjected to CAD in the ion trap in MS experiments.
Experimental section
Materials
This process was repeated until no more fragmentation could
be observed (up to MS experiments). All the ionized analytes
5
Unless otherwise stated, all commercially available chemicals
were purchased from Sigma-Aldrich (St Louis, MO, USA) and
were used as received. LC/MS grade water, methanol, and
acetonitrile were purchased from Fisher Scientific (Pittsburgh,
PA, USA).
and fragment ions were transferred into the high-resolution
orbitrap mass analyzer for high-resolution analysis. The repro-
ducibility of the relative abundances of the fragment ions was
±
15ꢀ.
HPLC separation was performed using a Surveyor Plus
HPLC equipped with a quaternary pump, a photodiode array
PDA) detector, and an autosampler. An organosolv lignin
Ethyl 3-(4-hydroxy-3-methoxyphenyl)-3-oxopropanoate and
5
,5′-bisvanillin were synthesized in house. The details of the
(
−
1
syntheses and the NMR spectra can be found in ESI.† The
organosolv poplar lignin sample was supplied by Dr Bozell of
the University of Tennessee Center for Renewable Carbon
solution was prepared at a concentration of 10 mg mL in
acetonitrile and water (1 : 1, v/v) and 10 µL of the solution was
loaded onto a reversed-phase Zorbax SB Phenyl column (4.6 ×
4
0
obtained using a previously published method. The organo-
solv poplar lignin sample was generated at a temperature of
250 mm, particle size 5 µm, Agilent Technologies, Santa Clara,
CA, USA) by using the partial loop injection mode. The mobile
phase solvents used were water (A) and acetonitrile (B) at a
flow rate of 600 µL min− . Both solvents were doped with 0.1ꢀ
1
1
60 °C, H
20 min.
2 4
SO concentration of 0.1 M, and a reaction time of
1
(
v/v) of formic acid. The nonlinear gradient used was as
Instrumentation
follows: 0 min, 70ꢀ A, 30ꢀ B; 6.0 min, 70ꢀ A, 30ꢀ B;
All experiments were performed using a Thermo Scientific 36.0 min, 20ꢀ A, 80ꢀ B; 36.1 min, 5ꢀ A, 95ꢀ B; 45.0 min, 5ꢀ
linear quadrupole ion trap (LQIT) mass spectrometer coupled A, 95ꢀ B; 45.1 min, 70ꢀ A, 30ꢀ B; 50.0 min, 70ꢀ A, 30ꢀ
with a high-resolution orbitrap mass analyzer. The instru- B. The column temperature was kept at 40 °C and the sample
ment was equipped with an electrospray ionization (ESI) tray was refrigerated at 4 °C. 400 µL of a wash solvent of 50 : 50
source that was operated in the negative ion mode. The LTQ (v/v) water : methanol was injected between each sample injec-
Tune Plus interface and Xcalibur 2.1 software were used to tion to prevent sample carryover between injections. After
operate the instrument. The linear quadrupole ion trap was HPLC separation, the analytes were ionized by using (−)ESI
filled with 2 mTorr of helium. Nominal pressure of approxi- under the following conditions: N sheath gas flow rate of 60
2
mately 1.5 × 10− Torr was maintained in the Orbitrap (arbitrary units), N
11
auxiliary gas flow rate of 20 (arbitrary
2
vacuum manifold, as read by a cold ion gauge (IKR 270, units), spray voltage of 3.5 kV, ion transfer capillary tempera-
Pfeiffer Vacuum, Asslar, Germany). A resolution of 60 000 ture of 275 °C, and capillary voltage of −10 V. The data-depen-
(
FWHM at m/z 200) was used for all high-resolution measure- dent scan functionality of the Xcalibur software was used to
n
ments. For (−)ESI HRMS analysis, a solution of the organo- perform the CAD MS analysis. The most abundant ions were
solv poplar lignin sample was prepared at a concentration of isolated and subjected to CAD. Their most abundant fragment
1
2
mg mL− in acetonitrile and water (1 : 1, v/v, 1 mL). The ions were then isolated and subjected to CAD. This process
4
solution was introduced into the mass spectrometer via a was repeated until MS stage of ion isolation/fragmentation
−
1
syringe drive at a flow rate of 10 µL min . To facilitate the was reached. All the analyte and fragment ions were analyzed
formation of a stable ESI spray, the sample solution was com- by using the high-resolution orbitrap mass analyzer.
bined with acetonitrile and water (50/50 (v/v)) eluting from a The reproducibility of the relative abundances of the ions
Finnigan Surveyor MS Pump Plus via a tee connector at a flow was ±15ꢀ.
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distinguish the degree of polymerization of the analytes in the
organosolv lignin sample. As seen in a high-resolution mass
Results and discussion
General compositional information was obtained for the orga- spectrum (Fig. 2), lignin monomers, dimers, trimers, tetramers
n
nosolv lignin sample by using (−)ESI HRMS. HPLC/(−)ESI MS
and some bigger oligomers were observed, with monomers
experiments based on CAD were carried out to provide struc- and dimers being most abundant.
tural information for the individual unknown compounds in
In addition to lignin-related compounds, carbohydrates,
the sample. In order to better understand the dissociation be- lipids, and other not lignin-related compounds may also exist
2
3
havior of deprotonated lignin degradation products, CAD of 16 in organosolv lignin samples. To better visualize the distri-
deprotonated lignin-related model compounds was examined.
bution of the different types of compounds in the sample, a
bubble chart (Fig. 3) was constructed that shows the relation-
ships of the m/z value and RDBE value for the ionized analytes.
The size of each bubble represents the relative abundance of
High-resolution MS analysis of the organosolv poplar lignin
sample
Accurate mass measurements (enabled by high resolution)
revealed the elemental compositions of the ionized analytes
and their degree of unsaturation and/or the number of rings
in them (ring and double bond equivalent (RDBE) = a + 1 − b/2
for compounds not containing nitrogen or halogen atoms,
where a = the total number of carbon atoms and b = the total
number of hydrogen atoms). Lignin-related compounds
contain almost exclusively C, H, and O atoms. Based on the
RDBE values, lignin-related ions can be classified into several
subcategories. Deprotonated lignin monomers have an RDBE
2
3
value of at least 4.5. This is due to the fact that they contain
one benzene ring with an RDBE value of 4. Deprotonation of
these compounds increases the RDBE by 0.5. Also, any double
or triple bonds in the side chains will increase the RDBE
values of deprotonated analytes by one or two, respectively.
Therefore, the RDBE values of deprotonated dimers (contain-
2
3
ing two aromatic rings) are equal to or greater than 8.5.
Fig. 3 Chemical composition of the organosolv poplar lignin sample
based on (−)ESI HRMS analysis. Lignin-related compounds and not lignin-
related compounds (most likely fatty acids) are shown inside green and
Similarly, the RDBE values of deprotonated trimers and tetra-
mers are at least 12.5 and 16.5, respectively. The m/z values of
deprotonated lignin-related compounds correlate with their red circles, respectively. For lignin-related compounds, green lines were
RDBE values. Therefore, the RDBE and m/z values were used to drawn to divide them into different categories based on their RDBE.
Fig. 2 High-resolution mass spectrum measured for the organosolv poplar sample by using (−)ESI HRMS. The approximate distributions of lignin-
related monomeric, dimeric, trimeric, and tetrameric as well as bigger compounds are shown.
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the ionized analyte (Fig. 3). The majority of the analyte ions Structural elucidation of unknown lignin degradation products
are positioned closely together to form a big “continent” of requires a fundamental understanding of how deprotonated
lignin-related ions (circled in green) while a few analyte ions lignin-related compounds fragment upon CAD. Previous
that are not related to lignin are visible as small “offshore studies elucidated the dissociation behavior of lignin model
3
7–39
38
39
islands” (circled in red). Determination of the elemental com- compounds with β-O-4,
β-5,
and/or 5–5 linkages.
positions of the analyte ions revealed that the C/O ratio of the Dissociation studies on simple lignin model compounds,
lignin-related compounds in the “continent” was 1.5–4.5. however, have only included compounds with aldehyde, car-
Other, not lignin-related compounds, such as lipids, fatty boxylic acid, and ester functionalities and a few compounds
2
3,36
acids and their esters, often have a low degree of unsaturation with two functionalities in the side chain.
Additional
(RDBE < 4.5) due to the lack of benzene rings. As can be seen deprotonated lignin model compounds, such as compounds
in Fig. 3, most of the analytes within the three red circles have with a hydroxy- or keto-functionality, as well as catechols,
RDBE values of less than 4.5, indicating that these analytes are were studied here to be able to more accurately interpret
not related to linin. Only a few compounds are located at the the fragmentation behavior observed for deprotonated
regions where the RDBE value is greater than 4.5. Ions of m/z unknown lignin-related compounds. Therefore, 16 deproto-
4
45 5
73 have the chemical composition C29H O and a C/O ratio nated lignin-related model compounds, including catechols
of 5.8, which is much greater than that of lignin-related com- and compounds with hydroxy and keto functionalities, were
pounds (C/O ratio 1.5–4.5). The large C/O ratio and the examined.
n
observed losses of water and CO
2
upon CAD of these ions
Model compound studies. HRMS experiments based on
suggest that they are likely to correspond to a fatty acid with a CAD were carried out for all of the ionized model compounds
high degree of unsaturation. On the other hand, the C/O ratio (Fig. 4), their fragment ions, their fragment ions, and so on
2
3
5
of carbohydrates is generally ∼1. Potential oxidation reac- until no further fragmentation was observed (up to MS ). The
tions at the hydroxyl groups of the carbohydrates will increase results are shown in Table S1† and are summarized below. The
2
3
the RDBE values but the C/O ratio will stay relatively MS and MS spectra of deprotonated ethyl 3-(4-hydroxy-3-
unaffected. Ions of m/z 667 have the chemical composition of methoxyphenyl)-3-oxopropanoate are shown as an example
C
23
H
23
O
23 and a C/O ratio of 1, indicating that they are ionized (Fig. 5).
carbohydrates. Thus, the C/O ratios in addition to the RDBE Deprotonated lignin model compounds with methoxy
values can be used to categorize the analytes into different groups readily lose methyl radicals and this is often the first
compound types (Fig. 3).
observed fragmentation event. For deprotonated lignin-related
compounds with several methoxy groups, losses of several
methyl radicals were often observed upon the initial consecu-
n
HPLC/HRMS analysis of the organosolv poplar lignin sample
To structurally characterize individual unknown compounds tive fragmentation events (Table S1†). Upon further CAD, elim-
n
in the organosolv lignin sample, an HPLC/(−)ESI HRMS
ination of one or more CO molecules were often observed.
3
6
2
method based on CAD of isolated analyte ions was employed. Occasionally, CO was lost after the loss of methyl radical(s).
Fig. 4 Model compounds studied.
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2
3
Fig. 5 CAD MS (top) and MS (bottom) spectra of deprotonated ethyl 3-(4-hydroxy-3-methoxyphenyl)-3-oxopropanoate at a normalized collision
energy of 35.
The fragmentations of deprotonated lignin model compounds Deprotonated lignin model compounds containing a car-
are discussed in detail below.
boxylic acid functionality have been reported to readily lose
3
6
2
Hydroxyl. The deprotonated lignin model compounds con- CO upon CAD. Indeed, all the deprotonated carboxylic acids
taining an aliphatic hydroxyl group predominantly lost water studied here, namely, 3,4-dihydroxyphenylacetic acid,
upon CAD, as is evident from the data measured for deproto- 4-hydroxy-3-methoxymandelic acid, 4-formylcinnamic acid,
nated coniferyl alcohol and 2-(4-hydroxyphenyl)ethanol and 4-hydroxyphenyl-pyruvic acid, showed the loss of CO
2
2
(Table S1†). Formaldehyde loss is also possible if a primary when subjected to CAD in the MS experiments. Therefore, the
2
hydroxy group is attached to an alkyl chain, which is evident loss of CO may indicate the presence of a carboxylic acid func-
from the data measured for deprotonated 2-(4-hydroxyphenyl) tionality in an unknown deprotonated compound. This loss
2
ethanol (Table S1†).
was usually observed in the MS experiments but sometimes
Aldehyde. According to a previous study, deprotonated other fragmentations were more competitive. For example,
lignin-related aldehydes lack specific fragmentation patterns deprotonated 2-ethoxy-3-(4-hydroxyphenyl)propionic acid and
3
6
unique to the aldehyde functionality. Some of the deproto- p-hydroxyphenyllactic acid both showed the loss of CO in the
2
3
2
36
nated model compounds studied here, namely, deprotonated MS experiments rather than in the MS experiments.
coniferyl aldehyde and 5,5′-bisvanillin, showed elimination of Catechol. Upon CAD, competitive losses of CO and H
an HCO radical (Table S1†), which likely involves the aldehyde deprotonated 3,4-dihydroxyphenylacetic acid were observed
2
O from
•
3
6
functionality.
However, based on previously published after the initial loss of the carboxylic acid moiety as CO
2
studies, many other deprotonated aldehydes, such as vanillin, (Table S1†). Deprotonated catechol also showed competitive
•
syringaldehyde, and sinapaldehyde, do not lose HCO upon losses of CO and H O upon CAD (Table S1†). Competition of
2
3
6
CAD. Therefore, deprotonated aldehydes are not straight- these two pathways may indicate the presence of a catechol
forward to identify as they fragment similarly to many other moiety in an unknown ion.
types of deprotonated compounds, such as ketones.
Keto. Deprotonated lignin model compounds with a keto
Carboxylic acid. Deprotonation of lignin-related compounds functionality can fragment via cleavages of bonds between the
with a carboxylic acid functionality in the alkyl side chain can carbonyl functionality and the two α-carbons (Fig. 6A and
occur at either the phenol or the carboxylic acid functionality, Table S1†). These fragmentations may be indicative of the
4
4–46
depending on the ESI solvents used.
Based on literature, presence of a keto functionality in an unknown ion. However,
the phenol is more likely to be deprotonated upon ESI when other dissociation pathways, such as methyl radical losses
4
4–46
the aprotic solvent acetonitrile is used.
Therefore, all from the methoxy groups, can be more competitive than these
deprotonated model compounds studied here are shown to be dissociation pathways. Quantum chemical calculations at the
deprotonated at the phenol site even though deprotonation at M06-2X/6-311++G(d,p) level of theory on deprotonated acetova-
the carboxylic acid moiety may also occur for some of them. nillone indicate that the loss of a methyl radical from the
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Fig. 6 (A) Pathways for the loss of small neutral molecules from deprotonated lignin model compounds with a keto functionality. (B) Calculated
Gibbs free energy surfaces for the loss of a methyl radical from the methoxy group and from the keto-functionality in deprotonated acetovanillone
at the M06-2X/6-311++G(d,p) level of theory. The free energies are shown in kcal mol−
1
.
methoxy group adjacent to the deprotonated site is energeti- deprotonated β-keto esters, the methyl 3-(4-hydroxyphenyl)-3-
cally more favorable than from the carbonyl functionality, as oxopropanoate and ethyl 3-(4-hydroxy-3-methoxyphenyl)-3-oxo-
expected (Fig. 6). Therefore, other dissociation pathways may propanoate, readily lose their alcohol moiety upon CAD. After
render fragmentations at the keto functionalities uncompeti- this, a CO loss was observed for both deprotonated β-keto
2
tive. Due to the existence of competitive dissociation pathways, esters upon CAD (Fig. 7 and Table S1†), which must involve a
deprotonated lignin model compounds with a keto functional- rearrangement. Similarly, deprotonated vanillin has been
•
ity may not be straightforward to identify.
reported to eliminate CO
2
after elimination of CH
3
, and based
Model compounds with two functionalities in the side chain. on quantum chemical calculations, a mechanism was deli-
4
7
The fragmentation patterns of deprotonated model com- neated for this complex rearrangement.
pounds with two functional groups in the side chain largely Dimeric lignin model compounds with an aldehyde functional-
depend on the type of the functional groups. The aliphatic car- ity and a 5–5 linkage. As mentioned above, the 5–5 linkage is
boxylic acid functionality is often lost in the first CAD event as quite recalcitrant and can stay intact after lignin degradation
3
9,48
CO
acid, 4-hydroxy-3-methoxymandelic acid, and 4-formylcin- break upon CAD of deprotonated lignin model compounds.
namic acid (Fig. 7 and Table S1†). CO2 can also be lost To better understand the fragmentations of simple deproto-
2
, as observed for deprotonated 4-hydroxyphenylpyruvic via an organosolv process.
Similarly, this linkage does not
3
9
•
together with another small molecule, such as CO or CH
is evident for deprotonated 4-hydroxyphenylpyruvic acid and lignin dimer with no functionalities in the side chains) and
-hydroxy-3-methoxymandelic acid, respectively. After the loss 5,5′-bisvanillin (G lignin dimer with aldehyde functionalities
of CO from deprotonated 4-formylcinnamic acid, CO can be in the side chains) were studied. Both ions lost water upon the
3
, as nated 5–5 type lignin model compounds, 2,2′-biphenol (H
4
2
lost from the aldehyde functionality. On the other hand, first CAD experiment (Table S1†). Due to the existence of two
Fig. 7 Pathways for the loss of small molecules upon CAD of deprotonated model compounds with two functionalities in the side chain.
990 | Green Chem., 2021, 23, 983–1000
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aldehyde functionalities and two methoxy groups in deproto- kages, which have a low abundance in plants, were not specifi-
•
•
nated 5,5′-bisvanillin, losses of CH , CO, and CHO, amongst cally examined. However, in cases for which the β-O-4, β-5, and
3
others, were also observed (Table S1†). Thus, no losses of 5–5 linkages could be ruled out, other linkages, such as 4-O-5
neutral molecules bigger than a monomer were observed. The or β-1, are likely present and were considered during structural
exclusive observance of small molecule losses for deprotonated elucidation.
3
9
lignin dimers is indicative of the presence of a 5–5 linkage.
When elucidating the structures of the lignin-related degra-
Organosolv lignin sample. The organosolv lignin sample dation products, the number of consecutive carbons in the
was subjected to HPLC (for details of the chromatographic side-chains was assumed not to exceed three because lignin is
method, see the Experimental section and ESI†) and the formed upon polymerization of the phenylpropanoid mono-
eluting compounds were ionized by (−)ESI in the linear quad- lignols that contain three consecutive carbons in their side
rupole ion trap mass spectrometer. The deprotonated com- chains, and polymerization via the formation of linkages such
pounds were transferred into the ion trap, isolated, and sub- as β-O-4, β-5, 5–5, and others does not increase the number of
n
jected to CAD in HRMS experiments to obtain structural the consecutive carbons in the side chains.
information. The knowledge obtained from the literature and
The m/z values and possible structures of some of the
in the model compound studies discussed above, combined deprotonated analytes are provided in the total ion current
n
with the CAD HRMS spectra measured for the deprotonated (TIC) chromatogram shown in Fig. 8. The CAD results, HPLC
unknown compounds, were used to elucidate the likely struc- retention times and the proposed structures of some analytes
tures for the major unknown compounds in the organosolv are listed in Table 1. All the remaining information is provided
2
3,36,38,49–51
sample.
in Table S2.† Structures were proposed for 62 compounds that
Structures were proposed for most of the unknown analyte generated adequate fragmentations upon CAD for structural
ions detected based on the retention times of the corres- elucidation. The structural elucidation of some representative
ponding neutral analytes, fragmentation patterns of the compounds in different types of analyte classes is discussed
ionized analytes, the elemental compositions of the ionized below.
analytes and their fragment ions, and their RDBE values. The
previously published fragmentation patterns diagnostic for the The unknown analyte ions of m/z 193 (C10
β-O-4, β-5, and 5–5 linkages were used to identify linkages in Table 1) are used here as an example. The RDBE value of 6.5
Structural elucidation of a monomeric lignin-related compound.
9 4
H O
, RDBE = 6.5,
37–39
the unknown ions.
Deprotonated lignin model com- indicates that these ions contain only one benzene ring
pounds with other linkage types, such as 4-O-5 and β-1 lin- (RDBE = 4) and possibly two double bonds (RDBE = 1 for
n
Fig. 8 HPLC total ion chromatogram obtained for the organosolv poplar sample analyzed using the HPLC/(−)ESI HRMS method. The m/z values
and proposed structures are shown for some of the major ionized analytes. One of the several possible isomeric structures is shown for the ions of
m/z 271 that likely correspond to an ionized fatty acid.
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Table 1 HPLC retention times (R.T.) of the compounds detected in the organosolv lignin sample, m/z values of the deprotonated compounds, their
a
2
4
b
measured elemental compositions, their ionic and neutral fragments formed upon CAD in MS to MS experiments and their relative abundances,
and the structures proposed for each analyte
R.T.
Elemental
Proposed structure
for the analyte
2
3
4
(min) m/z composition RDBE MS
MS
MS
Monomers
c
c
11.3
151
C
8
8
H
7
7
O
3
4
5.5
5.5
151-CH
3
(136) 100ꢀ
(152) 100ꢀ
136-CO (108) 30ꢀ
36-CO (92) 100ꢀ
1
2
•
6.6
167
C
H
O
167- CH
3
152-CO (124) 100ꢀ
1
67-CO
2
(123) 10ꢀ
2
152-CO (108) 20ꢀ
c
•
•
9
1
.8
181
C
9
H
9
O
4
5.5
6.5
181- CH
3
(166) 100ꢀ
(178) 100ꢀ
166- CH
3
(151) 100ꢀ
151-CO (123) 100ꢀ
136-CO (108) 100ꢀ
•
3.5
193
C
10
H
9
O
4
193- CH
3
178-C
2
H
2
O (136) 100ꢀ
1
1
1
78-CO (150) 10ꢀ
2 2
78-C H O–CO (108) 5ꢀ
78-2CO (122) 3ꢀ
•
•
13.8
223
C
11
H
11
O
5
6.5
223- CH
3
(208) 100ꢀ
208- CH
3
(193) 100ꢀ
–2CO (137) 70ꢀ 193-2CO (137) 70ꢀ
193-CO (165) 100ꢀ
•
2
08- CH
3
1
2
1
1
1
1
1
1
2 2
93-CO–C H O (123)
0ꢀ
93-3CO (109) 20ꢀ
2
93-CO–CO (121)
5ꢀ
2
93-CO (149) 10ꢀ
93-2CO–C H O (95)
2 2
0ꢀ
Dimers
•
•
25.5
299
301
C
17
16
H
15
13
O
5
6
10.5
10.5
299 CH
3
(284) 100ꢀ
284- CH
3
(269) 100ꢀ
(225) 10ꢀ
269-CO (241) 100ꢀ
269-H O (251) 15ꢀ
69-CO (225) 10ꢀ
69-CO–CO (197)
2
0ꢀ
2
84-CH
3
–CO
2
2
2
2
1
2
c
•
16.8
C
H
O
301- CH
3
(286) 100ꢀ
–CO (258) 50ꢀ
286-H
2
O (268) 100ꢀ
268-CO (240) 100ꢀ
•
•
•
3
01- CH
3
286- CHO (257) 90ꢀ
268- CHO (239) 20ꢀ
2
2
2
2
3
2
2
86-H
2
O–CO (240) 60ꢀ
268-2CO (212) 10ꢀ
•
86- OH (269) 50ꢀ
•
86- OH–H
2
(267) 45ꢀ
O– CHO (239)
•
86-H
5ꢀ
2
•
86-CO– CHO (229) 20ꢀ
•
86– CH
3
(271) 15ꢀ
•
•
18.3
401
C
21
H
21
O
8
11.5
401-H
2
H
O (383) 100ꢀ
(235) 50ꢀ
(386) 20ꢀ
O (371) 20ꢀ
383-CH
383- CH
3
O (352) 100ꢀ
3
352- CH
3
(337) 100ꢀ
•
4
4
4
01-C
01- CH
01-CH
9
10
O
3
(368) 85ꢀ
O (353) 60ꢀ
O–CO
•
3
383-CH
383-CH
2
2
2
2
(309)
2
0ꢀ
•
4
4
1
4
4
1
01-C
01-C
5ꢀ
2
H
H
4
3
383- CH
3
–H O (338) 10ꢀ
2
•
9
01-C
01-C
0ꢀ
9
H
H
10
9
10
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Table 1 (Contd.)
R.T.
Elemental
Proposed structure
for the analyte
2
3
4
(min) m/z composition RDBE MS
MS
MS
•
•
14.7
405
C
20
H
21
O
9
10.5
405-C11
H
10
12
O
5
(181) 100ꢀ
(223) 60ꢀ
– CH
3
181- CH
3
(166) 100ꢀ
3
166- CH (151) 100ꢀ
4
05-C
05-C11
9
H
H
O
4
•
4
12
O
5
(
4
(
166) 30ꢀ
05-C11
151) 20ꢀ
•
H
12
O
5
–2 CH
3
4
4
05-CO (377) 10ꢀ
•
05-C
9
H
10
O
4
– CH
3
(208) 10ꢀ
Trimers
•
•
21
497
641
C
26
H
25
O
10
14.5
15.5
497-C11
H
14
O (467) 20ꢀ
12
12
O
O
5
(273) 100ꢀ
(223) 65ꢀ
273- CH
3
(258) 100ꢀ
258- CH
3
(243) 100ꢀ
4
4
4
97-C15
97-CH
97-C11
H
2
H
5
•
O
5
– CH
3
(
4
(
258) 20ꢀ
97-C15
208) 10ꢀ
•
H
14
O
5
– CH
3
19.1
C
33
H
37
O
13
641-CH
2
O (611) 100ꢀ
611-CH
2
O (581) 100ꢀ
(223) 90ꢀ
O (593) 60ꢀ
581-C11
00ꢀ
581-CH
0ꢀ
581-C
40ꢀ
H
14
O
4
(371)
OH (549)
(427)
1
641-C22H
26
O
8
(223) 80ꢀ
641-C22
H
26
O
8
3
7
6
6
6
41-H
41-C11
41-H O–CH
2
O (623) 60ꢀ
H
611-H
611-CH
611-CH
2
8
H
O
10 3
12
O
5
(417) 50ꢀ
O (593) 30ꢀ
3
OH (579) 30ꢀ
O-CH OH (549)
2
2
2
3
2
0ꢀ
OH (579) 30ꢀ 611-C12
6
6
41-CH
41-C11
2
O–CH
3
H
H
16
O
O
5
(371) 20ꢀ
(347) 15ꢀ
H O
12 5
2
–CH O
611-C14
16
5
(
6
6
387) 15ꢀ
41-C14
H
H
16
O
O
5
(377) 15ꢀ
–CH
611-C10
611-C H O (427) 10ꢀ
9 12 4
H
14
O
4
(401) 12ꢀ
47-C14
16
5
2
O
(
6
347) 15ꢀ
41-2CH O (581) 10ꢀ
2
Tetramers
2.2 865
2
C
44
H
49
O
18
20.5
865-C11
H
12
O
5
(641) 100ꢀ
O (835) 50ꢀ
O (847) 45ꢀ
65-2H O (829) 10ꢀ
65-H O–CH O (817) 10ꢀ
65-C (803) 10ꢀ
65-C11
641-CH
641-C11
641-C22
2
O (611) 100ꢀ
8
8
8
8
8
8
65-CH
2
H
H
12
O
O
5
(417) 85ꢀ
(223) 80ꢀ
65-H
2
26
8
2
2
641-H O (623) 65ꢀ
2
2
2
H
6
O
2
H
12
O
5
O
5
O
5
–CH
–C11
–C11
2
O
(
8
(
8
611) 10ꢀ
65-C11
415) 10ꢀ
65-C11
417) 5ꢀ
H
12
H
14
O
5
5
H
12
12
H O
(
Fatty acid
26
271
C
15
H
27
O
4
2.5
271-H
2
O–CO
2
(209) 100ꢀ
One possible structure:
2
271-H O (253) 40ꢀ
a
The elemental compositions of the fragment ions were measured and the elemental compositions of the neutral molecules lost were derived
b
from the measured elemental compositions of the fragmenting ions and the fragment ions. The reproducibility of the relative abundances of
the fragment ions was ±15ꢀ. The fragment ions are shown in the order of their relative abundances. The information in the MS to MS columns
2
4
is shown in the following order: precursor ion m/z, elemental composition of the neutral fragment lost upon CAD, m/z of the product ion (in par-
c
enthesis), and the relative abundance of the fragment ion. Verified by comparing with authentic model compounds (Tables 1 and S1†) or pre-
3
6
viously published data on authentic model compounds.
•
3
each). Upon CAD of these analyte ions, loss of CH
3
was CO were observed in the MS experiment. The eliminated CO
2
observed in the MS experiment, suggesting that a methoxy is likely to originate from the aromatic ring, just as in the case
group bound to the benzene ring is present. The methoxy of deprotonated vanillin after elimination of CH . The elimi-
•
47
3
2
group is most likely located at C3 because most lignins are nated H CvCvO suggests the presence of a keto functionality
composed of the three basic monolignols (Fig. 1). After the in the side chain. Consideration of all this information leads
•
loss of CH , losses of H CvCvO, CO, 2CO, or H CvCvO + to the conclusion that the unknown analyte ions of m/z 193
3
2
2
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likely correspond to deprotonated 3-(4-hydroxy-3-methoxyphe- pathway involving the elimination of the charged end unit A
nyl)-3-oxopropanal. Also, cleavages of the two α-carbons of the (Scheme 2). On the other hand, upon CAD, fragment ions of
keto group results in the elimination of H
CvCvO + CO to form product ions of m/z 136 and 108, of the charge-remote unit B (Scheme 2). The observation of
respectively. these characteristic fragmentations associated with deproto-
Structural elucidation of dimeric lignin-related compounds. The nated compounds with a β-O-4 linkage supports the proposed
unknown analyte ions of m/z 405 (C20 , RDBE = 10.5, structure for the unknown analyte ions of m/z 405. Ions of m/z
Table 1) are used here as an example of a deprotonated β-O-4 181 were likely formed via a previously proposed cyclopropane
2
CvCvO and m/z 223 were generated via a pathway involving the elimination
H
2
21 9
H O
3
7
dimer. When subjected to CAD, these ions generated the most mechanism, which requires that the carbonyl is not at the
abundant fragment ions of m/z 181 via the loss of a neutral Cα-position. Otherwise, the elimination of the charged end
molecule of MW of 224 Da and the second most abundant unit A to form ions of m/z 181 cannot occur. Therefore, the
fragment ions of m/z 223 via the loss of a neutral molecule of hydroxyl group was assigned to the Cα-position and the carbo-
MW 182 Da. This fragmentation pattern matches that shown nyl group to the Cβ-position.
in Scheme 1A for deprotonated lignin model compounds with
The unknown analyte ions of m/z 401 (C H O , RDBE =
21 21 8
a β-O-4 linkage. If the unknown analyte ions of m/z 405 con- 11.5, Table 1) are used here as an example of a deprotonated
tained a β-O-4 linkage, a major fragment ion A (ions of m/z β-5 dimer. Based on a previous publication, deprotonated β-5
1
81) could be formed via the elimination of a neutral molecule dimeric model compounds fragment by elimination of the
B (MW 224 Da) and another major fragment ion B (ions of m/z charged end unit as a quinone methide-like neutral molecule
3
8
2
23) could be formed via the elimination of a neutral molecule (Q, Scheme 1B). This neutral molecule would have the
A (MW 182 Da). These findings indicate that the ions of m/z formula C H O, C H O , or C H O for the H-, G-, and
7
6
8
8
2
9
10 3
3
8
4
05 contain a β-O-4 linkage. When the most abundant frag- S-units, respectively (Scheme 1B). Upon CAD of the unknown
ment ions of m/z 181 were isolated and subjected to CAD in an analyte ions of m/z 401, ions of m/z 235 were formed via the
3
•
MS experiment, the loss of CH was observed to generate loss of a neutral molecule Q with the formula C H O , which
3
9
10 3
•
4
ions of m/z 166. These ions lost another CH
ment. The elemental composition (C , RDBE = 5.5) and and an S-type monomeric unit. After further examination of
fragmentation behavior of the ions of m/z 181 (Table 1) suggest the fragmentation of the ions of m/z 401 (Table 1), the ions of
that they correspond to deprotonated syringaldehyde. Based m/z 401 were concluded not to contain a β-O-4 linkage because
on the elemental composition of the unknown dimeric ions of the characteristic fragmentations shown in Scheme 1A for
m/z 405, their fragment ions, their fragmentation pattern and β-O-4 type compounds were not observed.
3
in an MS experi- suggests that the ions of m/z 401 may contain a β-5 linkage
9
9 4
H O
3
6
the finding that the unknown ions contain a β-O-4 linkage, the
Therefore, the ions of m/z 401 were concluded to contain a
structure shown in Scheme 2 was proposed for the unknown β-5 linkage. Fragment ions of m/z 235 fragmented by losing
•
2
ions of m/z 405. Based on the proposed structure, deproto-
CHO to form ions of m/z 206 in an MS experiment, indicating
nated syringaldehyde of m/z 181 was formed upon CAD via a that a formaldehyde functionality is present in the ions of m/z
•
2
3
35. Ions of m/z 235 also fragmented by losing CH to form
2
ions of m/z 220 in an MS experiment, indicating that a
methoxy group exists in the ions of m/z 235. When the elemen-
tal composition of the unknown ions of m/z 401, their frag-
ment ions, and their fragmentation pattern were considered,
the structure shown in Scheme 3 was proposed for these ions.
According to the mechanism shown in Scheme 1B, the gene-
ration of the ions of m/z 235 via the pathway involving the
Scheme 2 The structure proposed for the analyte ions of m/z 405 and
pathways for the formation of their major fragmentation products.
Scheme 3 Proposed structure of the analyte ions of m/z 401 and the
pathway for the generation of their major fragment ions of m/z 235.
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elimination of the charged end unit A requires the partici- ions of m/z 273 upon CAD, indicating that the ions of m/z 273
pation of the aliphatic hydroxyl group at the γ-position. contain a 5–5 linkage. Based on the elemental composition of
The observation of this fragmentation reveals that an aliphatic the unknown trimeric ions of m/z 497, their fragment ions,
hydroxyl group is located at the γ-position. Apart from the for- their fragmentation pattern and the finding that they contain
maldehyde functionality in the side chain of ring B, infor- a β-O-4 and a 5–5 linkage, the structure shown in Scheme 5
mation on the elemental composition and RDBE value of the was proposed for the unknown ions of m/z 497. Based on the
ions of m/z 401 indicate that another carbonyl group exists in proposed structure, fragment ions of m/z 273 are formed via
the side chain of ring B. The second carbonyl group is likely the elimination of the charged end unit A as a neutral mole-
directly connected to ring B based on the fragmentation pat- cule and ions of m/z 223 are formed via the elimination of the
terns of ions of m/z 401 shown in Table 1.
charge-remote end units B and C (Scheme 5). The formation of
3
7
The unknown analyte ions of m/z 299 (C H O , RDBE = ions of m/z 273 via the cyclopropane mechanism
1
7
15
5
1
0.5, Table 1) are used here as an example of a deproto- (Scheme 1A) requires that a hydroxyl group is located at the
nated 5–5 dimer. These unknown dimeric ions showed the α-position and a carbonyl group is located at the β-position in
•
2
loss of CH upon CAD in an MS experiment, followed by ions of m/z 497. In addition to above structural details, based
3
•
•
3
loss of CH
3
or both CH
3
and CO
2
upon CAD in an MS
on the elemental composition of ions of m/z 273, an aldehyde
functionality should exist in these ions. This functionality
3
experiment. When the fragment ions generated in the MS
experiment upon loss of CH were subjected to CAD, elim- could be attached to the 1-position of either ring B or ring
ination of CO was observed (MS ). On the other hand, the C. The structure shown in Scheme 5 represents one of the two
fragment ions formed upon loss of both CH
•
3
4
•
3
and CO
2
frag- possible structures. Based on the proposed structure, two
4
mented via loss of CO , CO and CO, or H O in an MS
phenol groups are present in the unknown ions of m/z 497 and
2
2
2
experiment (Table 1). No neutral fragments bigger than a either phenol group may get deprotonated upon ionization of
monomer were lost upon CAD, indicating that the ions of the trimer. If the deprotonated phenol group is that attached
•
m/z 299 contain a 5–5 linkage. Only two CH radicals were to the C ring, fragment ions of m/z 223 cannot be formed upon
3
lost throughout the CAD experiments, indicating that two CAD because the charge on the phenol group of ring C cannot
methoxy groups are present in the ions of m/z 299. The frag- migrate to the unit A via the cyclopropane mechanism
3
7
mentations and proposed structure for ions of m/z 299 are (Scheme 1A). On the other hand, if the phenol group on ring
shown in Scheme 4. A is deprotonated, this ion could form both fragment ions of
Structural elucidation of trimeric and tetrameric lignin-related m/z 273 and 223 upon CAD. Hence, the phenol group on ring
compounds. Upon CAD of trimeric unknown analyte ions of A is shown as deprotonated in Scheme 5.
m/z 497 (C26
H
25
O
10, RDBE = 14.5, Table 1), fragment ions of
In contrast to the above example, ionized oligomeric com-
m/z 273 and 223 were formed via the elimination of neutral pounds with no 5–5 linkages can lose more than two mono-
molecules of MW of 224 and 274 Da, respectively. This frag- meric units due to the labile nature of β-O-4, β-5, and 4-O-5 lin-
mentation pattern matches that shown in Scheme 1A for kages. This information was used to identify the unknown
deprotonated lignin model compounds with a β-O-4 linkage. If tetrameric analyte ions of m/z 865 (C44
49
H O18, RDBE = 20.5,
the unknown analyte ions of m/z 497 contained a β-O-4 Table 1). Upon CAD, these ions generated the key fragment
linkage, a fragment ion A (ions of m/z 273) could be formed via ions of m/z 641. Fragment ions of m/z 641 fragmented to form
the elimination of a neutral molecule B (molecule of MW 224
Da) and a second major fragment ion B (ions of m/z 223) could
be formed via the elimination of a neutral molecule A (mole-
cule of MW 274 Da). These findings indicate that the ions of
m/z 497 contain a β-O-4 linkage. The most abundant fragment
•
ions of m/z 273 lost CH
3
to form fragment ions of m/z 258 in
3
an MS experiment. These fragment ions fragmented by elimi-
nating another CH3 in an MS experiment (Table S2†). No
•
4
large neutral molecules were eliminated from the fragment
Scheme 4 Proposed structure of the analyte ions of m/z 299. Only
losses of small molecules were observed for this ion.
Scheme 5 Structure and major fragmentation pathways proposed for
the trimeric analyte ions of m/z 497.
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fragment ions of m/z 417 and 223 via losses of neutral mole- the analyte ions of m/z 865. The fragment ions of m/z 641 can
cules of MW of 224 and 418 Da, respectively. If the unknown eliminate the charge-remote end unit D to form ions of m/z
fragment ions of m/z 641 contained a β-O-4 linkage, a fragment 417. Ions of m/z 641 can also eliminate the charged-end unit B
ion A (ions of m/z 417) could be formed via the elimination of to form fragment ions of m/z 417, which can fragment by elim-
a neutral molecule B (molecule of MW 224 Da) and a second inating the charged end unit C to form fragment ions of m/z
major fragment ion B (ions of m/z 223) could be formed via 223. Alongside the losses of the monomeric or dimeric units,
2 2
the elimination of a neutral molecule A (molecule of MW 418 losses of small molecules, such as H O and CH O, were also
Da). These findings suggest that the fragment ions of m/z 641 observed. The locations of the hydroxyl groups and the carbo-
contain a β-O-4 linkage. After carefully examining the fragmen- nyl groups in the unknown analyte ions of m/z 865 were
tation patterns of ions of m/z 865, all the monomeric units deduced based on the fact that the elimination of the charged
3
7
were concluded to be linked via β-O-4 linkages. Based on the end unit via the cyclopropane mechanism requires that the
elemental composition of the unknown tetrameric ions of m/z α-position of the β-O-4 linkage does not contain a keto func-
8
65, their fragment ions, their fragmentation pattern and the tionality. The number of methoxy groups was determined
finding that they contain three β-O-4 linkages, the structure based on the elemental compositions of the fragment ions. An
shown in Scheme 6 was assigned for the unknown ions of m/z ethyl ether functionality is proposed to be bound to the C unit
8
65. Further explanation for this structural assignment is pro- based on the elemental compositions of the fragment ions.
vided below. The ethyl ether functionality may be a residue formed from a
Based on the proposed structure of the unknown analyte lignin–carbohydrate complex. Based on the structure proposed
ions of m/z 865 and the pathways shown in Scheme 1A, the for the tetrameric analyte ions of m/z 865 (Scheme 6), the
charge-remote end unit D was eliminated to form fragment neutral analyte of MW 866 Da consists of three S-units and
ions of m/z 641 (Scheme 6, pathway 1). The fragment ions of one H-unit, and thus is an S/H type lignin tetramer.
m/z 641 fragment by eliminating the charged end unit A to
All the other structural assignments for lignin-related com-
generate fragment ions of m/z 417, which dissociate to form pounds were made in a similar manner as for the detailed
ions of m/z 223 via elimination of the charge-remote end unit examples discussed above.
C. Fragment ions of m/z 641 can also eliminate the charge-
Structural elucidation of compounds not related to lignin. Apart
remote end units B and C together to generate ions of m/z 223. from lignin-related compounds, seven compounds not related
A second pathway for the generation of fragment ions of m/z to lignin were detected in the sample (Tables 1 and S2†). Six of
6
41 involves the elimination of the charged-end unit A from these compounds have an RDBE value of less than that of a
Scheme 6 Pathways proposed for the production of the major fragment ions of the tetrameric analyte ions of m/z 865.
996 | Green Chem., 2021, 23, 983–1000
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benzene ring (RDBE = 4). These six compounds are likely to be were identified as deprotonated vanillin, vanillic acid, syringal-
fatty acids based on their measured elemental compositions dehyde, and 5,5′-bisvanillin, respectively. The many unknown
and fragmentation patterns (possible structures are shown in analytes, several corresponding to different isomeric struc-
Tables 1 and S2†). However, the seventh deprotonated analyte tures, demonstrate that this organosolv lignin sample is very
ions (of m/z 473) have a greater RDBE value than expected for a complex.
fatty acid and a greater C/O ratio (chemical formula: C32
H
41
O
3
,
In order to perform a preliminary examination of the quan-
RDBE = 12.5, C/O ratio = 10.7, Table S2†) than lignin-related titative aspects of the above analytical method, an equimolar
compounds (C/O ratio in the range of 1.5–4.5). The identity of mixture containing 11 lignin model compounds, including
this analyte ion is still under investigation.
several monomers, three dimers with different types of lin-
An overview of the results. A total of 62 deprotonated com- kages, and a tetramer with only β-O-4 linkages, was studied
pounds generated adequate fragment ions upon CAD for struc- (Fig. S1 and S2†). The majority of the analytes (7 from 11) had
tural elucidation. Among these compounds, 15 are lignin a similar ionization efficiency (Fig. S1 and S2†), suggesting
monomers, 27 are lignin dimers, 12 are lignin trimers, one is a that the relative abundances of their deprotonated forms
lignin tetramer (ion of m/z 857), and seven are not lignin- reflect their relative molar amounts in the organosolv lignin
related compounds, most of them likely fatty acids. Lignin sample. However, isoeugenol, a monomeric lignin model com-
monomers and dimers (most with MW values of less than 550) pound, was not detected, indicating that it was not efficiently
account for the majority of the compounds detected. ionized due to competition from the other analytes in the
Oligomers larger than tetramers had very low abundances and mixture. This may be due to the lack of electron-withdrawing
did not generate enough fragment ions for structural substituents or internal hydrogen-bonding ability for isoeu-
elucidation.
genol. The β-O-4 (S–S) dimer also had a very low ionization
Based on the proposed structures, compounds with various efficiency for unknown reasons. On the other hand, the depro-
functionalities, such as hydroxyl, aldehyde, keto, and car- tonated 5–5 dimer, 2,2′-bisphenol, which bears two free
boxylic acid, were detected (Tables 1 and S2†). Lignin-related phenol groups, had an exceptionally high ionization efficiency,
compounds with two functionalities in the side chains were likely due to its ability to stabilize the negatively charged phe-
also found in this organosolv lignin sample. Monomeric nolic oxygen via internal hydrogen bonding. Similarly, the
lignin-related analyte ions of m/z 151, 167, 193, and one deprotonated β-O-4 (G–G–G–G) tetramer also had a high abun-
isomer in the isomeric group of ions of m/z 209 correspond to dance in the MS spectrum (Fig. S1†), again likely due to its
G-lignin degradation products. Ions of m/z 181 and one isomer ability of forming an internal hydrogen bond to stabilize the
in each of the isomeric groups of ions of m/z 209, 223, 225, negatively charged phenolic oxygen. This result is important
2
27, 253, and 295 correspond to S-lignin degradation products. as it indicates that the (−)ESI HRMS method used does not
Two isomeric ions of m/z 163 and ions of m/z 221 correspond discriminate against the bigger analytes in the mixture.
to H-lignin degradation products. Among the lignin dimers, 14 Last, a limitation of the method should be mentioned. The
contain a 5–5 linkage, eight contain a β-O-4 linkage, four assignment of the likely structures for the compounds in the
contain a β-5 linkage, and one likely contains a 4-O-5 linkage. organosolv lignin sample based on their retention times and
The labile nature of the most common β-O-4 linkage in lignin the elemental compositions and fragmentation patterns of their
likely contributed to the relatively low amount of compounds ions upon CAD is a very time-consuming process and requires a
detected with this linkage compared to the 5–5 linkage. β-O-4, comprehensive understanding of the dissociation mechanisms
5
–5, β-5, and/or possibly 4-O-5 linkages were observed in the of deprotonated lignin-related compounds, which may limit its
lignin trimers and tetramers. Despite the labile nature of the application. It is noteworthy, however, that the methodology
β-O-4 linkage, it is the most abundant linkage type in plants and the data presented here should greatly facilitate the chemi-
and thus some trimers and the detected tetramer contained cal characterization of other lignin degradation mixtures pre-
only β-O-4 linkages. However, the abundances of these oligo- pared by using other methods or prepared using a different
mers were low. No more than two of each of the 5–5, β-5, and type of biomass, as long as they contain similar compounds.
4
-O-5 linkages (Table S2†) were observed in the oligomers in
this study.
Two or more isomeric ions were successfully separated and
Conclusions
characterized for each of the isomeric groups of ions of m/z
1
63, 209, 343, 371, 385, 401, 473, and 609 (Table S2†). An organosolv poplar lignin sample was analyzed using (−)ESI
n
Particularly, four different isomers of ions of m/z 371 and HRMS and HPLC/(−)ESI HRMS based on CAD. After HPLC
three different isomers of ions of m/z 385 (Table S2†) were separation, the analytes in the sample were ionized by (−)ESI.
identified. Seven compounds were detected that were not The elemental compositions as well as the degree of unsatura-
lignin-related compounds. Most of them are likely to be fatty tion (number of double bonds and rings or RDBE) were deter-
acids. By comparing the observed fragmentation patterns with mined for each detected deprotonated analyte, which enabled
those of authentic deprotonated model compounds (Tables S1 the determination of their compound type. Many lignin-
3
6
and S2†) or previously reported fragmentation data,
related compounds, as well as a few compounds not related to
unknown analyte ions of m/z 151, 167 (C H O ), 181, and 301 lignin, were detected, with lignin-related compounds being the
8
7
4
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most abundant. Lignin monomers, dimers, trimers, tetramers,
and bigger oligomers were observed, with lignin monomers
and dimers constituting the majority of the compounds in the
mixture.
7 P. P. Peralta-Yahya, F. Zhang, S. B. del Cardayre and
J. D. Keasling, Microbial engineering for the production of
advanced biofuels, Nature, 2012, 488(7411), 320–328.
8 S. H. Ghaffar and M. Z. Fan, Structural analysis for lignin
characteristics in biomass straw, Biomass Bioenergy, 2013,
57, 264–279.
n
The CAD fragmentation behavior studied using HRMS
experiments on 16 deprotonated lignin-related model com-
pounds provided insights into the fragmentation behavior of
deprotonated unknown lignin-related compounds with various
9 K. Freudenberg, Biosynthesis and constitution of lignin,
Nature, 1959, 183(4669), 1152–1155.
functionalities and linkage types. These results and previous 10 C. Heitner, D. Dimmel and J. Schmidt, Lignin and Lignans:
3
7–39
studies
facilitated the structural elucidation of several
Advances in Chemistry, CRC Press, Boca Raton, FL, 2016.
unknown compounds. Structural information was obtained for 11 N. G. Lewis and L. B. Davin, The biochemical control of
6
2 ionized unknown compounds derived from the organosolv
monolignol coupling and structure during lignan and lignin
biosynthesis, in Lignin and Lignan Biosynthesis, American
Chemical Society, Washington, DC, 1998, vol. 697, pp.
334–361.
n
lignin sample by using HPLC/(−)ESI HRMS experiments.
Among these compounds, 15 were identified as lignin mono-
mers, 27 as lignin dimers, 12 as lignin trimers, one as a tetra-
mer, and seven were concluded not to be related to lignin. G-, 12 D. R. Gang, M. A. Costa, M. Fujita, A. T. Dinkova-Kostova,
H-, and S-lignin monomers were detected in the unknown
analyte ions. β-O-4, 5–5, β-5, and possibly 4-O-5 linkages were
identified to be present.
H. B. Wang, V. Burlat, W. Martin, S. Sarkanen, L. B. Davin
and N. G. Lewis, Regiochemical control of monolignol
radical coupling: a new paradigm for lignin and lignan bio-
synthesis, Chem. Biol., 1999, 6(3), 143–151.
1
3 R. Vanholme, B. Demedts, K. Morreel, J. Ralph and
W. Boerjan, Lignin biosynthesis and structure, Plant
Physiol., 2010, 153(3), 895–905.
Conflicts of interest
There are no conflicts to declare.
1
1
1
4 W. Boerjan, J. Ralph and M. Baucher, Lignin biosynthesis,
Annu. Rev. Plant Biol., 2003, 54, 519–546.
5 J. K. Weng and C. Chapple, The origin and evolution of
lignin biosynthesis, New Phytol., 2010, 187(2), 273–285.
6 N. G. Lewis and E. Yamamoto, Lignin: occurrence, biogen-
esis and biodegradation, Annu. Rev. Plant Biol., 1990,
Acknowledgements
The authors greatly appreciate the funding of this research by
the Center for Direct Catalytic Conversion of Biomass to
Biofuels (C3Bio), an Energy Frontier Research Center funded
by the U.S. Department of Energy, Office of Science, Office of
Basic Energy Sciences, under award number DE-SC0000997.
41(41), 455–496.
1
7 M. Staš, J. Chudoba, D. Kubička and M. Pospíšil, Chemical
characterization of pyrolysis bio-oil: application of orbitrap
mass spectrometry, Energy Fuels, 2015, 29(5), 3233–
3240.
1
8 L. Das, P. Kolar and R. Sharma-Shivappa, Heterogeneous
catalytic oxidation of lignin into value-added chemicals,
Biofuels, 2014, 3(2), 155–166.
References
1
T. Yoshikawa, T. Yagi, S. Shinohara, T. Fukunaga,
Y. Nakasaka, T. Tago and T. Masuda, Production of 19 W. J. J. Huijgen, G. Telysheva, A. Arshanitsa,
phenols from lignin via depolymerization and catalytic
cracking, Fuel Process. Technol., 2013, 108, 69–75.
D. M. Alonso, S. G. Wettstein and J. A. Dumesic, Bimetallic
R. J. A. Gosselink and P. J. de Wild, Characteristics of
wheat straw lignins from ethanol-based organosolv treat-
ment, Ind. Crops Prod., 2014, 59, 85–95.
2
3
4
5
6
catalysts for upgrading of biomass to fuels and chemicals, 20 J. J. Bozell, S. K. Black, M. Myers, D. Cahill, W. P. Miller
Chem. Soc. Rev., 2012, 41(24), 8075–8098.
D. M. Alonso, J. Q. Bond and J. A. Dumesic, Catalytic con-
version of biomass to biofuels, Green Chem., 2010, 12(9),
and S. Park, Solvent fractionation of renewable woody feed-
stocks: Organosolv generation of biorefinery process
streams for the production of biobased chemicals, Biomass
Bioenergy, 2011, 35(10), 4197–4208.
1
493–1513.
D. Mohan, C. U. Pittman and P. H. Steele, Pyrolysis of 21 M. Yáñez-S, B. Matsuhiro, C. Nuñez, S. Pan, C. A. Hubbell,
wood/biomass for bio-oil: a critical review, Energy Fuels,
006, 20(3), 848–889.
G. W. Huber, S. Iborra and A. Corma, Synthesis of transpor-
tation fuels from biomass: chemistry, catalysts, and engin-
eering, Chem. Rev., 2006, 106(9), 4044–4098.
P. Sannigrahi and A. J. Ragauskas, Physicochemical charac-
terization of ethanol organosolv lignin (EOL) from
Eucalyptus globulus: effect of extraction conditions on the
molecular structure, Polym. Degrad. Stab., 2014, 110, 184–
194.
2
J. Zakzeski, P. C. Bruijnincx, A. L. Jongerius and 22 D. Watkins, M. Nuruddin, M. Hosur, A. Tcherbi-Narteh
B. M. Weckhuysen, The catalytic valorization of lignin for
the production of renewable chemicals, Chem. Rev., 2010,
and S. Jeelani, Extraction and characterization of lignin
from different biomass resources, J. Mater. Res. Technol.,
2015, 4(1), 26–32.
1
10(6), 3552–3599.
998 | Green Chem., 2021, 23, 983–1000
This journal is © The Royal Society of Chemistry 2021

View Article Online
Green Chemistry
Paper
2
3 T. M. Jarrell, C. L. Marcum, H. Sheng, B. C. Owen,
C. J. O’Lenick, H. Maraun, J. J. Bozell and H. I. Kenttämaa,
Characterization of organosolv switchgrass lignin by using
high performance liquid chromatography/high resolution
and H. I. Kenttämaa, A fundamental tandem mass spec-
trometry study of the collision-activated dissociation of
small deprotonated molecules related to lignin,
ChemSusChem, 2016, 9(24), 3513–3526.
tandem mass spectrometry using hydroxide-doped nega- 37 J. Zhang, E. Feng, W. Li, H. Sheng, J. R. Milton,
tive-ion mode electrospray ionization, Green Chem., 2014,
6(5), 2713–2727.
4 X. Ouyang, X. Huang, B. M. S. Hendriks, M. D. Boot and
E. J. M. Hensen, Coupling organosolv fractionation and
L. F. Easterling, J. J. Nash and H. I. Kenttämaa, Studies on
the fragmentation mechanisms of deprotonated lignin
model compounds in tandem mass spectrometry, Anal.
Chem., 2020, 92(17), 11895–11903.
1
2
2
2
reductive depolymerization of woody biomass in a two-step 38 K. Morreel, H. Kim, F. C. Lu, O. Dima, T. Akiyama,
catalytic process, Green Chem., 2018, 20(10), 2308–2319.
5 J. Li, G. Henriksson and G. Gellerstedt, Lignin depolymeri-
zation/repolymerization and its critical role for delignifica-
tion of aspen wood by steam explosion, Bioresour. Technol.,
R. Vanholme, C. Niculaes, G. Goeminne, D. Inze,
E. Messens, J. Ralph and W. Boerjan, Mass spectrometry-
based fragmentation as an identification tool in lignomics,
Anal. Chem., 2010, 82(19), 8095–8105.
2
007, 98(16), 3061–3068.
39 H. Sheng, W. Tang, J. Gao, J. S. Riedeman, G. Li,
T. M. Jarrell, M. R. Hurt, L. Yang, P. Murria, X. Ma,
6 Q. Sun, R. Khunsupat, K. Akato, J. Tao, N. Labbé,
N. C. Gallego, J. J. Bozell, T. G. Rials, G. A. Tuskan,
T. J. Tschaplinski, A. K. Naskar, Y. Pu and A. J. Ragauskas,
A study of poplar organosolv lignin after melt rheology
treatment as carbon fiber precursors, Green Chem., 2016,
n
J. J. Nash and H. I. Kenttämaa, (−)ESI/MS procedure for
sequencing lignin oligomers based on a study of synthetic
model compounds with β-O-4 and 5–5 linkages, Anal.
Chem., 2017, 89(24), 13089–13096.
1
8(18), 5015–5024.
40 J. J. Bozell, C. J. O’Lenick and S. Warwick, Biomass fraction-
ation for the biorefinery: heteronuclear multiple quantum
coherence-nuclear magnetic resonance investigation of
lignin isolated from solvent fractionation of switchgrass,
J. Agric. Food Chem., 2011, 59(17), 9232–9242.
2
7 B. C. Owen, L. J. Haupert, T. M. Jarrell, C. L. Marcum,
T. H. Parsell, M. M. Abu-Omar, J. J. Bozell, S. K. Black and
H. I. Kenttämaa, High-performance liquid chromato-
graphy/high-resolution multiple stage tandem mass spec-
trometry using negative-ion-mode hydroxide-doped electro- 41 C. Lapierre, B. Pollet, M. Petit-Conil, G. Toval, J. Romero,
spray ionization for the characterization of lignin degra-
dation products, Anal. Chem., 2012, 84(14), 6000–6007.
8 M. Baucher, C. Halpin, M. Petit-Conil and W. Boerjan,
Lignin: genetic engineering and impact on pulping, Crit.
Rev. Biochem. Mol. Biol., 2003, 38(4), 305–350.
G. Pilate, J.-C. Leplé, W. Boerjan, V. Ferret and V. De Nadai,
Structural alterations of lignins in transgenic poplars with
depressed cinnamyl alcohol dehydrogenase or caffeic acid
O-methyltransferase activity have an opposite impact on
the efficiency of industrial kraft pulping, Plant Physiol.,
1999, 119(1), 153–164.
2
2
9 X. Li, J. K. Weng and C. Chapple, Improvement of biomass
through lignin modification, Plant J., 2008, 54(4), 569–581.
0 B. B. Hallac, Y. Pu and A. J. Ragauskas, Chemical trans-
formations of Buddleja davidii lignin during ethanol orga-
nosolv pretreatment, Energy Fuels, 2010, 24(4), 2723–2732.
1 R. El Hage, N. Brosse, P. Sannigrahi and A. Ragauskas,
42 J. Van Doorsselaere, M. Baucher, E. Chognot, B. Chabbert,
M. T. Tollier, M. Petit-Conil, J. C. Leplé, G. Pilate, D. Cornu
and B. Monties, A novel lignin in poplar trees with a
reduced caffeic acid/5-hydroxyferulic acid O–methyl-
transferase activity, Plant J., 1995, 8(6), 855–864.
3
3
Effects of process severity on the chemical structure of 43 F. Chen, Y. Tobimatsu, D. Havkin-Frenkel, R. A. Dixon and
Miscanthus, ethanol organosolv lignin, Polym. Degrad.
Stab., 2010, 95(6), 997–1003.
J. Ralph, A polymer of caffeyl alcohol in plant seeds, Proc.
Natl. Acad. Sci. U. S. A., 2012, 109(5), 1772–1777.
3
3
3
3
2 M. Nagy, K. David, G. J. P. Britovsek and A. J. Ragauskas, 44 Z. Tian, X. B. Wang, L. S. Wang and S. R. Kass, Are carboxyl
Catalytic hydrogenolysis of ethanol organosolv lignin,
Holzforschung, 2009, 63(5), 513–520.
3 F. Xu, J.-X. Sun, R. Sun, P. Fowler and M. S. Baird,
Comparative study of organosolv lignins from wheat straw,
Ind. Crops Prod., 2006, 23(2), 180–193.
groups the most acidic sites in amino acids? Gas-phase
acidities, photoelectron spectra, and computations on tyro-
sine, p-hydroxybenzoic acid, and their conjugate bases,
J. Am. Chem. Soc., 2008, 131(3), 1174–1181.
45 J. D. Steill and J. Oomens, Gas-phase deprotonation of
p-hydroxybenzoic acid investigated by IR spectroscopy:
solution-phase structure is retained upon ESI, J. Am. Chem.
Soc., 2009, 131(38), 13570–13571.
4 J. Ralph and R. D. Hatfield, Pyrolysis-GC-MS characteriz-
ation of rorage materials, J. Agric. Food Chem., 1991, 39,
1
426–1437.
5 G. van Erven, R. de Visser, D. W. H. Merkx, W. Strolenberg, 46 D. Schroder, M. Budesinsky and J. Roithova, Deprotonation
P. de Gijsel, H. Gruppen and M. A. Kabel, Quantification of
lignin and its structural features in plant biomass using
C lignin as internal standard for pyrolysis-GC-SIM-MS,
of p-hydroxybenzoic acid: does electrospray ionization
sample solution or gas-phase structures?, J. Am. Chem. Soc.,
2012, 134(38), 15897–15905.
1
3
Anal. Chem., 2017, 89(20), 10907–10916.
47 C. L. Marcum, J. S. Riedeman, T. M. Jarrell, J. J. Nash and
3
6 C. L. Marcum, T. M. Jarrell, H. Zhu, B. C. Owen,
L. J. Haupert, M. Easton, O. Hosseinaei, J. Bozell, J. J. Nash
2
H. I. Kenttämaa, Losses of CO and CO upon collision-acti-
vated dissociation of substituted 2-methoxyphenoxides
This journal is © The Royal Society of Chemistry 2021
Green Chem., 2021, 23, 983–1000 | 999

View Article Online
Paper
Green Chemistry
after methyl radical loss, Int. J. Mass Spectrom., 2020, 456,
16397–116402.
structural elucidation and characterization of synthetic and
natural products, Nat. Prod. Rep., 2016, 33(3), 432–455.
1
4
4
8 B. M. Upton and A. M. Kasko, Strategies for the conversion 50 S. Li and K. Lundquist, Acid reactions of lignin models of
of lignin to high-value polymeric materials: review and per-
spective, Chem. Rev., 2016, 116(4), 2275–2306.
β-5 type, Holzforschung, 1999, 53, 39–42.
51 K. Morreel, O. Dima, H. Kim, F. Lu, C. Niculaes,
R. Vanholme, R. Dauwe, G. Goeminne, D. Inze, E. Messens,
J. Ralph and W. Boerjan, Mass spectrometry-based sequencing
of lignin oligomers, Plant Physiol., 2010, 153(4), 1464–1478.
9 D. P. Demarque, A. E. Crotti, R. Vessecchi, J. L. Lopes and
N. P. Lopes, Fragmentation reactions using electrospray
ionization mass spectrometry: an important tool for the
1000 | Green Chem., 2021, 23, 983–1000
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