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 LIGNIN IN POLYMER
COMPOSITES

Omar Faruk
Mohini Sain

Amsterdam • Boston • Heidelberg • London • New York • Oxford

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William Andrew is an imprint of Elsevier
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understanding, changes in research methods, professional practices, or medical treatment may become necessary.
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 Dedicated
to
My Beloved Wife
“Shaila Shumi”
and
My Beloved Daughter
“Ornela Suhiya”

Omar Faruk, Canada


Umesh P. Agarwal USDA Forest Service, Forest
Products Laboratory, One Gifford Pinchot Drive,
Madison, WI, USA
Abdullah Al Mamun Institute for Materials
Engineering, Polymer Engineering, University of
Kassel, Kassel, Germany
Priyanka Bhattacharya Proton Power, Inc., Lenoir
City, TN, USA
Sabornie Chatterjee Chemical Sciences Division,
Oak Ridge National Laboratory, Oak Ridge,
TN, USA
Hoyong Chung Department of Chemical and
Biomedical Engineering, Florida State University,
FAMU-FSU College of Engineering, Tallahassee,
FL, USA
Norbert Eisenreich Fraunhofer-ICT, Germany
Gunnar Engelmann Fraunhofer Institute for
Applied Polymer Research IAP, Potsdam-Golm,
Germany
Omar Faruk Centre for Biocomposites and
Biomaterials Processing, Faculty of Forestry,
University of Toronto, Toronto, ON, Canada
Maik Feldmann Institute for Materials
Engineering, Polymer Engineering, University of
Kassel, Kassel, Germany
Johannes Ganster Fraunhofer Institute for Applied
Polymer Research IAP, Potsdam-Golm, Germany
Azadeh Goudarzi Department of Materials
Engineering, The University of British Columbia,
Vancouver, BC, Canada
Shayesteh Haghdan Department of Wood Science,
Forest Sciences Centre, The University of British
Columbia, Vancouver, BC, Canada
Hans-Peter Heim Institute for Materials
Engineering, Polymer Engineering, University of
Kassel, Kassel, Germany
Contributors
Emilia Regina Inone-Kauffmann Fraunhofer-ICT,
Germany
John F. Kadla Department of Forest Biomaterials,
North Carolina State University, Raleigh, NC, USA
Adel R. Kakroodi Centre for Biocomposites and
Biomaterials Processing, Faculty of Forestry,
University of Toronto, Toronto, ON, Canada
Muzaffer A. Karaaslan Department of Materials
Engineering, The University of British Columbia,
Vancouver, BC, Canada
Simon Kleinhans Institute for Materials
Engineering, Polymer Engineering, University of
Kassel, Kassel, Germany
Frank K. Ko Department of Materials Engineering,
The University of British Columbia, Vancouver, BC,
Canada
Mark T. Kortschot Department of Chemical
Engineering and Applied Chemistry, Advanced
Materials Group, University of Toronto,
Toronto, ON, Canada
Yingjie Li Department of Materials Engineering,
The University of British Columbia, Vancouver, BC,
Canada
Li-Ting Lin Department of Materials Engineering,
The University of British Columbia, Vancouver, BC,
Canada
Helmut Naegele Tecnaro GmbH, Germany
Mohammad Ali Nikousaleh Institute for Materials
Engineering, Polymer Engineering, University of
Kassel, Kassel, Germany
Numaira Obaid Department of Chemical
Engineering and Applied Chemistry, Advanced
Materials Group, University of Toronto, Toronto,
ON, Canada
Nikhil D. Patil Faculty of Forestry, University of
Toronto, Toronto, ON, Canada
xiii
xiv C ONTRIBUTORS

Juergen Pfitzer Tecnaro GmbH, Germany Nicolas R. Tanguy Faculty of Forestry, University
of Toronto, Toronto, ON, Canada
Scott Renneckar Department of Wood Science,
Forest Sciences Centre, The University of British Jimi Tjong Centre for Biocomposites and
Columbia, Vancouver, BC, Canada Biomaterials Processing, Faculty of Forestry,
University of Toronto, Toronto, ON, Canada
Annette Rüppel Institute for Materials
Engineering, Polymer Engineering, University of Newell R. Washburn Department of Chemistry,
Kassel, Kassel, Germany Carnegie Mellon University, Pittsburgh, PA,
USA; Department of Biomedical Engineering,
Mohini Sain Centre for Biocomposites and
Carnegie Mellon University, Pittsburgh,
Biomaterials Processing, Faculty of Forestry,
PA, USA
University of Toronto, Toronto, ON, Canada; Centre
of Advanced Chemistry, Adjunct, King Abdulaziz Ning Yan Faculty of Forestry, University of
University, Jeddah, Saudi Arabia Toronto, Toronto, ON, Canada; Department of
Chemical Engineering and Applied Chemistry,
Tomonori Saito Chemical Sciences Division, Oak
University of Toronto, Toronto, ON, Canada
Ridge National Laboratory, Oak Ridge, TN, USA
Daniel J. Yelle USDA Forest Service, Forest
Viola Sauer Institute for Materials Engineering,
Products Laboratory, One Gifford Pinchot Drive,
Polymer Engineering, University of Kassel, Kassel,
Madison, WI, USA
Germany
Lars Ziegler Tecnaro GmbH, Germany
Gregory D. Smith Department of Wood Science,
Forest Sciences Centre, The University of British
Columbia, Vancouver, BC, Canada
Nicole M. Stark USDA Forest Service, Forest
Products Laboratory, One Gifford Pinchot Drive,
Madison, WI, USA
 Editor’s Biography
Omar Faruk Mohini Sain
Dr Omar Faruk Mohini Sain is
completed his BS and dean and professor at
MS in chemistry at Faculty of Forestry,
the University of Chit- University of Toronto.
tagong, Bangladesh. He specializes in ad-
With a DAAD vanced nanocellulose
(German Academic technology, biocom-
Exchange Service) posites, and bio-nano-
scholarship, he joined composites. He is
at University of Kassel, cross-appointed to the
Germany. He achieved Department of Chemical Engineering and Applied
his PhD in mechanical Chemistry. He is a fellow of Royal Society of
engineering at 2005. Chemistry, UK. Besides, he is also an adjunct professor
He worked at the of the Chemical Engineering Departments at the
Department of Forestry, Michigan State University, University of New Brunswick, Canada; King
USA as a visiting research associate from 2006 to Abdulaziz University, Jeddah, Saudi Arabia; Univer-
2009. Since 2010, he was working at the Centre sity of Guelph, Canada, University of Lulea, Sweden,
for Biocomposites and Biomaterials Processing, honorary professor at Slovak Technical University
University of Toronto, Canada. He joined Ford and Institute of Environmental Science at the Univer-
Motor Co. Canada as research and development sity of Toronto, and collaborates with American and
engineer on January, 2015. He has more than European research institutes and universities. Prof.
70 publications to his credits which have been Sain holds several awards; most recent one is the Plastic
published in different international journals and Innovation Award and Kalev Pugi Award for his inno-
conferences. He also edited one book, titled Biofiber vation and contribution to industry. Author of more
reinforcement in composites materials published than 300 papers, 6 books and hi-cited researcher Prof.
from Woodhead Publishing Ltd. In addition, he Sain hugely contributed to the society at large by
is invited reviewer of 58 international reputed translating research to commercialization which led
journals, government research proposals, and book to three new companies making products ranging
proposals. from packaging to automotive to building construction.

xv

Lignin is a complex polymer abundantly found in
plants and it is the fibrous part of the plant. Tradition-
ally, lignin is used in a wide range of low-volume,
niche applications. Industrial lignins are currently
obtained as coproducts of the manufacture of cellu-
lose pulp for paper, as well as from other biomass-
based industries and there are various types of lignin
depending on their process and purity. Lignin market
is still limited in its application in a wide range of low-
volume, niche applications, but lignin can be used in
a wide range of applications such as in the manufac-
ture of vanillin, animal feed, dye dispersants,
micronutrients, resins, and cleaning chemicals. In
addition, low awareness about lignin among manu-
facturers is the key restraint to this market. It is also
found that there is a weak link between the industry
and research institutes which results in the low
exposure of manufacturers to the developments of
lignin in different applications. Furthermore, the
extraction and modification techniques and applica-
tion of lignin are still at a primary stage, which
hampers the lignin market also. Another obstacle of
lignin expansion in value-added applications is
mainly due to their low-purity standards, heteroge-
neity, smell and color problems of the existing
commercial lignins.
Currently, environmental pollution and increasing
awareness of limited resources, there is growing
Preface
opportunity in the use of lignin as a substitute for
fossil-based raw materials and it could be used in
the manufacture of a wide range of products such
as plastics, chemical products, and carbon fibers.
Recently extensive ongoing research focusing on
lignin drawbacks is increasing the application scope
of lignin in the market.
In recent years, there have been a number of
review papers published on lignin covering lignin
chemistry, modification, polymer composites from
lignin, oxidative upgrade of lignin, biocomposites
and nanocomposites with lignin, industrial lignin
production and applications, and carbon fibers
from lignin. This book focuses specifically on
lignin-based polymer composites (thermoplastic,
thermoset, and biopolymer, rubber, nano, carbon),
lignin-based aerogels, lignin-based foaming mate-
rials, as well as sources and types of lignin, lignin
interunit linkages and model compounds, extraction
of lignin, characterization and properties of lignin,
and applications of lignin. The book will be helpful
to researchers, engineers, chemists, technologists,
and professionals who would like to know more
about the development and potential of lignin and
lignin-based composites.
Omar Faruk and Mohini Sain
xvii

Shayesteh Haghdan, Scott Renneckar and Gregory D. Smith
Department of Wood Science, Forest Sciences Centre, The University of British Columbia, Vancouver, BC, Canada
O U T L I N E
1. Introduction to Lignin
2. Lignin Functions
3. Sources of Lignin
3.1 Wood
3.2 Pulp and Paper Industry
1. Introduction to Lignin
The term lignin is derived from the latin name
lignum meaning wood (Mccarthy and Islam, 1999).
It was first isolated from wood in a scientific report
by the French scientist Payen (1838) and later given
its current name in 1857 by Schulze. Lignin was
initially described as an incrustant of cellulose, and
this point is insightful as lignification occurs after the
deposition of the polysaccharide framework. In an
extremely simplified view it is analogous to the
matrix material for a fiber-reinforced composite.
Lignin has several functions with the cell wall such as
changing the permeability and thermal stability, but it
has the primary function to serve as a structural
material that adds strength and rigidity to plant
tissue. In the sense lignin distinguishes lignocellu-
losic biomass from other polysaccharide-rich mate-
rials, by reinforcing the polysaccharide scaffolding of
the cell wall. Its performance is so effective that it
allows trees to outcompete other plants for sunlight
forming the largest organisms on the planet.
As lignin constitutes 15e40% of dry weight of
woody plants, it is the most abundant aromatic
polymer on the earth and the second most abundant
organic polymer after cellulose. Based on yearly
biomass growth rates, the overall production of
lignin is on the order of 5e36  108 tons (dos Santos
Lignin in Polymer Composites. http://dx.doi.org/10.1016/B978-0-323-35565-0.00001-1
Copyright © 2016 Elsevier Inc. All rights reserved.
1 Sources of Lignin
1 3.3 Sugarcane Bagasse 6
3.4 Agricultural Residues 7
4
4. Lignin Potential 8
5
5 References 8
6
et al., 2014). Hence, lignin has the potential to be an
important source of aromatic chemicals for the
chemical industry, arising from the conversion of
modern era CO2, and its efficient utilization solves
a potential puzzle in creating valuable by-products in
a biorefinery scheme. This reasoning is because if
wood is converted to the billion ton scale for biofuels
and biochemicals, then there will be greater than
300 million tons of lignin potentially available. To
put this in perspective it is roughly the size of the
global polymer market.
As mentioned above, lignin is an aromatic poly-
mer. The monomeric precursors have a phenolic ring
with a three carbon side chain providing a basic nine
carbon structure commonly referred to as a C9-unit
and/or phenylpropane unit as shown in Figure 1.
The side chain is terminated with a primary
hydroxyl group on the Cg, while the Ca and Cb are
connected together with an unsaturated bond. The
phenolic ring is methoxylated (eOCH3) to various
degrees, dependent upon the species; p-coumaryl
alcohol, coniferyl alcohol, and sinapyl alcohol have
none, one, or two methoxyl groups at the 3- and
5-positions, respectively. Based on the lignin mono-
meric composition involved in polymerization, the
resulting lignin is classified into three types: (1) lignin
that contains mainly coniferyl alcohol is called
guaiacyl (G) lignin and is found predominantely in
1
2 L IGNIN IN P OLYMER C OMPOSITES

Figure 1 Examples of C9 monomers: p-coumaryl alcohol, coniferyl alcohol, and syringyl alcohol.

gymnosperms; (2) lignin formed from sinapyl alcohol
is called syringyl lignin (S) and mixtures of G and S
are found in angiosperm lignin; and (3) lignin which
incorporates p-coumaryl alcohol is p-hydroxyphenyl
(H) lignin, and the three types of monomers are
commonly found in grasses. Overall hardwoods have
a G:S ratio that approaches 1:2 and softwoods have
approximately 95% G lignin (Lin and Dence, 1992).
One simple way of determining this ratio is based on
the methoxy content of the lignin, while other tech-
niques can be used to identify the specific C9 struc-
tures either directly using nuclear magnetic
spectroscopy or determined from the derivatized
thioacidolysis products using gas chromatography.
The monomeric units are built into the macro-
molecule lignin from the oxidative radical coupling
of these substructures (Adam et al., 2011; Dinis et al.,
2009; De la Cruz, 2014; Bowyer et al., 2007).
Lignification is initiated when a phenolic hydroxyl
hydrogen atom is abstracted by the enzyme peroxi-
dase to form a phenoxy free radical, typically
referred to as dehydrogenative polymerization, as
described by Freudenberg and Neish (1968). This
phenoxy-free radical will then delocalize to both
aromatic and side chain carbon atoms by the process
of resonance stabilization (Figure 2).
Coupling of these radicals may form ether link-
ages, carbonecarbon bonds, and bonds occasionally
to more than one other phenylpropane unit. Based on
the stability of the radical at each location, there is
a higher probability that certain carbons will host the
free radical. This, in turn, will provide preferred
linkages between lignin units. It has been determined
that approximately 50% of these bonds are b-O-4
ether type (De la Cruz, 2014; Bowyer et al., 2007)
with other bonds associated such as b-5, b-1, b-b,

Figure 2 Resonance stabilization of radicals that lead to interunit linkages in lignin.

1: S OURCES OF L IGNIN
Figure 3 Examples of interunit connections in lignin. Note, the diagram is to simply illustrate possible linkages
and this structure is not a representative “segment” of lignin.
and 5-5 (Figure 3). It is evident that the Cg retains its
hydroxyl group through this process, providing the
native lignin opportunity to also retain a certain level
of hydrophilicity. Furthermore, as indicated in
Figure 3, the benzylic carbon (at the Ca) is typically
highly unstable and undergoes reactions with nucle-
ophilic compounds. These reactions can lead to the
formation of a secondary hydroxyl group at the Ca by
reaction with water which is a reactive site for link-
ages to carbohydrates creating ligninecarbohydrate
complexes. These linkages formed to the poly-
saccharides are both ether and ester linkages
dependent upon the functional groups of the mono-
saccharide (Fengel and Wegener, 1983).
Analysis of lignin impacts several changes to its
native state making it difficult to have an exact
structure of lignin within the cell wall. However,
there are isolation methods that can be used to get
a better idea of the characteristics of a less severely
modified lignin. One such method involves the
resizing of wood into a fine flour through ball milling,
using cellulose-degrading enzymes to remove the
bulk of the polysaccharides and then an acidolysis
reaction to break several of the lignin carbohydrate
linkages (Wu and Argyropoulos, 2003). The isolation
of this enzymatic mild acidolysis lignin (EMAL)
preserves several aspects about lignin and is used as
a standard to compare other lignin isolation methods.
3
When analyzed using quantitative nuclear magnetic
resonance (NMR) spectroscopy, it reveals that the
quantity of functional groups is based on the quantity
of lignin (Granata and Argyropoulos, 1995). It is also
revealed that most lignins have w4 mmol aliphatic
hydroxyl groups attached to the side chain per gram
of lignin and 0.2e1 mmol free phenolic groups per
gram of lignin (Pu et al., 2011). Furthermore, it is
possible to analyze lignin within the whole cell wall
without the need for isolation. This approach requires
the milling of wood into a fine powder and then using
special solvents for cell wall dissolution. Dissolved
wood material is analyzed using two-dimensional
(2D) 13Ce1H heteronuclear single quantum coher-
ence NMR spectroscopy (Mansfield et al., 2012). The
technique can determine the relative concentrations
of interunit lignin structures providing an insight into
the structure of lignin.
As may be inferred from the above description of
isolating EMAL, there are two processes that occur
during delignification. The first involves breakage of
key ligninecarbohydrate linkages. This bond
breakage will allow extractability of the lignin as
seen during mild acidolysis of lignin where dilute
hydrochloric acid is used to break these linkages.
The second process involves cleavage of some of
the interunit bonds of lignin that may reduce
the molecular weight. Hence, delignification
4 L IGNIN
technologies dramatically impact lignin function-
ality and molecular weight. As a result, the proper-
ties of lignin related to its solubility are modified.
Several studies have shown that lignin can be frac-
tionated by using several solvents with largely
different solvent parameters (Moerck et al., 1986). A
small fraction of lignin may be soluble in a nonpolar
solvent like toluene, nonhydrogen bonding solvents
like dichloromethane, while other fractions are
soluble in more polar solvents like aliphatic alco-
hols. The different solubility of the fractions shed
light on the heterogeneous nature of lignin even if it
is isolated from a softwood lignin that contains 95%
guaiacyl lignin. Delignification can modify the
lignin to appreciative degrees by the addition of the
reactants such as sulfur or alcohol to the lignin or
increase the molecular weight (or change the inter-
unit linkages) by reactions of lignin with itself
during delignification. These isolation processes
result in lignin that contains acidic groups, lose
aliphatic hydroxyl groups, increase the free phenolic
groups by breakage of the b-O-4 linkage, and
contain more carbonecarbon bonds between units
(which are referred to as condensed structures).
Because of the variety of intermolecular linkages
between the precursors, lignin is also a highly
heterogeneous polymer (Fengel and Wegener, 1983;
Baucher et al., 1996). The variety of bonds results in
a branched lignin polymer with a potentially cross-
linked three-dimensional structure (Schmidl, 1992).
The recent literature has suggested that lignin may be
more uniform than previous thought, with either
linear chain topologies (Crestini et al., 2011) or even
being capped by specific lignin monomers (Sangha
et al., 2014). However, it is clear that lignin poly-
merization is left up to the fates of thermodynamics
as the polymerization process occurs outside the
control of the cell cytoplasm (Ralph et al., 2004).
Hence, the cellular control is limited to the produc-
tion and release of the monomers involved in ligni-
fication and the enzyme-activated dehydrogenated
monomers will polymerize with the growing lignin
chain.
Another important aspect of polymeric materials is
their molecular weight. Absolute knowledge about
lignin’s molecular weight has been difficult to ach-
ieve as removing lignin to analyze it automatically
impacts the lignin structure. Additionally, lignin
molecular weight measurements suffer from issues
surrounding solubility, aggregation, adsorption, and
fluorescence making it one of the most difficult
IN P OLYMER C OMPOSITES
polymers to analyze. Early experiments looking into
the molecular weight of lignosulfonates suggested
that delignification occurred like breaking a network
polymer (Gorning, 1971). However, several different
isolated lignins have been analyzed with modern
analytical equipment, and lignin usually has
a bimodal distribution with number-average molec-
ular weight between 3000 and 10,000 and weight-
average molecular weight from 8000 to 80,000
dependent upon solution conditions, and lignin type
(Guerra et al., 2007). Many technical lignins
described in the literature either have lower molec-
ular weights because of fragmentation of lignin into
oligomers or much greater molecular weights. When
delignification occurs, a number of reactions can
occur but many involve radical formation and
subsequent radical coupling where lignin fragments
can become bonded, increasing the molecular weight.
Additional heterogeneity of lignin involves how
the structure of this polymer differs within a given
cell wall dependent on its location in the wall, plant
species (hardwood vs softwood), and growth
conditions, especially related to reaction wood
formation (Campbell and Sederoff, 1996; de Wild
et al., 2010). These differences arise from the
monomeric composition available during the ligni-
fication reactions. Overall, one can see the
complexity of trying to analyze a lignin that may be
different in various locations of the cell wall. Ter-
ashima has shown that lignin microstructure is
dependent upon its location in the cell wall. In the
cell wall corners, lignin forms spherical clusters,
while lignin in the secondary cell wall is greatly
reduced in dimensions surrounding the microfibril
lamellar structure of the cell wall layers as shown in
Figure 4 (Terashima et al., 2004). Work with the
Raman spectroscopy has indicated that lignin in
the secondary wall has a preferred orientation along
the microfibril structure (Atalla and Agarwal, 1985).
2. Lignin Functions
Lignin is part of the structural framework in
plants, forming part of the primary load-bearing
element of the cell wall. From an evolutionary
point of view, lignin has been credited as the terres-
trial adaption that permits significant vertical growth.
As an essential part of the cell, lignin supports the
plant by imparting rigidity to the cell wall. The plant
withstands natural environmental stresses because of
1: S OURCES OF L IGNIN
Figure 4 Cell corner region. Large globular modulus form aggregates at random, left and lignin modules (arrows)
are growing along cellulose microfibrils (CMFs) at an almost regular distance, right.
this cell wall building block (Lopes et al., 2011;
Sarkanen and Ludwig, 1971). Lignin not only
provides stiffness to the plant but in conjunction with
the heteropolysaccharides, it provides flexibility
which is fundamental for an appropriate response to
dynamic loads from wind and snow. In addition to
mechanical support, lignin modifies the poly-
saccharide network to make it resistant to outside
organisms. Lignin helps to protect woody tissue from
the microbial and fungal attack encasing the carbo-
hydrate structure, providing reduced accessibility of
enzymes for hydrolysis. Limited solubility and
complexity of the lignin make it resistant to degra-
dation by most microorganisms (Prasongsuk et al.,
2009; Bholay et al., 2012; Berg and Meentemeyer,
2002; Crow et al., 2009; Verma and Dwivedi, 2014).
One can imagine if lignin only contains a single
linkage that would become the cell wall’s Achilles’
Heal. However, with the variety of linkages wood-
destroying organisms require the breakage of both
aryl carbon bonds and aryl ether bonds needing to
expand the cost of production of specific enzymes or
develop nonspecific pathways for delignification. In
addition, lignin is less hydrophilic than the poly-
saccharides helping to change the permeability of the
cell wall by sealing it and enabling water transport
through the vascular tissue (Ten and Vermerris,
2013). Finally, the aromaticity of lignin lends itself to
enhance the thermal stability of wood providing char
layer. This has been exploited with isolated technical
lignin, turning it into carbon fiber material by
controlled high-temperature heating. While lignin is
seen as more thermally stable because of it, the native
structure of lignin is greatly impacted by the thermal
modification and researchers should be cautioned
5
that lignin structure can change during processing at
temperatures used in the production or some ther-
moplastic materials. Such modification includes
depolymerization, loss of the Cg hydroxyl, and
formation of new acidic groups.
3. Sources of Lignin
The source from which lignin is obtained, the
extraction methods and the secondary treatments
applied, have strong impacts on its physical and
mechanical properties (Garcia et al., 2011; Khanam
et al., 2006). Lignin can be derived from various
sources such as wood, pulp and paper, sugarcane
bagasse, and cereal straws using a variety of pulping
methods. In terms of weight, the lignin content in
woody plants from gymnosperms and angiosperms is
the highest with the order of 30e40% while other
sources only contain around 3e25% (Smolarski,
2012). The pulp and paper sector produces a large
amount of lignin (Jungmeier, 2010; Doherty et al.,
2011) with even greater potential from future ligno-
cellulosic biorefineries. Minimal lignin is recovered
from grass, branches, leaves, and solid waste in urban
and rural areas where their lignin content is estimated
to be less than 15% (Wang et al., 2011; Philippidis
and Hatzis, 1997).
3.1 Wood
Lignin has nonuniform thickness in the middle
lamella, and in the primary and secondary cell walls
depending on the plant species and cell type
(Figure 5). Approximately 70% of the total lignin
content of the cell wall is concentrated in the
6 L IGNIN
thickest layer of the secondary cell wall. Lignin
quantity and quality vary naturally among wood
species with 19e28% in hardwoods (angiosperms)
and 24e33% in softwoods (gymnosperms) (Fengel
and Wegener, 1983). In fact, hardwoods, in general,
contain more hemicellulose and less lignin than
softwoods (Bjornsson, 2014). Not only the amount
of lignin varies between hardwoods and softwoods,
the relative concentration also differs in the location
within a tree. The juvenile wood has a higher lignin
content than latewood (De la Cruz, 2014; Bowyer
et al., 2007). Lignin content is also variable within
populations of plants of the same genus. For
example, the average content of lignin ranges from
25% in Pinus monticola to 30% in Pinus palustris
within the genus Pinus. Reaction wood forms
mechanical stress and has different lignin content in
comparison with the normal wood depending on the
conditions and species (Tiimonen, 2007).
Lignin in hardwoods is syringyleguaiacyl type
and in softwoods is typically guaiacyl with limited
p-hydroxyphenyl lignin in both types. Both of these
structures have differences arising from the interunit
linkages. Guaiacyl lignin can undergo coupling
reactions at the 5-position of the phenylpropane unit,
and this provides a significant place for branching and
cross-linking reactions, especially occurring during
Figure 5 Wood cell wall including middle lamella (ML),
primary cell wall (P), layers of the secondary cell wall
(S1, S2, S3), and warty layer (W) (Sjostrom, 1993).
IN P OLYMER C OMPOSITES
delignification process. The presence of syringyl units
makes hardwood lignin more readily removed during
the pulping process by limiting lignin forming
condensed structures at the open methoxy position. In
the case of compression-wood lignin, it is more
difficult to hydrolyze because it contains a higher
proportion of condensed p-hydroxyphenyl units
(Campbell and Sederoff, 1996; Novaes et al., 2010).
3.2 Pulp and Paper Industry
Softwood and hardwood species, and certain types
of annual plants, have commercial interest as a source
of cellulose fibers for the production of paper and
board products. Lignin is not easy to isolate in the
native form from plant material (Leisola et al., 2012).
The pulp and paper industry is the primarily
commercial source of lignin, however, the delignifi-
cation process modifies lignin to various degrees. In
technical fiber liberation processes, such as alkaline
or sulfite pulping, huge quantities of lignin are dis-
solved as alkali lignin and lignosulfonates, respec-
tively. Large amounts of lignin are made available
annually from the pulp and paper industry as by-
products of the delignification process. These sulfite
or sulfate lignins have varying levels of covalently
bonded sulfur resulting in the polymer with different
characteristics than the original lignin (Sainsbury,
2013; Khanam et al., 2006; Lora and Glasser, 2002).
Typically, lignin impurities include low-molecular-
weight sugars and resin acids that are removed during
the purification process. For example, kraft lignin is
usually purified from kraft black liquors which are
complex mixtures of fibrous materials and dissolved
organics such as lignins, hemicelluloses, sugars, acids,
and resins and also inorganic salts such as ash (Wall-
berg et al., 2003). The recent technology in enhancing
lignin recovery from black liquor by CO2 acidification
has been transferred to the industry and has created
a readily available dry lignin powder stream (Ohman
et al., 2013; Kouisni et al., 2012). As shown in Table 1,
in all commercial pulping processes the lignin
extraction methods will affect the value of the products
that can be derived from it and will alter the structure of
the lignin in comparison with the native one (Smo-
larski, 2012).
3.3 Sugarcane Bagasse
Sugarcane bagasse is the fiber that remains after the
sugars have been extracted. As an agro-industrial
residue, sugarcane bagasse (Saccharum officinarum)
1: S OURCES OF L IGNIN
Table 1 Lignin Purity with Its Associated Sources (Holladay et al., 2007)
Lignin Type Lignin Puritya
Lignosulfonates Medium (some reducing sugar
(up to 20 wt%) and sulfur)
Kraft Medium (some ash and sulfur)
Organosolv High (sulfur free)
a
Presence of residual carbohydrates, ash, and proteins depends on feedstock and process.
is another source of lignin raw material. It is a by-
product of the sugarcane industry with approxi-
mately 32e34% cellulose, 19e24% hemicellulose,
25e32% lignin, 6e12% extractives, and 2e6% ash
(Sakdaronnarong and Jonglertjunya, 2012; Rezende
et al., 2011; Pandey et al., 2000). Chemical compo-
sition of sugarcane bagasse is similar to the other plant
cell walls. Each class of plants, grasses, softwoods,
and hardwoods produces a lignin rich in one type of
the phenylpropane repeat unit. Sugarcane bagasse
lignin has a higher proportion of H-type lignin, p-
hydroxyphenyl, and hence a lower methoxy content
than softwood and hardwood lignins (Doherty et al.,
2007). Approximately 250e280 kg of bagasse is
generated from processing each ton of sugarcane
which roughly yielded 54 million tons of bagasse
annually (Canilha et al., 2012). Currently, a large
amount of bagasse is burnt as a low-grade fuel for
energy recovery, and only a limited quantity has been
used to make pulps, board materials, and composites.
It is estimated that 200 million tons of lignin is
produced annually from bagasse (Singh et al., 2005).
3.4 Agricultural Residues
The advantages of using agricultural residues are
threefolds: economic, environmental, and techno-
logical. Unlike wood pulps, agricultural pulps can be
produced using more environmentally benign pro-
cessing and bleaching methods (Clancy-Hepturn,
1998). In the wood pulping process, most of the pulp
is bleached using chlorine or chlorine-based chem-
icals while straw can be treated with minimal addi-
tions of chlorine-free chemicals which results in no
production of toxic chemicals. Moreover, agricul-
tural residues have generally a more porous structure
and a lower lignin content than woody plants which
facilitate their pulping process. Examples of the
7
Potential Products
Dispersants, agricultural chemicals, emulsion
stabilizer, industrial binders, carbon black, ink
and pigments, and concrete additives
Dispersants, carbon fibers, emulsifiers,
activated charcoal, and binders
Aromatic polyols, new diacids, activated carbon,
phenolic resins, carbon fibers, vanillin, phenol
derivatives, and antioxidants
agricultural residues are rice and wheat straws which
are discussed in this section. Rice straw also called as
cereal straw is another source for lignin production.
Total world production of rice straw, Oryza sativa, is
525 million tons per year.
About 90% of the rice straw is produced in Asia.
China has the greatest capacity for pulping rice straw.
It has approximately 30e35% cellulose, 25e30%
hemicellulose, 15e28% lignin, and 4e7% ash
(Marques et al., 2010). The high ash content arises
from the silica in the cell wall limiting the ability to
burn this material without causing a significant envi-
ronmental nuisance. High silica content in rice straw,
as high as 18% in clean rice straw, makes the pulping
process more costly due to increased chemical
recovery difficulties and costs. In addition to rice
straw, wheat straw, Triticum aestivum, is annually
generated in abundance of 529 million tons per year.
When farmed intensively, wheat straw can be
produced in a larger scale. The amount of lignin of
wheat straw varies between 5% and 17% depending
on the geographical location of the plants which is
comparable to hardwoods (Buranov and Mazza,
2008). Wheat straw has one of the highest cellulose
contents of all of the agricultural fibers. Straw papers
are known to possess good printing qualities and are
made from pulp requiring low energy relative to that
required to process wood pulp. Researchers found that
wheat straw must be pulped under conditions of less
energy and fewer chemicals in comparison with the
wood pulps to maximize pulp yields (Clancy-Hepturn,
1998). To make the strongest product, papermakers
will likely combine some stronger hardwood, kenaf,
or hemp pulp with straw pulp. Despite the advantages
of using agricultural residues, the high collection,
transportation, and handling costs associated with
these resources, as well as their seasonal availability,
limit their applications in paper production.
8 L IGNIN
4. Lignin Potential
Currently around 50 million tons of lignin is
produced per year by the pulp and paper sector but only
2% of that is used for applications other than combus-
tion and energy production (de Wild et al., 2014; Fengel
and Wegener, 1983; Schmidl, 1992; Thielemans et al.,
2002). There are limitations in utilization due to the
lignin structure, heterogeneity, and the industrial pro-
cessing costs for delignification (Holladay et al., 2007;
Vanholme et al., 2010; Neutelings, 2011; El Hage et al.,
2009; Chiang, 2005). To improve upon these limita-
tions, different types of modifications are done to
increase its chemical reactivity and uniformity, reduce
the brittleness of lignin-derived polymers, increase its
solubility in organic solvents, and improve the ease of
processing the lignin (Argyropoulos, 2012). Lignin
potentials can also be considered in the production of
additives, resins, and coating materials (Jungmeier,
2010; Vishtal and Kraslawski, 2011). This renewable
aromatic polymer can equally be replaced with
synthetic polymers and aromatic chemicals (Zakzeski
et al., 2012). High antioxidant capacity of lignin and its
applications in polymer enhancement have been
experimentally proved (Piazza et al., 2014). The find-
ings of research on lignin depolymerization showed
that due to the ligninmacromolecular structure of lignin
with many phenolic units, lignin depolymerization can
be used as a large-scale source for production of valu-
able chemicals (Kleinert et al., 2009; Meier et al., 1992;
Horácek et al., 2012). Lignin also has potential to be
used in the fabrication of composite materials.
El Mansouri et al. (2007) showed that exterior-grade
particleboards made with lignin-based adhesives
successfully met the requirements of international
standards. The potential of lignin in polymeric
composites will be discussed in later chapters.
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15567030903226249.
de Wild, P.J., Huijgen, W.J.J., Gosselink, R.A., 2014.
Lignin pyrolysis for profitable lignocellulosic bio-
refineries. Biofuels, Bioproducts and Biorefining 8
(5), 645e657. http://dx.doi.org/10.1002/bbb.
de Wild, P.J., van der Laan, R.R., Wilberink, R., 2010.
Thermolysis of lignin for value-added products. In:
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Society. Energy Research Centre of the Nether-
lands, Tenerife, Canary Islands, Spain, pp. 1e27.
Wu, S., Argyropoulos, D.S., 2003. An improved
method for isolating lignin in high yield and
purity. Journal of Pulp and Paper Science 29 (7),
235e240.
Zakzeski, J., Jongerius, A.L., Bruijnincx, P.C.A.,
Weckhuysen, B.M., 2012. Catalytic lignin valoriza-
tion process for the production of aromatic chemicals
and hydrogen. ChemSusChem 5 (8), 1602e1609.
http://dx.doi.org/10.1002/cssc.201100699.
 2 Extraction and Types of Lignin
Hoyong Chung 1 and Newell R. Washburn 2,3
1
Department of Chemical and Biomedical Engineering, Florida State University,
FAMU-FSU College of Engineering, Tallahassee, FL, USA
2
Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA
3
Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA

O U T L I N E

1. Introduction 13 2.5 Steam Explosion Lignin (Hydrothermal

Process) 20
2. Commercial-Grade Lignins 15
2.6 Dilute Acid Lignin 21
2.1 Kraft Pulping 16
2.2 Sulfite Pulping 16 3. Conclusions 22
2.3 Soda Process 18
References 22
2.4 Organosolv Lignin 19

1. Introduction comprising primarily of G-units with small concen-

Lignin is the second most abundant terrestrial
biopolymer after cellulose and is the largest renew-
able source of aromatic groups in nature. Lignin is
found in plant cell walls and is an important struc-
tural component of woody plants. The main functions
of lignin in plants are to provide physical strength,
to form water-conducting vascular networks using
hydrophobic interactions and to protect plants from
microorganisms and insects. Chemically, lignin is
composed of a random network of phenylpropane
groups. The three basic structural monomer units are
coumaryl alcohol, coniferyl alcohol, and sinapyl
alcohol, as shown in Figure 1. In nature, these
monomer units oxidize to phenoxy radicals by
peroxidase and then undergo polymerization through
multiple reactive sites to form a complex three-
dimensional polymer. This biosynthesis is referred
to as dehydration polymerization, and the resulting
monomer units within lignin are referred to as
p-hydroxyphenyl (H), guaiacyl (G), and syringyl
(S). Differences in lignin according to various plant
sources can be manifested in differences in mono-
mer concentrations (Vanholme et al., 2010), with
gymnosperms, such as the various types of pines,
trations of H-units. In contrast, angiosperm dicots,
including many hardwoods, have a mixture of G and
S-units, which reduce branching concentrations and
can improve lignin processibility.
As a result of the random radical polymerization of
phenylpropane monomers, lignin adopts complex
three-dimensional structures with various types of
functionality. As shown in Figure 2, important CeO
linkages are b-O-4, a-O-4, and 4-O-5; and CeC links
are b-5, 5-5, b-1, and beb linkages (Koch, 2008;
Calvo-Flores and Dobado, 2010). Also, common
functional groups in lignin include methoxyl, phenolic
hydroxyl, aliphatic hydroxyl, and other carbonyl
groups (Chakar and Ragauskas, 2004).
Figure 1 Monomeric lignin building blocks: p-coumaryl
alcohol, coniferyl alcohol, and sinapyl alcohol.

Lignin in Polymer Composites. http://dx.doi.org/10.1016/B978-0-323-35565-0.00002-3

Copyright © 2016 Elsevier Inc. All rights reserved. 13
14
Figure 2 Schematic model of the lignin structure with important linkages (Koch, 2008; Calvo-Flores and Dobado,
2010; Laurichesse and Averous, 2014).
Chemical characterization of lignin, lignin deriv-
atives, and other lignin-based materials is not a
simple task due to the three-dimensional architecture,
diversity of chemical linkages and functional groups,
difficult isolation, and poor solubility in many
organic solvents (Figure 2). This is further compli-
cated by many different types of lignin depending on
the plant source and processing method. Numerous
analytical approaches have been employed to char-
acterize the structure of lignin and other lignin-based
materials.
Nuclear magnetic resonance spectroscopy is one of
the most useful techniques to determine the chemical
structure of lignin (Ralph et al., 1999; Ralph and
Landucci, 2010). Lignin contains a high fraction of
methoxy groups and aromatic hydrogens, allowing
these groups to be readily determined by 1H NMR.
However, 1H NMR is not suitable for characterizing
the most abundant reactive group, the hydroxyl group.
Hydroxyl group content is best determined after
acetylation of hydroxyl groups by acetic anhydride
in the presence of pyridine (Bonner and Mcnamara,
1968; Chung and Washburn, 2012). The acetoxy
groups produced in this reaction correspond to
L IGNIN IN P OLYMER C OMPOSITES
hydroxyl groups of lignin and appear at chemical
shifts of approximately 2 ppm (Lundquist, 1992;
Chung and Washburn, 2012). After acetylation, the
phenolic hydroxyl group can be determined selec-
tively by relatively fast and selective deacetylation
of phenolic acetyl groups (Månsson, 1983). Alkyl-
ation of the hydroxyl group was performed to deter-
mine hydroxyl groups in 1H NMR spectra (Adler
et al., 1987). While 1H NMR provides quantitative
information, certain peaks, such as those due to
methoxy groups, can appear broad and featureless
because of the diversity of chemical environments
(Liitia et al., 2003). Two-dimensional 1H-13C NMR
spectroscopy can provide detailed information on
connectivity. Model lignin complexes provided
detailed information necessary for peak assignments
(Ede and Ralph, 1996), and numerous pulse sequences
have been developed to elucidate the connectivity of
functional groups and identify specific linkages
(Ede and Brunow, 1992; Kilpelainen et al., 1994).
For example, the syringyl-to-guaiacyl (S/G) ratio
was followed during kraft pulping using hetero-
nuclear single quantum correlation NMR, which
provided information on changes due to oxidation
2: E XTRACTION AND T YPES OF L IGNIN 15

during processing (Ibarra et al., 2007). A different
approach that provides information on alcohol
functionality utilizes 31P NMR in which hydroxyl
groups were converted to phosphityl groups by
2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane
to do 31P NMR analysis (Granata and Argyropoulos,
1995; Froass et al., 1998). For 31P NMR analysis,
phosphitylation was carried out by another agent,
1,3,2-dioxaphospholanyl chloride (Argyropoulos,
1994; Froass et al., 1996). Most of the lignins from
the processing methods described here have been
characterized by NMR spectroscopy.
Size-exclusion chromatography (SEC) analysis of
lignin is an important analysis to determine molecular
weight and molecular weight distribution, which are
critical data for materials development (Himmel et al.,
1995; Hortling et al., 1995). However, the reliability
of SEC analysis can be limited due to the many
functional groups in lignin, such as hydroxyl groups
and carboxylic acids, and the lignin analyte can have
strong interactions with the column support materials
or between other lignin species (Sarkanen et al., 1981,
1982, 1984; Iversen, 1985). Therefore, the hydroxyl
groups are acetylated and carboxylic acid groups
are methylated prior to the SEC analysis to exclude
possible noncovalent interactions, such as hydrogen
bonding and electrostatic aggregation, from the lignin
(Himmel et al., 1995; Kim et al., 2007). Lignin is also
characterized by various morphological methods such
as thermal analysis and Raman/IR analysis (Agarwal
and Atalla, 1999, 2010; Glasser Wolfgang, 1999).
The chemistry and material properties of lignin
have been reviewed extensively (Goldstein et al.,
1983; Lewis et al., 1999; Chakar and Ragauskas,
2004; Calvo-Flores and Dobado, 2010; Hatakeyama
and Hatakeyama, 2010; Duval and Lawoko, 2014) as
well as chemical methods for modifying and blending
it (Wang et al., 1992; Meister, 2002; Liitia et al.,
2003; Mohanty et al., 2009; Doherty et al., 2011;
Washburn and Chung, 2012). In this chapter, we
present extraction methods and types of commercial-
grade lignin from a raw biomass. A better under-
standing of types and production methods of lignin is
an important starting point to study lignin as new
materials, biofuel, and renewable sources of fine
aromatic chemicals.
2. Commercial-Grade Lignins
Lignin is biosynthesized from monomeric substi-
tute phenyl propylene units, coumaryl alcohol, con-
iferyl alcohol, and sinapyl alcohol, as lignocelluloses
as well as nonconjugated lignin. Because the ligno-
celluloses and lignin are intimately incorporated in
plant structures, a variety of processing methods have
been developed to isolate lignin. Table 1 demon-
strates diverse sources of lignin from raw biomass

Table 1 Polymeric Components of Raw Biomass and Plant-based Materials (Suchy and Argyropoulos, 2002;
Koch, 2008)

Source (%) Lignin Hemicellulose Cellulose Extract

Hardwood 16e24 25e35 43e47 2e8
Softwood 25e31 25e29 40e44 1e5
Bagasse 20 30 40 10
Coir 43e49 10e20 32e43 4
Corn cobs 15 35 45 5
Corn stalks 35 25 35 5
Cotton 1 2 95 0.4
Flax (retted) 2 21 71 6
Flax (unretted) 3 12 63 13
Hemp 6 22 70 2
Jute 13 14 71 2
Sisal 11 14 73 2
Wheat straw 15 50 30 5
16
and plant-based materials such as cotton and
bagasse. Raw wood species, hardwood, and soft-
wood contain a high percentage of lignin, between
25% and 40%. Lignin and other polymeric compo-
nents of each wood species differ as shown in
Table 2. Glucomannan and glucuronoxylan are typical
types of hemicellulose in each woody species. Over
the all woody species, the portion of the lignin is at
least 20%. Therefore, it is necessary to isolate two
important plant-based polymers, lignin and cellulose,
from raw biomass resources through an efficient,
industrial-scale method. Each commercial isolation
method has trade-offs in terms of reagents and energy
required that affect the molecular weight and purity
of the final product, as well as the extent to which
new chemical functionality is introduced as a result
of processing. The pulp and paper industry is the
major producer of commercial-grade lignin as a by-
product of cellulose-rich pulp/paper manufacturing
but cellulosic ethanol facilities are currently coming
online. It is critical for researchers to understand how
lignin processing has changed the chemistry and
properties of the material. Six common commercial-
grade lignin-producing methods are discussed here.
2.1 Kraft Pulping
The kraft pulping process is the most commonly
used chemical pulping methods to produce cellulose
fibers from raw wood and plant sources (Sixta et al.,
2008). The global production using kraft pulping is
the largest among all production methods with
131.2 M ton in 2000 (Sixta et al., 2008), making kraft
lignin the most common form (Smook, 2002). Kraft
pulping is a method of chemical pulping that uses
chemical reactions to degrade and dissolve the lignin
in order to separate pure cellulose from the wood
(Sixta et al., 2008). An important step of kraft pulping
is a treatment of biomass with sodium hydroxide and
sodium sulfide to cleave ether linkage by sulfide and
bisulfide ions (Sjöström, 1993). In this process,
phenolic groups are converted to quiononemethide
groups by sodium hydroxide and then hydrogen
sulfide ion attacks a-carbon atoms of ether linkages
forming a benzylthiolate anion. Next, the b-phenolate
anion is released from the benzylthiolate anion to
produce free phenolic groups. The free phenolic
groups yield quiononemethide again, and the same
process is repeated to cleave ether bonds continuously
and produce lower molecular weight, soluble frag-
ments. Lignin carbonecarbon bonds can be formed at
L IGNIN IN P OLYMER C OMPOSITES
the last stage of the process; this generally involves
a condensation reaction and yields a very robust
lignin complex (Saake and Lehnen, 2000; Sixta et al.,
2008) (Schemes 1 and 2). Kraft lignin is hydrophobic
at neutral pH and contains approximately 1% sulfur
(measured in atomic%) in the form of aliphatic thiol
groups (El Mansouri and Salvado, 2007). Also other
important functional groups in kraft lignin (expressed
in w/w%) are methoxy group (14%), aliphatic
hydroxyl group (10%), phenolic hydroxyl group
(2e5%), and carboxylic acid group with composi-
tions that can vary depending on the plant source and
processing conditions (4e7%) (El Mansouri and
Salvado, 2007; Doherty et al., 2011). However, as
a by-product in paper production, most kraft lignin is
still burned as a fuel to produce power in the pulp mill
and to recover inorganic components (Saake and
Lehnen, 2000; Mohanty et al., 2009; Doherty et al.,
2011).
2.2 Sulfite Pulping
The process of sulfite pulping is the reaction of
wood or biomass with sulfur dioxide (SO2), which
reacts with water to form sulfonic acid, and bases such
as calcium, sodium, magnesium, or ammonium at
high temperatures, 125e150
C, for 3e7 h. This
process is acid-catalyzed to cleave a-ether linkage
and b-ether linkages during the pulping process
(Shimizu et al., 1998). The lignin can be dissolved and
separated in aqueous solution due to sulfonic acids
which introduced to a-carbon of ether linkage (Saake
and Lehnen, 2000). The resulting lignin has func-
tional groups of lignosulfonic acid, lignosulfonate,
carboxylic group together with phenolic/aliphatic
hydroxyl groups (Vishtal et al., 2011). Due to the high
content of sulfonate, the produced lignin is water
soluble. Lignin produced by the sulfite process has
broad molecular weight distribution and relatively
high ash content. The carboxylic acid content is
approximately 4%, while the sulfonate content can be
13% (El Mansouri and Salvado, 2007). Thus, the
materials applications are limited to the area of
animal feeds, particle boards, surfactants, adhesives,
cement additives, and stabilizer/dispersing agents
(Vishtal et al., 2011; Laurichesse and Averous, 2014).
Due to the high content of sulfur, approximately
5e6%, most of the obtained lignin by the sulfite
pulping process is used as a fuel for the pulp mill and
to recover inorganic components (Saake and Lehnen,
2000; Doherty et al., 2011) (Scheme 3).
Table 2 Polymeric Components of Various Softwood and Hardwood Species (Sjöström, 1993; Koch, 2008)

Species Common Names Extractives Lignin Cellulose Glucomannan Glucuronoxylan Polysaccharides

Softwoods
Abies balsamea Balsam fir 2.7 29.1 38.8 17.4 8.4 2.7
Pseudotsuga Douglas fir 5.3 29.3 38.8 17.5 5.4 3.4
menziesii
Tsuga Eastern hemlock 3.4 30.5 37.7 18.5 6.5 2.9
canadensis
Juniperus Common juniper 3.2 32.1 33.0 16.4 10.7 3.2
communis
Pinus radiate Monterey pine 1.8 27.2 37.4 20.4 8.5 4.3
Pinus sylvestris Scots pine 3.5 27.7 40.0 16.0 8.9 3.6
Picea abies Norway spruce 1.7 27.4 41.7 16.3 8.6 3.4
Picea glauca White spruce 2.1 27.5 39.5 17.2 10.4 3.0
Larix sibirica Siberian larch 1.8 26.8 41.4 14.1 6.8 8.7
Hardwoods
Acer rubrum Red maple 3.2 25.4 42.0 3.1 22.1 3.7
Acer saccharum Sugar maple 2.5 25.2 40.7 3.7 23.6 3.5
Fagus sylvatica Common beech 1.2 24.8 39.4 1.3 27.8 4.2
Betula verrucosa Silver birch 3.2 22.0 41.0 2.3 27.5 2.6
Betula papyrifera Paper birch 2.6 21.4 39.4 1.4 29.7 3.4
Alnus incana Gray alder 4.6 24.8 38.3 2.8 25.8 2.3
Eucalyptus Blue gum 1.3 21.9 51.3 1.4 19.9 3.9
globulus
Acacia mollissima Black wattle 1.8 20.8 42.9 2.6 28.2 2.8
18 L IGNIN IN P OLYMER C OMPOSITES

Scheme 1 Reaction of b-aryl ether linkages in nonphenolic phenylpropane units.

Scheme 2 Reaction of sulfide with phenolic phenylpropane units. Styrenic products are capable of further reac-
tions to form a complex polymer mixture.

Scheme 3 Mechanism of lignin isolation during the sulfite pulping process.

2.3 Soda Process As shown in Scheme 4, an a-ether bond can be

The soda process is the oldest pulping method
that uses sodium hydroxides as the chemical
reagents of the pulping process, and is now being
used to process nonwood material, which includes
mainly annual plants and agricultural waste, such as
wheat straw, sisal, kenaf, hemp, and bagasse. In this
process, biomass reacts with concentrated sodium
hydroxide (ca. 1 M) under high temperature (ca.
170
C) and high pressure (ca. 10 psi) (Gierer, 1980).
hydrolytically cleaved by this method, resulting in
lower molecular weight fragments with increased
solubility, making it an effective process for
delignification.
Anthraquinone (AQ) is often added to the reaction,
which oxidizes carbohydrates and forms anthrahy-
droquinine (AHQ) (Lundquist et al., 1981). The AHQ
dianion can cleave the b-O-4 ether linkage easily
during the process by reacting with quinine methides
formed from soda reactions via nucleophilic attack,
2: E XTRACTION AND T YPES OF L IGNIN
Scheme 4 Cleavage of the a-ether bond in lignin
during the soda process.
thus regenerating the AQ. This reaction is shown in
Scheme 5.
Addition of AQ can significantly increase the
delignification rates; Lunquist et al. reported a
reduction in cooking time from 250 min for soda to
90 min for soda-anthraquinone in studies on spruce
wood (Lundquist et al., 1981). Furthermore, soda
lignin has been used as a precursor for raw materials
that is considered as being closer to natural lignins in
a broad range of applications because of the absence
of sulfur functionality (Gierer et al., 1979; Saake and
Lehnen, 2000).
2.4 Organosolv Lignin
Organosolv processes represent a large family of
methods used to delignify biomass, and there are
several excellent reviews on these (McDonough,
1993; Young and Akhtar, 1998). Common features
include mixtures of water and organic solvents to
remove lignin from the wood or other biomass as well
as the use of acid or base catalysts (Pye and Lora,
1991; Stockburger, 1993; Akhtar and Young, 1997;
Zhang et al., 2007). Ethanol, methanol, formic acid,
or acetic acid have been used, and it is known that the
organosolv process is more effective in treating
annual plants and hardwoods (Pye and Lora, 1991;
Lora et al., 1993; Creamer et al., 1997). Organosolv
lignin is free of sulfur, and the molecular weight is
low compared to other lignin production methods,
generally around 5 kDa, although this will depend on
the organic solvent used, with carboxylic acids often
Scheme 5 Cleavage of the b-O-4 ether linkage in the presence of anthrahydroquinine in the soda process.
19
being less effective at preventing recombination
than alcohols. However, the use of organic solvents
increases the cost of processing and can have a
greater environmental impact, although the lack
of odor of this process compared to the kraft process
and its compatibility with agricultural residues
provide some benefits in this regard (Pye and Lora,
1991; Stockburger, 1993; Akhtar and Young, 1997;
Zhang et al., 2007). Many organosolv processes
have been investigated extensively and tested in pilot-
scale plants, including Alcell, Acetosolv, and Milox.
Most involve an acid catalyst or are performed in an
acidic solvent, which allows use of lower tempera-
tures than traditional pulping methods, and they
generally avoid use of sulfur- and chlorine-based
chemicals.
The Alcell method is based on ethanol processing
using a sulfuric acid catalyst, which is being actively
studied for the pretreatment of lignocellulosics for
ethanol production. Ethanol more efficiently solubi-
lizes lignin cleavage products formed as a result of
catalyst action than water. While originally devel-
oped for hardwoods, recent studies have focused on
adapting this for softwoods, such as pine (Pan et al.,
2005), as well as agricultural residues (Cybulska
et al., 2012). Optimized conditions for softwood
were found to be 170
C, 60 min, 1.1% H2SO4, and
65% ethanol/35% water from which 79% of the
lignin was recovered (Pan et al., 2007). As deter-
mined using 13C and 31P NMR measurements, the
major reaction products from processing pine were
formed through cleavage of the b-O-4 linkages as
well as ester bonds (Sannigrahi et al., 2010). Char-
acteristic molecular weights for these lignins are
Mw w2000 g mole1 with a polydispersity of w2.0.
Interestingly, ethanol organosolv products tend to be
richer in phenolic hydroxyl groups, making them
highly effective at radical scavenging (Pan et al.,
2006).
The Acetosolv process uses a cooking medium
of 93% acetic acid and <1% hydrochloric acid as
a catalyst. The addition of a mineral acid catalyst allows
20
for lower digestion temperatures, often below 140
C
instead of 170
C or greater without it. The kinetics of
the Acetosolv process have been studied for both hard-
wood (eucalyptus) and softwood (pine) (Vazquez et al.,
1997). The proposed mechanism is based on a sequence
of first-order reactions involving formation of soluble
lignin species through lignin hydrolysis, lignin conden-
sation in solution, and subsequent reprecipitation.
Effective delignification was observed at 180
C and
0.1% HCl, whereas increasing the HCl concentration to
0.2% allowed for a temperature reduction to 140
C, but
longer reaction times can actually reduce yield due to
lignin recombination.
The Milox (or Formacell) process takes its name
from the Milieu Pure Oxidative process developed
by the Finnish Pulp and Paper Research Institute
(KCL) (Sundquist, 1986). It is based on use of per-
oxyformic acid to perform tandem delignification
and bleaching. Peroxyacids react with nucleophiles
having p-electrons via oxygen transfer, and in
acidic media the strong electrophile þOH is formed.
A diversity of functional groups result from this
oxidation reaction, including quinones, carboxylic
acids, and alkenes, and formation of recombination
by-products is minimized through efficient solubili-
zation in the water/formic acid medium.
Generation of peroxyformic acid is accomplished
by reacting formic acid with hydrogen peroxide
according to the reaction:
which has a forward rate constant at 22
C that is 50
times greater than the analogous reaction of acetic acid
and hydrogen peroxide (Suchy and Argyropoulos,
2002). Peroxyformic acid can be readily separated
from the reaction mixture by distillation, and has been
applied to the reactor as a concentrated solution.
However, these concentrated solutions can be explo-
sive, and more recent approaches rely on generating it
in situ. Seisto et al. (Seisto and PoppiusLevlin, 1997)
established multistage processes based on impregna-
tion of biomass with formic acid and introduction of
hydrogen peroxide following by addition of water and
cooking at up to 120
C for periods ranging from 60 to
180 min. This was applied to the hybrid grass Mis-
canthus  giganteus, and a comparison with native
lignin showed that the weight and number average
molecular weights (Mw and Mn) had decreased from
L IGNIN IN P OLYMER C OMPOSITES
31742 g mole1 and 2681 g mole1 to 7636 g mole1 and
1417 g mole1, respectively, as a result of the Milox
processing. In addition, the concentration of carbox-
ylic acid sites increased from 0.18 mmol ge1 to
0.35 mmol ge1 and total phenolic groups increased from
0.84 mmol ge1 to 1.31 mmol ge1, while the concen-
tration of aliphatic hydroxyl groups was reduced
from 5.54 mmol ge1 to 1.66 mmol ge1. Clearly even
organosolv processing can have significant impact on
the chemical functionality of lignins.
Alkaline conditions have also been explored in
organosolv processes. For example, the Organocell
process first impregnates biomass with a mixture
of water and methanol, or sometimes ethanol, at
temperatures of 110e140
C. Then alkali, such as
sodium hydroxide, and anthraquinone are added in
the digester where the pulping liquor, containing up
to 30% methanol by volume, is heated to 170
C at
pressures of order 10 bar for 2 h. It is thought that the
methanol may directly participate in the delignifica-
tion by forming methyl ether groups at carbocation
sites where lignin condensation could occur. This
would maintain a low-molecular weight of the lignin
fragments compared to those formed by kraft or
sulfite pulping.
2.5 Steam Explosion Lignin
(Hydrothermal Process)
The steam explosion process requires highly
compressed water, 200e2000 psi, and high temper-
ature, 180e230
C, during the short processing time
of 1e20 min in the presence of a catalyst (Wayman
and Lora, 1978, 1979, 1980; Datar et al., 2007; Zhang
et al., 2007; Ruiz et al., 2008). Hydrolysis, aryl ether
cleavage and homolytic cleavage of CeC bond,
demethoxylation, alkylation, and condensation reac-
tions occur during the steam process (Wang et al.,
2009; Kang et al., 2011). While the aromatic groups
in the lignin are not degraded by steam explosion
process, b-O-4 ether linkages and CaeCb bonds are
being cleaved during the process. The resulting lignin
has a similar character as organosolv lignin, low
molecular weight, and good solubility in organic
solvents (Wayman and Lora, 1978; Shimizu et al.,
1998). Steam explosion has great potential because
this process is the first step to produce biofuel prior to
fermentation of polysaccharide (Glasser and Wright,
1998). A few examples of lignin subunits that can be
2: E XTRACTION AND T YPES OF L IGNIN 21

Figure 3 Proposed subunits of lignin through a steam explosion (hydrothermal) process.

produced by hydrothermal process are demonstrated
in Figure 3.
2.6 Dilute Acid Lignin
Lignin can be produced by pretreatment of
biomass feedstock with various acids: sulfuric
acid (Brennan et al., 1986; Converse et al., 1989;
Thompson et al., 1992; Esteghlalian et al., 1997),
nitric acid (Brink, 1994), hydrochloric acid (Israilides
et al., 1978; Goldstein et al., 1983), phosphoric
acid (Israilides et al., 1978), oxalic acid (Chaturvedi
and Verma, 2013), and peracetic acid (Chaturvedi
and Verma, 2013). For example, dilute sulfuric acid
(0.5e1.4%) treats biomass under high temperature,
165e195
C, for 3e12 min followed by a water-
washing step (Guo et al., 2008; Ruiz et al., 2008).
The dilute acid treatment can produce lignin under
relatively mild acidic conditions with high solubility
of obtained lignin as well as low production cost
(Selig et al., 2007). The most important step of dilute
acid treatment is acid hydrolysis of raw biomass. The
acid hydrolysis let the raw biomass release mono-
meric sugars and soluble oligomers from the cell wall

Figure 4 Dilute acid pretreated biorefinery process and lignin recovery (Azadi et al., 2013; Ragauskas et al., 2014).
22
matrix to yield the porous structure of biomass
without affecting hemicellulose and lignin. The
porous hemicellulose and lignin structure offers easy
access to enzymes that may produce a biofuel
precursor, xylose and other fermentable sugars
(Mohanty et al., 2009; Chaturvedi and Verma, 2013;
Badiei et al., 2014). Following processing, the lignin
remains as a solid residue (Ragauskas et al., 2014)
(Figure 4). The overall conversion rate, however, is
quite low and the resulting lignin contains sugar
derivative impurities (Guo et al., 2008; Ruiz et al.,
2008; Mohanty et al., 2009).
3. Conclusions
The diversity of plant sources and processing
methods results in a broad range of lignins available
for use in materials applications. In addition to
established products, such as kraft lignin and
lignosulfonates, newer forms of lignin derived from
steam explosion, organosolv, or dilute acid treat-
ment broaden the range of chemical characteristics.
However, the range of molecular weight and varying
concentrations of aliphatic hydroxyl, phenolic,
carboxylate, and sulfonate groups can make lignin
choice a design variable when developing materials
based on it. Knowing the source and processing
methods provides an important starting point for
predicting the characteristics of a given lignin, and
numerous analytical methods have been estab-
lished to perform a definitive characterization.
With knowledge of these attributes, rational design
of a next generation of lignin-based materials is
possible.
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 3 Lignin Interunit Linkages and Model Compounds
Faculty of Forestry, University of Toronto, Toronto, ON, Canada
2
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, Canada
O U T L I N E
1. Introduction
2. Common Linkages
3. Model Structure of Lignin
4. Model Compounds of Lignin
5. Advanced Characterization Technique
5.1 Nuclear Magnetic Resonance Spectroscopy
5.1.1 1H NMR
5.1.2 13C NMR
5.1.3 31P NMR
1. Introduction
Lignin is an integral component of plant cell walls,
and it is one of the most abundant organic substances
on earth, next to cellulose. Lignin is a polymeric
material that consists of the cross-linked component
of three monolignols: coumaryl alcohol (H), con-
iferyl alcohol (G), and sinapyl alcohol (S) (Zakzeski
et al., 2010). The proportions of monolignols
distinctly differ in every plant, for example, plant
species and plant geographical locations, among
others. For example, while softwood dominantly
contains coniferyl alcohols as compared to sinapyl
alcohol, they are found in equal proportions in
hardwood (Fengel and Wegener, 1984). Lignin is
generated in large quantities by the pulp and paper
industry as a by-product, and thus is a readily avail-
able source of renewable organic substances.
However, it is commonly burned as a low-grade fuel
and its industrial usage is limited. Nevertheless, in
the past few decades there has been extensive work to
exploit the advantageous structural properties of
Lignin in Polymer Composites. http://dx.doi.org/10.1016/B978-0-323-35565-0.00003-5
Copyright © 2016 Elsevier Inc. All rights reserved.
Nikhil D. Patil 1, Nicolas R. Tanguy 1 and Ning Yan 1,2
1
27 5.1.4 HSQC Analysis 36
5.2 Molecular Mass Spectrometry 38
28
5.2.1 Matrix-Assisted Laser Desorption/
29 Ionization Time-of-Flight Mass
Spectrometry 39
30
5.2.2 Electrospray Ionization Mass
30 Spectrometry 41
30
6. Conclusions 43
31
33 References 43
35
lignin, which translated in its usage as a substitute/
partial substitute to petroleum-based aromatic
chemicals and composites. Some examples of these
developments include lignin-based polymers (Koike,
2012), lignin-based carbon fiber (Kadla et al., 2002),
and lignin fiber composite materials (Thielemans
et al., 2002). The development of lignin-based
materials has been difficult as compared to that of
cellulose, largely due to the large molecular size of
lignin and to the lack of understanding of its struc-
ture. However, the increasing sophistication of
spectroscopic instruments and the pioneering work of
several research groups have considerably improved
our understanding of lignin structure in recent
years. Traditionally, there have been many practical
difficulties in investigating the structure of the
lignin polymer, because of its low solubility in
a commonly recognized solvent and rigidity toward
most chemicals. In an attempt to investigate the
structure of lignin, many researchers are using
similar approaches, which involve breaking the
polymer into smaller subunits by various processes
27
28 L IGNIN IN P OLYMER C OMPOSITES

like ozonolysis, pyrolysis, hydrocracking, oxidation, Table 1 Approximate Percentage of Linkages Found
thioacidolysis, and biodegradation (Tang et al., 2010; in Softwood and Hardwood Lignin (Chakar and
Faix et al., 1991; Tien and Kirk, 1983; Ventorim Ragauskas, 2004; Adler, 1977)
et al., 2014; Lapierre et al., 1991). These smaller
subunits are then analyzed using advanced spectro- Approximate Percentage
scopic and spectrometric techniques. In this book Linkage Type Softwood Hardwood
chapter, we will first recapitulate the common
beOe4 45e50 60
linkages and structural features of lignin polymer.
Then, we will discuss the advantages and limitations 5e5 18e25 5
of the different techniques used routinely for the be5 9e12 6
structural characterization of lignin. Finally, we will 4eOe5 4e8 7
review the latest achievements toward understanding
be1 7e10 7
the structural features of lignin polymer.
beb 3 3

2. Common Linkages
The common interunit linkages and functional
groups present in a lignin sample have already been
elucidated. Based on structural investigation studies,
the most familiar linkages found in lignin are
beOe4, 5e5, be5, 4eOe5, be1, and beb and are
shown in Figure 1 (Zakzeski et al., 2010). The
relative percentage of each linkage in lignin depends
upon biomass sources, as illustrated in Table 1, and
was found to vary depending on the technique used
to isolate lignin. However, it is established that the
beOe4 is largely prominent among all other link-
ages, and turn out to be more than 50% in most plant
species. Additionally, in plants, the lignin polymer is
covalently bonded to carbohydrates through various
linkages believed to be phenyl glycoside bonds,
esters, and benzyl ethers; the intricate structure is
known as ligninecarbohydrate complexes (LCC)
(Lapierre et al., 1991). Hence, the isolation of lignin
from plant biomass through pulping techniques
remains a difficult task, as processes involve the
cleavage of ligninecarbohydrate covalent bonds and
the depolymerization of lignin. In pulping and
biomass pretreatment processes, depolymerization
largely occurs through the cleavage of beOe4
linkages. However, the quantity of beOe4 linkages
in lignin varies depending on the proportion of S, G,
and H units. The methoxy groups at the 3rd and 5th
position of the aromatic ring in the S unit prevent the
formation of be5, 5e5, or dibenzodioxocin link-
ages, and thus the S/G/H ratio directly affects the
percentage of beOe4 linkages in lignin.

β-O-4 5-5 β-5 4-O-5

β-1 β-β dibenzodioxocin spirodienone

Figure 1 Common linkages in lignin.

3: L IGNIN I NTERUNIT L INKAGES AND M ODEL C OMPOUNDS 29

Consequently, the degradability of lignin is related
not only to its isolation process, but also to its source
(Thielemans et al., 2002).
3. Model Structure of Lignin
Once basic knowledge was obtained on the
chemical structure of lignin’s monomeric units as
well as the most common linkages, researchers were
able to design model structures that mimic the
chemical structure of native lignin. Traditionally,
these model structures have been designed via the
structural characterization of lignin after chemical
pretreatment or synthesizing artificial lignin polymer
by dehydrogenation polymerization. The structural
information obtained is usually implemented into
computational models to generate larger structures.
Freudenberg et al. pioneered this field of work in
1963 with the construction of the first model structure
of softwood lignin (Freudenberg, 1964). The model
was fabricated by investigating the structure of
enzymatic dehydrogenation of coniferyl alcohol, and
this led to a polymeric material having a very similar
structure to that of lignin. This first model provided
crucial information on the linkages in native lignin
and was later refined in a series of papers and is
shown in Figure 2 (Freudenberg and Harkin, 1964;
Freudenberg, 1965; Freudenberg and Neish, 1968).
Following this, several research groups made
significant contributions to the fabrication of a more
representative model structure of lignin, among
which some of the most notable are Adler (1977),
Sakakibara (1980), and Nimz (1974). In contrast to
the approach followed by Feudenberg et al., these
groups used the characterization results of the
degradation product of lignin to construct a model
structure. While these preliminary models were found
to give relatively accurate information on lignin
structure, they did not account for the heterogeneous
structure of lignin in plant cell walls (Matsukara and
Sakakibara, 1969) and for the bonding/structural
configuration between lignin and carbohydrates
(Yaku et al., 1976; Eriksson et al., 1980).
In order to obtain a more accurate picture of the
structure of native lignin, researchers have then
aimed at characterizing LCC (Iversen and
Wännström, 1986; Gierer and Wännström, 1986;
Karlsson and Westermark, 1996; Lawoko et al.,
2003; Lawoko, 2013). First, these efforts have
allowed to determine the nature of the association
between lignin and carbohydrates in plants. The
results suggested the presence of covalent bonds
between carbohydrates and lignin and three major
types of chemical bonds were proposed: benzyl
esters and ethers and phenyl glycoside linkages
(Fengel and Wegener, 1984; Lawoko et al., 2003;
Lawoko, 2013). Secondly, the characterization of
LCC demonstrated the presence of two distinct lignin
structures that occur depending on the nature of the

CH3O
OH

O OCH3
O
HO HO
OCH3 CHO
HO
OH
OCH3 O
HO OH
HO
OH
HO O
O OH H3CO
O
OCH3
OCH3
O
O OH
HO
CH3O OCH3 OH
OH O
O OH
O
OCH3 OH
HO O
OH OCH3
O OH
HO H3CO

CHO

HO
O
OCH3
HO

Figure 2 Model structure of spruce lignin (Adler, 1977).

Image adapted with permission from reference. Copyright 1977 Springer-Verlag.
30
carbohydrate bonded to the lignin molecule (Lawoko
et al., 2005). These results indicated that the fiber
wall possesses a more complex pattern than what was
previously suspected. While these results shed some
light on the chemical structure of lignin, there have
been large controversies in the findings due to the
effects of the isolation methods and fiber morphology
on the structure of LCC (Gierer and Wännström,
1986; Lawoko et al., 2003). For example, LCC
isolated via the pulping process provided evidence
of the formation of additional bonds between
carbohydrates and lignin (Gierer and Wännström,
1986). Although more successful procedures were
recently reported in the literature to dissolve LCC
with keeping the complex structural integrity
(Lawoko et al., 2003; Lawoko, 2013), these diffi-
culties have considerably slowed down the under-
standing of the structure of native lignin, and it is
clear that there is much work needed prior to the
design of model structures that accurately represent
the native lignin.
4. Model Compounds of Lignin
The depolymerization of lignin polymer into its
lower molecular-weight oligomers and monomers is a
remarkable approach to generate bulk chemicals and
biofuels. However, the use of lignin as a precursor for
the synthesis of polymeric materials is difficult. The
lignin separated from biomass by numerous pretreat-
ment processes generally contains nonwoody impu-
rities, has a highly heterogeneous structure, and
remains insoluble in most organic solvents. Also,
aromatic lignin macromolecules containing phenolic
and aliphatic hydroxyl groups can catalyze unwanted
polymerization reactions or it may likewise degrade
upon heating. In short, it is particularly tough to
predict the reactivity and to characterize lignin
polymer.
On the other hand, the study of lignin model
compounds bearing similar linkages and function-
ality of native lignin seems to be relatively practical.
Firstly, characterization of model compounds and
purification of products are much simpler than
complex lignin polymer. Secondly, chemical reac-
tions of model compounds are apparently more
predictable, issues of selectivity are addressed with
enough certainty, and mechanistic questions are
accountable. Figure 3 illustrates examples of lignin
model compounds.
L IGNIN IN P OLYMER C OMPOSITES
After previous failed attempts to manufacture
polyurethanes using unmodified and modified
lignins, Detoisen and group studied the reaction
between hexamethylene diisocyanate (HDI) and
phenolic lignin model compounds like vanillin,
phenol, p-hydrobenzaldehyde, guaiacol, eugenol,
and isoeugenol to examine how well condensation
reaction proceeds and to generalize the specificity of
the reactions (Detoisen et al., 1985). The depoly-
merization of b-aryl ether has been extensively
studied in the past and mainly focused on oxidative
and reductive cleavage (Ren et al., 2013). Yelle et al.
investigated regioselective reactivity of different
hydroxyl groups in lignin model compounds (b-aryl
ether, phenylcoumaran, and pinoresinol) toward
phenyl isocyanate. This study helped to elucidate the
extent of reaction between isocyanates and cellulose,
hemicellulose and lignin in loblolly pine (Yelle et al.,
2011). Wool and coworkers demonstrated that
renewable functionalized lignin model compounds
can be incorporated in the synthesis of homopoly-
mers and block copolymers to eventually replace
petroleum-based materials (Holmberg et al., 2014).
Likewise, lignin model compounds play an important
role to investigate the detailed synthesis patterns of
the reaction and predict how well high-molecular-
weight lignin polymer will behave.
5. Advanced Characterization
Technique
5.1 Nuclear Magnetic Resonance
Spectroscopy
Over the years, nuclear magnetic resonance
(NMR) spectroscopy has proven itself to be one of the
most useful methods to investigate the structure of
lignin. The detailed structural analysis of functional
groups and linkages in the complex lignin polymer is
well documented by researchers, and NMR spec-
troscopy results showed satisfactory agreement with
structural details obtained by wet chemical method,
IR spectroscopy, computational studies, etc. (Tien
and Kirk, 1983; Chakar and Ragauskas, 2004).
Moreover, in recent years the NMR technique has
been consistently used for routine investigation of the
structure of lignin. In this section, we focus on 1H
NMR, 13C NMR, 31P NMR, and heteronuclear single
quantum coherence (HSQC) spectroscopy. The NMR
spectroscopic methods have considerably evolved
3: L IGNIN I NTERUNIT L INKAGES AND M ODEL C OMPOUNDS
Figure 3 Molecular structure of selected lignin model compounds.
over the past few years due to extensive research,
which has resulted in the introduction of modern two-
dimensional heterocorrelated NMR (HSQC) spec-
troscopy. This technique played a major role in the
elucidation of the structure of lignin.
5.1.1 1H NMR
The use of 1H NMR for the structural investigation
of lignin was first reported in 1964 by Ludwig et al.
They compared the 1H NMR spectra of a variety
of monomeric and dimeric model compounds to those
of acetylated dioxane lignin, milled wood lignin
(MWL), and brauns lignin from spruce and hemlock
(Ludwig et al., 1964a,b). These publications shed
considerable light on the chemical structure of lignin
by providing additional evidence of various interunit
linkages including b-aryl ether, phenylcoumaran, and
5,5-biphenyl in lignin. A semiquantitative estimation
of various functional groups including benzylic
hydroxyls, aliphatic and aromatic hydroxyls, and total
aliphatic hydrogens in lignin was also conducted and
the results obtained agreed well with earlier
estimations.
This work has allowed the design of a first data-
base of the chemical shifts associated with the
functional groups and linkages found in lignin (Lai
and Sarkanen, 1971). This database was further
extended and used to compare the structural differ-
ences of different model compounds and lignin from
different tree species (Lundquist, 1979a,b, 1980,
1981; Lundquist et al., 1983). While these studies
provided vital information on lignin structure, at the
time NMR spectroscopy suffered from a poor reso-
lution and high signal-to-noise ratio, which hindered
31
more thorough investigations. These technological
limitations were progressively overcome in the
following years, notably with the introduction of
Fourier-transform-NMR and increased magnetic
field strength, and allowed the identification of other
functional groups in lignin (e.g., formyl groups and
benzaldehyde units).
Perhaps one of the largest contributions to the
routine use of 1H NMR for lignin characterization was
from Lundquist’s group who extensively investigated
the structure of MWL from birch and spruce
(Lundquist, 1979a,b, 1980, 1981; Lundquist et al.,
1983; Lundquist and Olsson, 1977). His group notably
established the use of 1H NMR as a robust technique
for estimating the frequency of the occurrence of
various functional groups in lignin including aliphatic/
phenolic hydroxyl, formyl, methoxy, and carboxylic
acid groups (Freudenberg, 1964; Eriksson et al., 1980).
A typical 1H NMR spectrum of acetylated MWL
from birch is shown in Figure 4. The chemical shifts
associated with protons appear from 6.0 to 8.0 ppm
for aromatics; 4.2e6.2 ppm for interunit ether link-
ages; 3.6e3.8 ppm for aromatic methoxy group; and
0.5e2.0 ppm for aliphatic protons. The acetylation of
lignin is by far the most common method for struc-
tural investigation with 1H NMR spectroscopy. This
procedure increases the solubility of the lignin
polymer in organic solvents, which considerably
improves the spectral resolution, as shown in
Figure 4. For example, Ha and Hb peaks that overlap
in nonderivatized MWL are properly separated after
acylation. It is important to note that proton NMR
peaks of lignin are not particularly sharp, but still
provide significant spectral information (Lundquist,
1979a, 1980).
32 L IGNIN IN P OLYMER C OMPOSITES

δ 9 8 7 6 5 4 3 2 1 0

Figure 4 1H NMR spectrum of acetylated milled wood lignin from birch in chloroform-d (Lundquist, 1979a)
(Permission in progress).

The assignment of typical functional groups and Integration of 1H NMR spectra is a powerful tool to
linkages to the corresponding chemical shift is given estimate the relative amount of functional groups and
in Table 2. Extensive databases can be found various linkages in lignin. Based on the spectrum in
elsewhere. Figure 4, the authors integrated the peaks at d 1.95,
2.02, 2.13, and 2.29 that correspond to acetyl groups
Table 2 Signal Assignments in the 1H NMR Spec- to quantify aliphatic and phenolic hydroxyl groups.
trum of Acetylated Milled Wood Lignin from Birch They integrated the peaks ranging from 6.3 to
Lignin (Lundquist, 1979a) (Permission in progress) 8.0 as a reference by assuming that the sum corre-
sponds to 2.3 protons per phenylpropane unit. The
Chemical results indicated the presence of 1.5 acetyl groups per
Shit phenylpropane unit in MWL lignin from spruce.
Assignment d (ppm)
Similarly, this method can be extended to the quan-
Aliphatic acetate 1.95, 2.02, tification of interunit linkages in lignin. For example
2.13 and based on the spectrum in Figure 4, the peaks at
Aromatic acetate 2.29 d 6.01 and 4.6 that correspond to Ha and Hb protons of
Hb in beb structures 3.08 the beOe4 structure were integrated to estimate the
relative amount of beOe4 linkages in the lignin
Proton in the methoxyl group 3.76
sample. The results indicated that MWL from
Hg in several types of structures 4.18e4.43 spruce contains 40e50% of beOe4 interunit
Hb in beOe4 structures 4.60 linkages.
Ha in be5 structures 5.44 This approach is also useful to obtain qualitative
and quantitative information on other linkages and
Ha in beOe4 and be1 structures 6.01 functional groups in lignin (Lundquist, 1979a;
Aromatic protons in syringyl units 6.60 Lundquist and Olsson, 1977), and thus is adequate to
Aromatic protons in guaiacyl units 6.94 compare the effects of chemical processes, chemical/
Aromatic protons located ortho to 7.50
biological treatments, and lignin origin on the
carbonyl groups chemical structure of lignin (Capanema et al., 2004).
While a considerable amount of information on lignin
Formyl protons in cinnamaldehyde 9.67
can be obtained through 1H NMR characterization,
units
this technique has intrinsic limitations. The 1H NMR
Formyl protons in benzaldehyde 9.86 spectra of lignin typically reveal substantial over-
units lapping of peaks. This phenomenon is due to the
3: L IGNIN I NTERUNIT L INKAGES AND M ODEL C OMPOUNDS
restricted chemical shift region, only extending from
d 0 to 10 ppm. In addition, the high-molecular-weight
of lignin restricts the random motion of polymer
segments, which reduces the relaxation time and
leads to peaks that are broad and poorly defined.
Even the 1H NMR characterization of simple trimer
model molecules of lignin has exceptionally complex
spectra due to the numerous stereochemical varia-
tions, which eventually leads to broader peaks (Ralph,
1993). Thus, 1H NMR spectra are not adequate for
a detailed investigation of the structure of complex
lignin polymer but they are a useful tool to study low-
molecular-weight fragmented MWL or simple
synthetic lignin molecules having regular linkages.
13
5.1.2 C NMR
13
C NMR is an intrinsically better choice than 1H
NMR for a comprehensive investigation of the
carbon skeleton in complex lignin polymers. Firstly,
the chemical shift ranges are considerably wider and
extend from 0 to 220 ppm. Secondly, proton decou-
pling removes the splitting of peaks resulting from
the neighboring H atoms. Therefore, each carbon
atoms or set of equivalent carbon atoms in a molecule
shows single set lines (Lüdemann and Nimz,
1973). Compared to 1H NMR, higher-concentrated
samples are needed for 13C NMR because this
Table 3 13C NMR Chemical Shift Assignments for Lignin and Quantification of the Spectral Region
(Holtman et al., 2004)
Spectroscopic
Range d (ppm) Spectral Region
195e190 Carbon from carbonyl
type structures
160e141 Aromatic CeO bond
141e125 Aromatic CeC bond
125e103 Aromatic CeH bond
57e54 Methoxyl content
125e103 Degree of
condensation
MWL, milled wood lignin; CEL, cellulolytic enzyme lignin.
33
reduces the S/N ratio and a spectrum is obtained in
less time. Nevertheless, with an increase in concen-
tration, lignin becomes less soluble and the viscosity
of the solution increases. The use of 13C NMR for the
structural investigation of lignin was pioneered by
Linz and Lüdemann in 1974 (Lüdemann and Nimz,
1973, 1974; Nimz et al., 1974). Following a similar
approach to Ludwig’s group, they first assigned the
chemical shifts of carbon atoms from various model
compounds and then characterized MWL from beech
and spruce.
Currently, extensive databases of the 13C NMR
chemical shifts of lignin are available in the literature
and more information on the individual chemical shifts
found in lignin is available elsewhere (Kishimoto
et al., 2008; Ralph et al., 2004). Briefly, the chemical
shifts associated with different sets of equivalent
carbon are located in different regions of the spectrum,
as shown in Table 3. For example, the chemical shifts
found from 160 to 100 ppm represent carbon atoms
from aromatics, and this region is subdivided into
three regions: aromatic carbon attached to oxygen
(160e141 ppm), alkyl group (141e125 ppm), and
hydrogen (125e103 ppm). It should be noted that the
aliphatic region, located from 50 to 90 ppm of 13C
NMR spectrum, has poor resolution and suffers from
overlapping peaks. This issue can be partially allevi-
ated by acetylation of lignin samples (Nimz et al.,
Number of Moieties
per Aromatic Ring
Examples MWL CEL
0.12 0.08
2.02 2.06
1.53 1.42
2.57 2.64
0.96 1.17
0.43 0.36
34 L IGNIN IN P OLYMER C OMPOSITES

1981). In addition, Landucci (1991) reported various 6.12, which is contributed from the aromatic carbon
factors to increase the sensitivity and resolution of the and double bonds of coniferyl alcohols and con-
13
C NMR spectra of lignin. iferaldehydes (Holtman et al., 2004). In addition,
Traditionally, the use of 13C NMR to quantify these analyses may be the source of serious bias
functional groups and linkages in lignin has been when quantifying moieties in processed lignin,
conducted by comparing the integration of the altered or genetically modified lignins as the
different structural group peaks in the spectrum with resulting materials contain degraded side chains and
the aromatic region from 160 to 100 ppm. This fragmented aromatic rings.
approach was found to be suitable as lignin is The need for a more accurate method to quantify
a polymer composed of phenylpropane (C9 or C6C3) the different moieties is evident. A methodology
repeating units. Hence, the quantitative results are to alleviate this issue was recently reported by
reported as moieties/100 phenylpropane units. Argyropoulos et al. A known amount of an internal
Following this method, Kadla’s group compared the standard was added to the dissolved lignin sample
amount of various linkages and functional groups in prior to NMR characterization in order to provide an
MWL and cellulolytic enzyme lignin (CEL) from absolute reference in the quantification of different
loblolly pine. MWL 13C NMR spectra are shown in moieties in treated lignin (expressed in millimoles
Figure 5 and quantitative results are shown in per gram of sample) (Xia et al., 2001). The results
Table 3 (Holtman et al., 2004). The results indicated agreed well with that of 31P NMR spectroscopy
that a higher amount of condensed structures and and that obtained by other analytical methods. Thus,
a lower amount of beOe4 linkages were found in this method appears to be a promising tool for the
MWL compared to CEL. However, in this study, the unbiased quantification of a large range of moieties
reference integration of aromatic region was set at in native or treated lignin.

MWL-Ac
28
20 21

21 28
MWL
20

27
DMSO

90 80 70 60 50
78 26

18
9 19

17 25
10
23
6 22
12 21
13 14 24
11 28
15
16 20
1 23 5
29 30
4

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

Figure 5 Quantitative 13C NMR spectra of milled wood lignin (MWL). Comparative aliphatic region spectra of the
acetylated MWL (MWL-Ac) and MWL (Holtman et al., 2004).
Reprinted with permission from reference. Copyright (2004) American Chemical Society.
3: L IGNIN I NTERUNIT L INKAGES AND M ODEL C OMPOUNDS
By conventional methods, the quantitative deter-
mination of interunits linkages in lignin using the
inverse gate decoupling technique is an exceedingly
time-consuming process (Landucci, 1991; Holtman
et al., 2004). However, this issue can be alleviated by
adding minute amounts of chromium(III) acetyla-
cetonate, a relaxation agent, in the NMR solvent to
shorten the spin-lattice relaxation time of the carbon
nuclei, thus allowing data to be collected more
quickly (Nimz et al., 1981).
In general, the chemical shifts in the 13C NMR
peaks of aromatic carbons in model compounds do
not perfectly match with those of isolated lignin, but
the chemical shifts of artificial lignin do match with
those of isolated lignin.
31
5.1.3 P NMR
31
P NMR is a reliable technique that can be used as
an alternative to 1H NMR for the quantitative investi-
gation of hydroxyl groups in lignin (Argyropoulos
et al., 1993). This technique allows the estimation
of syringyl, guaiacyl, and p-hydroxyphenyl-free
phenolic groups, as well as primary groups and
secondary groups that were not accessible using
other spectroscopic techniques (Argyropoulos,
1994a,b). It is important to note that the 31P NMR
technique is much more sensitive than 31C NMR but
less sensitive than 1H NMR as 31P isotopes have 100%
abundance, a relatively high magnetogyric ratio, and
a nucleus with spin of 1/2. 31P spectra typically have
a large range of chemical shifts, therefore separating
the peaks for different classes of hydroxyl groups is
achieved. In addition, data can be acquired more
quickly even with small amounts of sample.
In this method hydroxyl groups in the lignin are
quantitatively derivatized by excess of phospholane
reagents under mild reaction conditions. The
derivatization step is shown in Figure 6. Verkade
et al. using lignin model compounds derivatized
different types of hydroxyl groups using five distinct
phosphorous-containing reagents. The results
revealed that the chemical shifts usually do not
mutually overlap and 2-chloro-4,4,5,5-tetramethyl-
1,3,2-dioxaphospholane (TMDP) was found to be
Figure 6 Schematic representation of the derivatization step of the lignin hydroxyl group using TMDP.
35
the best reagent (Wroblewski et al., 1988). The
database of model compounds derivatized by TMDP
is available elsewhere (Wroblewski et al., 1988; Faix
et al., 1994). Most of the work on 31P-derivatized
lignin reported in the literature used TMDP to label
the hydroxyl group, and 1.6:1 pyridine/CDCl3 was
used as the NMR solvent (Granata and Argyropoulos,
1995; Pu et al., 2011). This reagent proves to be
helpful in quantifying hydroxyl groups using an
appropriate internal standard. As previously re-
ported by Argyropoulos et al., TMDP happened to
be more stable than other phospholanes due to the
presence of dimethyl groups on the vicinal carbon of
pinacol.
The quantitative estimations of total phenolic
hydroxyl groups and total hydroxyl groups obtained
by 31P NMR are comparable to that obtained by
1
H NMR and wet chemistry (Argyropoulos, 1994a;
Faix et al., 1994). Following the establishment of
this technique, it has been widely used to investigate
the structural effects of various pulping processes
on the structure of lignin, including CEL, MWL,
Kraft, and steam treatment (Guerra et al., 2006; Froass
et al., 1996; Pereira Ramos, 2003). A typical quanti-
tative 31P NMR spectrum of TMDP-derivatized three
round robin lignins obtained by different processes is
shown in Figure 7 (Granata and Argyropoulos, 1995).
The NMR peaks of hydroxyl groups that belong to
aliphatic, syringyl, guaiacyl, p-hydroxylphenyl units,
and carboxylic acid are well resolved from each other;
however, aliphatic and secondary hydroxyl peaks
overlap and were not projected separately. Cyclo-
hexanol was used as an internal standard in this
experiment; N-hydroxy compounds have been re-
ported as a more suitable internal standard as they do
not interfere with the chemical shift of lignin.
Recent years have seen the emergence of novel
quantitative characterization protocols with 31P
NMR. Particularly, the derivatization followed by
reductive cleavage (DFRC) of lignin after derivati-
zation has attracted large interest from lignin chem-
ists (Guerra et al., 2006). The selective cleavage of
aryl ether linkages induced by DFRC leads to the
release of phenolic hydroxyl groups, and thus the
estimation of condensed and uncondensed units
36
Internal Standard Condensed Phenolic Units
Syringyl
Guaiacyl and Demethylated
Phenolic Units
Aliphatic OH Phenolic Units
Sucroline Acid Hydrolyzed
152 150 148 146 144 142 140
31
P (ppm)
Figure 7 Quantitative 31P NMR spectra of three
TMDP-derivatized commercial lignins and cyclohexa-
nol as internal standard (Granata and Argyropoulos,
1995).
Reprinted with permission from reference. Copyright
(1995) American Chemical Society.
linked through aryl ether linkages in lignin as the
phenolic hydroxyl released can be quantified (Guerra
et al., 2008).
5.1.4 HSQC Analysis
NMR spectroscopic methods have considerably
evolved over the past few years due to extensive
research, which has resulted in the introduction of
modern two-dimensional heterocorrelated NMR
spectroscopy. This technique has provided another
powerful tool for the structural investigation of lignin
as the interunit linkages and the aromatic peaks that
overlap in 1D NMR spectra can be distinguished
(Lundquist, 1979b). Particularly, the 13C-1H HSQC
spectroscopy has attracted large interest to explore
previously unknown lignin subunits, LCC linkages
and confirmed some linkages elucidated by 1D NMR
(Yuan et al., 2011; Kilpelainen, 1994).
Ralph and Sun groups largely contributed to this
field through multiple publications focusing on the
identification of unknown structures via the charac-
terization of synthetic model compounds and lignin
from different plant species by HSQC (Yuan et al.,
L IGNIN IN P OLYMER C OMPOSITES
2011; Marita et al., 1999; Lu and Ralph, 2003; Ralph
et al., 1998; Boerjan et al., 2003; Wen et al., 2012,
Carboxylic Acids 2013, 2015). Figure 8 shows 13C-1H HSQC NMR
spectra of lignin separated by two approaches: ionic
Alcell Organosolv
(Mixed Hardwoods)
liquid-organic solvent mixture lignin and alkaline
ethanol solvent lignin (AEL) (Sun et al., 2013). The
HSQC sequence in Figure 8 correlates the chemical
shift of 1H with the chemical shift of the directly
Induline Kraft
(Mixed Softwoods)
bonded 13C. The two most important regions in the
spectra are the side chain (dH/dC 2.7-5.1/47-91) and
the aromatic (dH/dC 6.0-7.5/99-126).
Signals for methoxyls and beOe40 linkages were
noticeable in the side chain region. The HSQC adds
(Bagasse) new peaks to the spectra that were overlapping in its 1D
counterpart. Unusual lignin linkages in both the frac-
tions were observed and were identified as acylated g
carbon of beOe40 units. The HSQC spectrum also
138 136 134 revealed a distinct peak that represents beDeXylp of
LCC. The 1H/13C chemical shift correlations of most
common linkages are listed in Figure 9 and Table 4
(beOe40 , beb0 , be50 ). The aromatic region mostly
consists of syringyl (S) and guaiacyl (G) units; Ca-
oxidized S0 units were also observed at dH/dC 7.27/
106.7. The quantitative estimation of the S/G ratio
calculated from HSQC spectra was 3.25 for AEL and
1.20 for ionic liquid lignin fractions. And the presence
of spirodienone substructures was represented based
on minor signals.
Recently, Sun’s group investigated different
methodologies for the preparation of whole lignin,
that is, virtually intact lignin, from eucalyptus via
HSQC (Wen et al., 2015). The isolation and char-
acterization of whole lignin has attracted more
interest over the past decade to obtain a more
representative picture of native lignin. The existing
methodology for the isolation of lignin is time-
consuming, has unsatisfactory yield, and does not
provide panoramic information on the syringyl/
guaiacyl units within the plant cell walls (Lu and
Ralph, 2003). The authors reported a novel method
for isolating higher amounts of lignin from wood
and investigated the structural changes compared
with other traditionally used methods: CEL and AL.
In this novel method, the dissolution of lignin
was achieved by a mild alkaline treatment (4%
NaOH, 25  C, 24 h) followed by enzymatic hydro-
lysis. The results indicated that this methodology
favored the dissolution of a significant higher yiel-
d of lignin from plant cell walls compared with
CEL and AL but the structural features observed
were similar.
3: L IGNIN I NTERUNIT L INKAGES AND M ODEL C OMPOUNDS 37

Figure 8 2D HSQC NMR spectrum

(a, b) alkaline ethanol lignin (AEL) and
(c, d) ionic liquid lignin (ILL) (Sun
et al., 2013) (Permission in progress).

Figure 9 The main structural

units and acetylated
substructures of lignin (A)
beOe40 linkages; (A0 )
beOe40 linkages with
acetylated g carbon; (A00 )
beOe40 linkages
p-coumaroylated g carbon;
A A' A'' H (H) p-hydroxyphenyl unit; (B)
beb; (C) be5; (G) guaiacyl
unit; (S) syringyl unit, and
(S0 ) Ca-oxidized S0 units
(Sun et al., 2013).

B C G S S'
38 L IGNIN IN P OLYMER C OMPOSITES

Table 4 Assignment of Peaks in the HSQC Spectra of the Lignin Fractions (Sun et al., 2013)
(Permission in progress)

Labels dH/dC (ppm) Assignment

Bb 3.13/54.1 HbeCb in beb0 substructures (B)
eOMe 3.74/56.2 HeC in methoxyls
Ag 3.48/60.1 HgeCg in beOe40 substructure (A)
(A0 , A00 )g 3.98/63.5 HgeCg in geacetylated beOe40
substructure (A0 /A00 )
Bg 4.18/71.6 and 3.82/71.6 HgeCg in beb0 substructures (B)
(A, A0 , A00 )a 4.89/72.3 HaeCa in beOe40 substructure linked
to an S unit (A/A0 /A00 )
Cg 3.42/63.2 HgeCg in be50 substructures (C)
Ba 4.64/85.4 HaeCa in beb0 substructures (B)
Ab(S) 4.22/85.8 and 4.03/86.4 HbeCb in beOe40 substructure linked
to an S unit (A)
S2,6 6.72/104.7 H2,6eC2,6 in syringyl units (S)
0
S 2,6 7.28/106.7 H2,6eC2,6 in Caeoxidized (C]O)
phenolic syringyl units (S0 )
G2 6.96/111.7 H2eC2 in guaiacyl units (G)
G5 6.80/113.3 and 6.76/115.1 H5eC5 in guaiacyl units (G)
G6 6.89/119.6 H6eC6 in guaiacyl units (G)

HSQC has also proven to be helpful to determine
the relative proportions of interunit linkages in lignin.
Kilpelainen’s group correlated the quantitative NMR
spectra of wood lignin by modulating the polariza-
tion transfer delays of HSQC using the lignin model
compound internal standard (Heikkinen et al., 2003).
In 2005, the accuracy of the technique was further
increased by replacing the polarization transfer steps
with Carr Purcell Meiboom Gill type sequences
(Koskela et al., 2005). Another quantitative method
published by Zhang is based on selecting suitable
internal standard reference signals from the lignin
polymer (Zhang and Gellerstedt, 2007). Crestini et al.
compared different HSQC methods that reported
a quantitative analysis, and the results suggest that
the analysis based on quick, quantitative HSQC was
faster whereas the HSQC time-zero method is
simpler to implement (Sette et al., 2013). Altogether
HSQC has been extremely reliable in investigating
the structure of lignin.
5.2 Molecular Mass Spectrometry
The mass spectrometer (MS) is one of the most
important analytical tools used to predict the struc-
ture of unknown materials and performs a quantita-
tive study of complex mixtures. The three essential
components of mass spectrometry instruments are
ion source, mass analyzer, and detector. The ion
source is classified into two categories: gas-phase
source and desorption source. In the gas-phase ion-
source, a low boiling point sample is first vaporized
under high vacuum and then ionized, whereas in the
desorption ion-source nonvolatile or thermally less
stable samples are directly converted to gaseous ions.
The selection of the ion-source type depends on the
nature of the sample and the kind of data required.
Further, the accelerated ions get separated by a mass
analyzer according to their mass-to-charge ratio by
employing a magnetic or electric field. This chapter
is mainly focused on different types of mass spec-
trometry, which have been used to investigate
3: L IGNIN I NTERUNIT L INKAGES AND M ODEL C OMPOUNDS
structures of lignin oligomers and to determine the
molecular weight of the lignin polymer. Mass spec-
trometry techniques are also commonly combined
with gas chromatography (GC) or pyrolysis GC to
determine the mass of lignin fragments such as
monomers and dimers (Harman-Ware et al., 2013). In
this method, a solid/liquid sample is vaporized by
heating and undergoes fragmentation or degradation
during this step. However, soft ionization methods
are largely preferred as they allow the analysis of the
whole lignin polymer.
Soft ionization methods have been widely used to
obtain molecular weights of large polymers such as
cellulose, protein, and DNA. Methods like laser
desorption, fast atom bombardment, and plasma
desorption techniques are suitable to determine the
molecular weight of lignin, as the polymer generally
undergoes less fragmentation during ionization. In
this regard, the literature is rich with examples
where matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF
MS), thermospray MS, electrospray ionization MS,
and fast-atom bombardment have been employed to
study the whole lignin polymer (Awal and Sain,
2011; Angelis, 1996; Metzger et al., 1992).
5.2.1 Matrix-Assisted Laser Desorption/
Ionization Time-of-Flight Mass
Spectrometry
In 1985, Karas and Bachmann described the role
of introducing an absorbing matrix to facilitate the
ionization of large and thermally labile biomole-
cules in laser desorption mass spectrometry (Karas
et al., 1985, 1987). These observations eventually
led to the development of MALDI-TOF MS, in
which an analyte is mixed with excess matrix
material, deposited on sample holder, and dried
before being introduced in the MS. The sample is
irradiated by the laser, which induces desorption and
ionization of the matrix/analyte (Karas and Krüger,
2003). The most commonly used matrices are
2,5-dihydroxybenzoic acid, nicotinic acid, and
3,5-dimethoxy-4-hydroxycinnamic acid (Bocchini
et al., 1996). Recently, Ul’yanovskii and group
investigated an effective way to prepare samples
and the choice of the matrix to obtain high-quality
mass spectra (Kosyakov et al., 2014) and it was
found that better spectra are obtained using ionic
liquids as matrices as compared to conventional
crystalline matrices. MALDI coupled with TOF can
39
detect absolute molecular weight distribution of
unlimited mass range even with small amounts of
sample.
The application of MALDI-MS was put forward by
Metzger et al. (1992) to characterize MWL from
birch. The molecular weight distribution of lignin
ranges from a few hundreds to 16,000 Da, having
center of gravity at 2600  100 Da. The number-
average molecular weight, M n, from MALDI-MS
shows satisfactory agreement with previously re-
ported gel permeation chromatography results
(Metzger et al., 1992). The MALDI-MS spectra
reveal important structural information of the lignin
polymer. Firstly, the fine structure of the MALDI-MS
spectra illustrates information about a series of olig-
omers from trimers to nonamers in the range
600e1800 Da, and the hyperfine structure confirmed
that birch lignin is typically made of syringyl and
guaiacyl monomeric units. Srzic et al. (1995) reported
the analysis of model lignin, spruce and birch lignin
by negative-ion laser desorption Fourier-transform
mass spectrometry. The spectra from spruce and
model lignin illustrate that spacing between the m/z
peaks is 444, which shows the presence of trimer
building blocks. De Angelis and group reported
a structural investigation of model lignin synthesized
from the dehydrogenation of coniferyl alcohol using
horseradish peroxide and H2O2. In this study, aliquots
of the reaction mixture were quenched over different
times and analyzed by MALDI-TOF MS. The time-
dependent studies reveal that initially higher molec-
ular weight lignins are formed and then successively
degrade to smaller oligomers (Angelis, 1996). Simi-
larly, Bocchini et al. (1996) illustrate the information
on different linkages and the oligomeric distribution
of natural and synthetic lignin subject to
MALDI-TOF MS. Mattinen et al. (2011) report the
elucidation of the structure of synthetic lignin ob-
tained by polymerization of the lignans by Melano-
carpus albomyces laccase (MaL).
Recently, Pizzi and group published a series of
papers on the MALDI-TOF MS analysis of glyoxa-
lated lignin for wood adhesives (Navarrete et al.,
2013, 2012). The mass spectrometry results shown in
Figure 10 demonstrates important structural features
of spray-dried glyoxalated lignin; MS analysis
proved to be extremely helpful to examine the lig-
nineglyoxal reactions and elucidate the change in
the lignin structure after spray drying (Navarrete
et al., 2012). The MALDI-TOF MS spectra shown in
Figure 10 demonstrate that the peaks were regularly
40 L IGNIN IN P OLYMER C OMPOSITES

Figure 10 MALDI-TOF spectra of spray-dried glyoxalated lignin at different m/z ranges (Navarrete et al., 2013)
(Permission in progress).
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observer might have perceived that she was slightly marked with the
small-pox. Her skin was delicately fair, and her beautiful flaxen hair
clustered in heavy ringlets, less showery than generally worn
according to the fashion of the time, over her forehead and neck. Her
eyes were blue, swimming in softened light, and her countenance
was overspread by a regard so tender yet so full of modesty, that
she gained at the same moment the love and esteem of all who
gazed upon her; and yet, when the occasional lighting up of her
features as the King addressed her, died away, they became pale
and sad. Her smile was followed by a pensive expression, which
accorded but ill with the festivity around her.
‘Ah, times are changing!’ said the abbe, as he gazed at her; ‘and
that fair lady’s reign is nearly over. I question whether La Montespan,
with all her witcheries, will love him half so well though.’
‘Who is it?’ asked Jean.
The abbe appeared slightly astonished at the ignorance of his new
acquaintance, as he replied—
‘Who could it be but Louise de la Vallière? Ah! hers was a curious
destiny. Picked out by Louis to cover his attention to his sister-in-law
Henriette, she has supplanted her. But it does not seem likely that
the liaison will last much longer. Montespan has his heart.’
As he spoke, Mademoiselle de la Vallière rose from her seat and
crossed over to speak to Madame de Maintenon, who was sitting on
the parapet of the basin that received the water from the fountain.
She limped as she walked along, and Jean saw that she was lame.
‘She seldom dances,’ continued the garrulous abbe, ‘on account of
her defect; and so she does not care always to be present at the
balls. I can conceive the reason of her not being at the play.’
‘How was that?’ inquired the Gascon.
‘Because the King’s sentiments appear to be somewhat changed
since our Molière was commanded to write the Princesse d’Elide. He
was then madly in love with La Vallière, although at the time she
resisted all his entreaties. What else could these lines mean?’
And Jean flinched as the abbe again commenced a piece of
declamation, quoting from the piece in question in a monotonous
tone of dulness suited to the subject—
 The homage which is offer’d to a countenance refined
Is an honest indication of the beauty of the mind;
And scarcely possible it is, if love be not innate,
That a young prince should come to be or generous or great:
And this above all other regal qualities I love,
This sign alone the tenderness of royal hearts can prove!
To one like you, a bright and good career we may presage,
When once the soul is capable of loving, at your age.
Yes, this immortal passion, the most noble one of all,
An hundred goodly virtues training after it can call;
The most illustrious actions are engender’d by its fires,
And all the greatest heroes have experienced its desires.’13

Jean bowed respectfully at the termination of each line, as if he fully

concurred in the sentiments it conveyed, but was very glad when it
was over.
‘Ha! the music has ceased,’ said the abbe; ‘and there will be a
masque, and some fireworks on the Bassin de Neptune, and the
étang beyond. That will be also a trial for La Vallière. The last fetes
at night were in her honour, and they are going to use the old
machines newly decorated. It will be a renaissance of the Ile
Enchantée.’
The company retired to the banks of turf which surrounded the
Salle de Bal, Louis, and a few immediately attached to him only
remained below, amongst whom were of course La Montespan and
La Vallière. When the floor was cleared, a cavalcade of heralds,
pages, and squires, all richly clad in armour, and dresses
embroidered with thread of silver and of gold, marched into the
bosquet, the music of Lulli’s band of twenty-four violins being
exchanged for that of martial instruments. When they had taken their
places, a large car, made to imitate the chariot of the Sun, was
slowly moved into the ballroom by concealed means, conveying the
Sun, surrounded by the four Ages of gold, silver, iron, and brass; the
Seasons, the Hours, and other mythological characters. On arriving
opposite the point where Louis was sitting, the colossal machine
halted, and Spring addressed a complimentary oration to the King,
involving also some flattering sentences for Madame de Montespan
and Mademoiselle de la Vallière—but more especially for the former.
When this had finished, the young person who had played the
character of Spring descended from the car, and having offered
some rare bouquets to Louis and his favourites, took her place
amongst the company. She was the only performer in the masque
who did this, being the lovely Françoise de Sévigné—the daughter of
Madame de Sévigné—now about eighteen years of age. She had
been requested, on account of her extreme beauty and propriety of
expression to play the part,—since, in the fetes at Versailles, it was
not usual for the dames de la cour to figure.
This portion of the masque having finished, the various
mythological personages descended as well, but it was only to bring
in a number of long tables, which they placed before the company on
the lowest turf-benches of the amphitheatre. These they spread with
cloth of gold, and thus gave the signal for another large piece of
mechanism to enter, representing a mountain, on which were seated
Pan and Diana. When it stopped, these deities opened various parts
of it, and aided by the others, brought out an exquisite collation,
which they placed upon the tables, the music playing all the time. At
the first sight of the banquet, the abbe bustled off to find a place at
the tables; and Jean Blacquart, not wishing to lose the caste which
he imagined he had acquired, and knowing that he could not join the
feasters, turned upon his heel into the gardens, to see if anywhere
he could discover Maître Picard.
Few who had seen Marie de Brinvilliers, as she mingled in the
dances which had been taking place before the appearance of the
pageant, would have conceived that any other feelings but those of
mirth and excitement amidst the glittering throng by which she was
surrounded were paramount in her bosom. There was the same kind
expression—so terrible in its quietude had her heart at that time
been laid open—the same sweet features, almost girlish in their
contour (for although she was now thirty years of age, she could well
have passed for eighteen), which all admired so much. And when
she smiled, the witchery that played around her rosy mouth, as her
parted lips displayed that most beautiful set of teeth, whose dazzling
whiteness had been the theme of more than one court epigram,
captivated by its spell all who came within its magnetic influence. Of
all that lovely throng of women who graced the court of Louis
Quatorze—the bevy of fair dames, so many of whom swelled the
conquests of that heartless, selfish, roué monarch—the Marchioness
of Brinvilliers was the most fascinating. And this fair creature, who
now, in the light of her peerless beauty, of which she seemed
unconscious, moved gracefully in the dance—this fearful woman—
had broken up a home; deserted her children at an age when a
mother’s guidance was all they needed, with an unnatural
indifference towards her offspring that one might have sought for in
vain amidst the lowest animals; and adding parricide as a coup to
her already dark career, was yet but on the verge of the terrible line
she had marked out to be pursued. Woman, in her love and
gentleness, in her ministering care and patient endurance, when all
the holiest attributes of her sex exist in her character, approaches far
nearer to the angel than her companion, man. Alas! it is equally true,
that in the absence of these characteristics she sinks far deeper in
approximating to the demon!
Gaudin de Sainte-Croix had studiously avoided Marie in the Salle
de Bal. The reports which had crept about Paris rendered them both
cautious, for the present, of their deportment, although they were
about to set all restraint at defiance. Whilst she was dancing, he had
walked out into the gardens of the palace, that the night air might
come cold and refreshing upon his brow, fevered with the events of
the last few hours. He had told her as he left where she would find
him when the dance concluded; and he now sauntered towards the
rendezvous in question.
There formerly existed in the gardens of Versailles, at the right
angle of the central body of the palace, where the north wing now
stands, a fountain and cavern of marvellous construction, called the
Grotto of Thetis. The chapel at present occupies its site, built by
Louis in 1699, when, under the influence of Madame de Maintenon,
his pursuits changed from those of the most unbridled licentiousness
to the extreme of devotion. The statues which it contained, with
some fragments of its structure, may be seen at the present day by
the visitor to Versailles, in the bosquet of the Bains d’Apollon. Three
arcades, closed by iron doors of exquisite workmanship, formed the
entrance to this grotto, on one of which a representation of the sun,
gilt and highly polished, was so artfully contrived as to catch the rays
of the real setting sun, and throw an almost magic light into the
interior. All the artists that Louis XIV. had about him were employed
in turn to ornament this delicious retreat. Perrault had designed the
architecture, and Lebrun the figures, with the exception of the
principal group, which was by Girardon, still existing, and
represented Apollo attended by his nymphs, in the midst of the
sheets of water flowing on all sides over rock-work of mother-of-pearl
and coral.14
It was in this retreat, lighted by a few illustrated shades, which cast
a subdued warm light upon the groups of statuary and plashing
water, that Gaudin awaited the Marchioness. Nor was he long in
expectation. Little time elapsed before Marie’s step was heard upon
the terrace, and she entered the grotto. Gaudin took her hand and
led her to a seat. There was still no trace of emotion from the late
terrible intelligence: her hand was cool, and her step equal and
unfaltering. On the other hand, Sainte-Croix was pale and agitated:
he might have felt less than the Marchioness, but his outward
demeanour was a clearer index to his feelings.
‘Why do you not speak, Gaudin?’ asked Marie, as her lover had
remained some minutes in silence: ‘and you are pale as this cold
marble! What has occurred?’
‘It is the ghastly light of the lamp,’ said Gaudin. ‘I am well—quite
well—could I be otherwise when all has prospered?’
‘I will tell you what you are thinking of,’ returned the Marchioness,
as she riveted her basilisk eyes upon Sainte-Croix: ‘I should be but a
poor enchantress if I could not read your inmost thoughts. It is the
reaction of your spirit, Gaudin. The cord has been stretched too
tightly, and it has broken; you know that a fearful tie has now bound
us to each other, and for the first time you feel that I am a clog upon
your free actions.’
‘You are mistaken, Marie,’ replied Sainte-Croix with energy,
although every word of the Marchioness thrilled through him. ‘I may
call Heaven to witness—’
‘Heaven!’ exclaimed his companion, interrupting him, and
clutching his arm with nervous force, as a sneer played over her
beautiful lips,—‘do not invoke that power again, Gaudin: what have
we to do with Heaven now? I put as little faith in your protestations
made before it, as you do in its testimony to your truth. We are both
without its pale,’ she added coldly.
‘What can I say, then, that you will trust me? Is there any oath I
can take that will give my asseverations weight with you, Marie?
How will you believe me?’
Gaudin half knelt before her as he spoke, and the large drops of
agony stole over his brow. He saw that the Marchioness was trying
her power over him, now that they had been so fearfully bound to
each other,—that she was playing with his feelings, until they could
be broken, and rendered servilely subservient to her will.
‘What oath will you have me take?’ he continued, as he threw the
whole intensity of his soul into every word. ‘Marie!—answer me, I
implore you,—if not from love, from pity at what I have undergone. If
you will not think of me as I believed you did, look on me as an
animal that was in pain and suffering from an evil you had caused.
What means this fearful revulsion of your feelings?’
He grasped her hands whilst he spoke, until the Marchioness felt
them as though they had been in a vice of hot iron. But she returned
no answer. That fearful aggravation which woman can exert with
such crushing power,—that frigid and apparently insensible
demeanour, the colder in proportion as the heart she has drawn into
her toils is anguished and convulsed, was driving Gaudin to
distraction. ‘Marie!’ he again cried, ‘do you not believe in the love
which I bear for you?’
‘It is not love, Sainte-Croix,’ at length she replied. ‘A liaison like
ours has little love to nourish its continuance; passion and jealousy
can be its only ties of endurance, and sooner or later it must end in
misery. It is my turn now to say—let us part, for ever.’
‘Part!’ cried Gaudin rapidly—‘never! What fearful change has
passed over your feelings? How can I assure you of my truth, Marie.
Think on what I underwent for your sake in the gloomy cells of the
Bastille. Look at me now—at your feet, so blindly, servilely in your
power, that I could hate myself for such concession, had not my
reason taken flight before your influence over me. Be satisfied with
the crime—by committing which both our souls are lost—as a
sufficient safeguard of our future attachment; if you will take no more
human assurance. Believe in me, if not from truth, from mutual guilt,
and reign my sole, adored one.’
Subdued by his overcharged feelings, his head fell upon the lap of
the Marchioness as he uttered the foregoing words with wild and
impassioned energy, and he burst into tears. It is a strange sight,
that of a man weeping: and when Marie saw a man like Gaudin de
Sainte-Croix thus overcome and at her feet, she was for the moment
affected. But she returned no answer; and would have remained
silent until her companion in guilt and passion again spoke, had not
a sudden interruption diverted her attention. A short hurried moan,
which, low as it was, teemed with anguish, sounding from the group
of figures as though one of the statues had uttered it, caused her to
start affrighted from the coral bank on which she was seated. Sainte-
Croix also heard it even through his excitement, and started to his
feet; whilst the Marchioness rushed immediately behind the statues
to discover the cause. There was another cry of alarm, and she
returned leading forth Louise Gauthier. The girl had sought a retreat
from the glare and tumult of the crowd within the grotto, previous to
Sainte-Croix’s arrival, and on his approach had retired behind the
statues to conceal herself, imagining until he spoke that he was
some lounger who had entered merely from curiosity, and would
soon depart.
The calm expression on the features of the Marchioness for once
gave way to a withering look of hate and jealousy. Gaudin started
back as the words, ‘Louise Gauthier here!’ burst almost involuntarily
from his lips; and then, paralysed by the sudden apparition of the
trembling Languedocian, he remained silent.
The Marchioness was the first to speak.
‘So!’ she exclaimed, quivering with emotion, in a voice almost
stifled by her anger; ‘this was the reason that you named the grotto
for a rendezvous, and it appears I came too soon. There—take your
latest conquest—the servant of Madame Scarron. She is yours—we
meet no more.’
With a glance of contempt at Louise, she threw her arm away,
and, impelling her towards Sainte-Croix, was about to leave the
grotto, when Louise caught hold of her robe and tried to draw her
back.
 ‘Stop, madame,’ she cried, ‘you are wrong. I was here by accident,
—on my soul, and by our Lady, this is the truth.’
There was an earnestness of appeal in her voice that caused the
Marchioness to stop. And perhaps her asseveration might have
derived additional force from the manner in which she called that
power which the others dared not look to, to witness her sincerity.
‘But you have met before,’ said Marie, after gazing at Louise for an
instant with the strangest of expressions; ‘you know each other.’
‘It was long ago,’ replied Louise despondingly, as she looked at
Sainte-Croix: ‘I would not have sought him; and yet, after what I have
heard,—for not a syllable of your conversation has escaped me,—
perhaps Providence sent me here to save him—to save you both.’
As she spoke she advanced towards Gaudin, and took his hand.
There was no attempt on the part of the Marchioness to stop her.
Her curiosity was singularly roused as she watched the progress of
this strange interview.
‘Do not speak to me, Louise,’ exclaimed Sainte-Croix, with averted
face, and struggling with his feelings. ‘Leave me, I beseech you.’
‘I am going to leave you, Gaudin,’ she replied; ‘and I shall never
trouble you more. I did not willingly intrude upon you now, for I knew
that all had long since passed away between us—even the
recollection of what once was. I am sorry that we have met.’
‘You have my thanks for this interposition, girl,’ said the
Marchioness; ‘for my eyes have been opened through it. Monsieur
de Sainte-Croix,’ she added coldly to Gaudin, ‘there is little
confidence, it appears, between us. I should be sorry to come in
upon an old attachment. This lady can still be yours.’
‘Heed her not, Marie,’ cried Sainte-Croix, after a powerful effort to
master his feelings. ‘I had no other motive in concealing this from
you than the wish to spare you. Believe in me still. This has been
madness—infatuation—call it what name you will, but you are the
only one I ever loved.’
And he advanced towards the Marchioness; whilst Louise, pale as
death, gasped forth hurriedly—
‘This is indeed cruel; but even now you have yet to learn what
woman can put up with from affection. You know your secrets are in
my possession.’
 ‘You threaten us!’ said Marie furiously.
‘Far from it,’ replied the other; ‘I would save rather than destroy
you. Gaudin! I am ignorant what fearful influence has spellbound
your better feelings; but I know that such is not your nature. Have I
the slightest power—discarded, heart-broken as I am—that can
snatch you from these fearful toils?’
‘Our absence will be remarked,’ observed the Marchioness coldly
to Sainte-Croix; ‘let us rejoin the court.’
‘Hear me,’ cried Louise, seizing Gaudin’s hand, ‘for the last time
perhaps on earth—hear me, Gaudin. By the recollection of what we
once were to each other, although you scorn me now, and the
shadowy remembrance of old times, before these terrible
circumstances, whatever they may be, had thus turned your heart
from me, and from your God. There is still time to make amends for
all that has occurred. I do not speak for myself, for all those feelings
have passed; but for you alone. Repent, and be happy,—for happy
now you are not.’
 The Good and Evil Angels

Gaudin made no reply, but his bosom heaved rapidly, betraying his
internal emotion. Once he turned towards Louise Gauthier as if to
speak: the words died on his tongue.
‘This is idle talk,’ said the Marchioness, as she drew Sainte-Croix
to her side. ‘If you would not be taken for our accomplice, girl, you
will keep silent as to what you have heard. Sainte-Croix, you are
stupefied by this person’s raving. Will you not come with me,
Gaudin?’
She seized his hand, and rapidly changing the tone of anger she
had adopted to one of softness and affection, gazed tenderly at her
lover, as her fair countenance resumed its tranquillity, and her eyes,
beaming with gentleness and light, looked into Sainte-Croix’s, with
an expression that thrilled his very soul.
 ‘Marie!’ cried Gaudin faintly, ‘take me where you list. In life or after
it,—on earth or in hell, I am yours—yours only.’
A flush of triumph passed over her face as she led Sainte-Croix
from the grotto, leaving Louise Gauthier clinging to one of the
statues for support—so pale, that she might have been taken for
another figure of the group, but for the violent emotion that agitated
her slight and trembling frame.
 CHAPTER XVII.
THE GASCON GOES THROUGH FIRE AND WATER TO ATTRACT ATTENTION
—THE BROTHER AND SISTER

During the stormy interview we have just narrated, the festivities

were proceeding with unflagging splendour. The repast in the
Bosquet de la Salle de Bal had finished, and the company were now
thronging along the Tapis Vert, towards the Bassin de Neptune,
whereon some magnificent fireworks were to be displayed. Beyond
this the canal was illuminated by coloured lights placed round its
edge, and quivering in the water by reflection; and a number of small
boats, similarly decorated, passed to and fro, until they were almost
lost in the distance. A species of vast tent, open towards the water,
had been erected at the extremity of the Tapis Vert for the reception
of Louis and his court; the inferior guests, who were not supposed to
be sensible of any difference of temperature, stood about upon the
grass, wherever the best view of the feu d’artifice was to be
obtained,—for to witness this portion of the fete the people were
admitted to the gardens indiscriminately; the royal guard, however,
forming a sufficiently impregnable barrier to keep them from intruding
too closely upon the presence of the monarch and his favourites.
Amongst the crowd was Jean Blacquart, who had escaped from
the abbe, and having discovered Maître Picard, was pressing
forward to obtain a front place, where his martial dress and gay
ribbons could be seen to the best advantage, even at the risk of
being pushed into the basin. Several of his old acquaintances were
near him—bourgeoisie of the Quartier Latin, and students at the
schools. Amongst these latter Philippe Glazer had mounted on to
one of the urns, which stood on pedestals surrounding the basin, for
the double purpose of obtaining a better view of the exhibition, and
addressing, from time to time, those amongst the crowd whom he
knew, and a great many more whom he did not; and as the court had
not yet arrived, his verbal tournaments with such as he chose to joke
with, or at, produced great mirth amongst the bystanders.
 ‘Maître Picard,’ cried Philippe, ‘take care of your feather; you are
burning it against the lamp.’
The little bourgeois, who was below, turned hurriedly round, and
took off his hat to look at it. Of course nothing was the matter. The
people began to laugh.
‘Pardon, bourgeois,’ continued Philippe; ‘I mistook your red face
for a flame, as it was reflected in your halberd. I forgot you had been
used for a lamp yourself before now. Do you remember the
“Lanterne” in the Rue Mouffetard? I’m afraid the rain almost put you
out.’
‘Polisson!’ cried Maître Picard very angrily, as he recalled the
adventure. ‘I shall trounce you and your graceless fellows yet. You
will all come to the gallows.’
‘Of course we shall—the day you are hung,’ replied Glazer. ‘You
may count upon our attendance.’
There was another burst of laughter from the bystanders, and
Maître Picard waxed wrathful exceedingly. He turned the halberd
upside down, and made a blow at Philippe with the long wooden
handle of it. But the student, as he was perched upon the urn,
caught up his sword in its scabbard, and warded off the blow, so that
it was turned on one side, and the pikestaff descended with all its
weight upon the head of Jean Blacquart, who was directly
underneath, crushing his fine hat, and nearly sending him into the
water.
‘Ohé, messieurs!’ shouted Philippe, without giving the bourgeois
time to recover himself. ‘The King! the King! He is coming to the
pavilion.’
‘The King! the King!’ echoed the people, imagining, from Glazer’s
elevated position, that he could see what was going on. Maître
Picard immediately bustled through the crowd, and the mob pushing
after him effectually prevented him for the time from returning; which,
however, he attempted to do as soon as he found the announcement
was a false alarm.
‘That was a spiteful blow, Blacquart, and, of course, done on
purpose,’ continued Philippe to the Gascon, who was, with a rueful
countenance, rearranging his hat. ‘Maître Picard is jealous of you.’
 ‘The women certainly do come to the shop very often when I am
sitting in the parlour,’ replied Jean, whose temper was smoothed at
once by what he considered a compliment. ‘Madame Beauchesne,
the young widow of the Rue Hautefeuille, is smitten, I am sure; but,
betwixt ourselves, talks to Maître Picard as a cloak to her true
sentiments. Mass! what a neck and shoulders she used to display!’
‘And why does she not now, Jean?’
‘Pardieu! the curé of Saint Etienne-du-Mont attacked her suddenly
during mass for going to church gorge découverte. He told her from
the pulpit that such display was wrong, for priests were mortal after
all. How the congregation shouted again with laughter!’
‘I will swear that you are here to captivate some of the court
ladies,’ continued Theria.
‘Nay, hardly that,’ replied the Gascon conceitedly, as he cocked his
hat and drew himself up as high as he could; ‘although I did fancy De
Montespan eyed me as I stood by the door in the theatre. She has a
goodly presence.’
Glazer was about to make some reply, calculated to draw forth a
fresh outpouring of Jean’s Gascon conceit, when he was interrupted
by a stranger, who advanced hastily towards the spot where
Blacquart was standing, and at once addressed him. His dress was
little suited to the festival. He wore large riding-boots, which were
dusty, as though he had just come from a journey. His dress too was
disordered, his hair carelessly arranged, and his general appearance
sufficiently marked to attract attention amongst the gay crowd about
him, even in the semi-obscurity of the illumination.
‘Are you on guard here, monsieur?’ he said to Blacquart, scarcely
noticing his eccentric accoutrements, which might have prevented
him from asking the question.
Jean was flattered at being evidently taken for a real soldier. He
boldly admitted at once that he was.
‘Can you tell me if the Marchioness of Brinvilliers is at Versailles
this evening?’
‘She is,’ returned Jean. ‘I saw her arrive with Madame Scarron—
de Maintenon, as they now call her. And not ten minutes back she
crossed the Tapis Vert on the arm of M. Gaudin de Sainte-Croix.’
 The stranger uttered a subdued oath, as Blacquart pronounced
the name.
‘Which way were they going?’ he asked quickly.
‘Towards the pavilion,’ answered Jean. ‘I have no doubt you will
find them there by this time.’
The new-comer returned no answer, but turning hastily away,
passed on to the pavilion, which had been erected at the edge of the
basin. It was hung with lamps, and he could discern the features of
all the company who were assembled in it. His eye ran anxiously
along the lines of plumed and jewelled head-dresses, until at last his
glance fell upon Marie and Sainte-Croix, who were seated in a
corner of the building near one of the entrances. He started slightly
as he saw them; and then hurriedly tracing a few lines upon his
tablets, he pointed the Marchioness out to one of the pages, who
were in waiting at the pavilion, and told him to give the message to
her. The boy immediately obeyed his orders. As the Marchioness
read the note her features underwent a rapid change; but the next
instant they recovered their wonted unfathomable calmness; and
whispering a few words to Sainte-Croix, she rose from her seat and
left the pavilion. Gaudin waited until she had quitted the building, and
then, as if moved by a sudden impulse, followed her.
As she reached the outer entrance she found the stranger waiting
to receive her. It was her brother. She held out her hand to greet him;
but he refused to take it, and retreating a step or two, raised his hat,
as he received her with a cold salute.
‘François!’ exclaimed the Marchioness; ‘what brings you here! Has
anything happened to our father? Tell me!’
‘He is dead, Marie!’ replied her brother, with a solemn
earnestness, that would have shivered the feelings of any other
human being but the one he addressed. ‘I have left the body not an
hour and a half ago, to bring you the intelligence in the midst of the
heartless glitter of Versailles.’
‘Dead!’ repeated the Marchioness, feigning the same surprise with
which she had received the self-same words from Sainte-Croix such
a short time previously. ‘Dead! and I was not there!’
‘No, Marie!’ returned François d’Aubray; ‘and I come to find you at
Versailles—in this licentious court, not with females in whom you
might have confided your reputation, after what has already
occurred, but with the man by whose wretched acquaintance with
you the last days of your father’s life were poisoned.’
Marie started at the words: could it be possible that the cause of
death was suspected?
‘Ay, poisoned,’ continued her brother, ‘as fatally as though real
venom had been used, instead of this abandoned heartlessness.’
The Marchioness breathed again.
‘To whom do you refer?’ she asked coldly.
‘To Monsieur de Sainte-Croix,’ replied her brother.
‘Who is here to answer any charge you may have to make against
him, monsieur,’ interrupted Gaudin, who just now joined the party.
‘You shall have the opportunity afforded you, monsieur,’ replied
François d’Aubray; ‘but this is neither the time nor the place. Marie,
you will return with me immediately to Paris.’
‘With you, François?’
‘This instant! I have your father’s dying words yet echoing in my
brain, committing you to our care. Are you ready?’
‘Surely the Marchioness of Brinvilliers is her own mistress?’
observed Gaudin, scarcely knowing how to act.
‘She will obey me, monsieur,’ replied the other. ‘Come, Marie; you
know me.’
As he spoke he seized his sister’s arm, and bowing to Sainte-
Croix, drew her away.
‘You still live in the Place Maubert, I believe,’ he continued: ‘you
will receive a message from me in the morning. Viens!’
He spoke in a tone of authority that Marie felt was only to be
disputed by an instant encounter between François and Sainte-
Croix, where they were then standing. So, throwing an expression
full of intense meaning to Gaudin, she allowed her brother to lead
her along the Tapis Vert, towards the entrance of the palace. Gaudin
saw them depart, and then going to the stables had his horse
resaddled, and rode at a desperate pace back to Paris, passing the
calèche in which the Marchioness had been placed by her brother on
the road.
Meanwhile the King and his immediate suite had arrived at the
pavilion, and the fireworks were about to commence. Water-serpents
and floating pieces of fire were already whizzing and spinning about
on the surface of the basin; and one or two men had crossed the
water from the opposite side of the fountain to the well-known group,
where they were arranging the cases for the grand bouquet. Philippe
saw this from his perch upon the urn, and determined to turn the
Gascon’s vanity to some account.
‘Your dress is really very handsome, Jean,’ he observed. ‘It is a
pity that its beauty is lost in the mob.’
‘I think so myself, indeed,’ replied Blacquart; ‘but I have been
allowed no opportunity of showing it off. At court everything goes by
interest; and—hem!—I can excuse a little jealousy on the part of the
Garde Royale.’
‘Now, if they will let you light the feu d’artifice,’ said Philippe, ‘you
will be seen by everybody.’
‘But how can I get to do it?’ asked Blacquart.
‘Come with me,’ said Glazer.
And tumbling from his post, purposely, on the head of Maître
Picard, who had returned to his position, he shot amongst the crowd,
before the bourgeois could contrive to aim another blow at him, and,
followed by Jean, got to the other side of the fountain. Here he
claimed acquaintance with one of the artificers, who, it appeared,
had been under his care at the Hôtel Dieu with an accident; and by
his interest Jean was furnished with a link, and directed what to do,
being inducted into the group along a slight temporary bridge of
boards.
In the interim before the grand piece was lighted, Jean arranged
and rearranged his cloak and hat a hundred times; and when at last
he applied the light to the quickmatch, and the horses began to blow
out fire from their nostrils, apparently in the centre of the water, and
the points of Neptune’s trident also went off in a brilliant discharge of
sparks, Jean was in ecstasies. The people applauded; all of which
he took to himself, and would even have bowed in return to them,
had not the presence of the King restrained him. But he felt satisfied
that, in the glare of the fire, he was plainly visible to all, and this for
the time consoled him.
But his evil genius was about to triumph. A number of changes
had taken place in the bouquet, when suddenly, and simultaneously
from every point of the statues, a column of fire shot up high in the
air, and fell again in a shower of flame upon the group, threatening to
exterminate the Gascon in its descent. His first impulse was to
retreat to the planks and get to the edge of the basin, but a
formidable blazing wheel, forming the back-work of the entire piece,
cut off his flight, so that he was driven back again. Thicker and
thicker fell the flakes, as the tawdry dabs of lace which hung about
his dress caught fire; and his thin, half-starved feather, which gained
in height what it lost in substance, also took light. Philippe Glazer,
who had foreseen all this, set up a loud huzza, in which those near
him joined; the remainder fancied that the figure of the Gascon, as
he danced amidst the glowing shower, was a part of the exhibition,
and intended to represent one of the allegorical personages who
always figured in the masques and tableaux of the period. But at last
he could bear it no longer. His cloak was just bursting into a flame
when, in the agony of his despair, he threw himself into the basin,
amidst the renewed hilarity of the spectators, including Louis himself,
who, with La Montespan, and even the pale pensive La Vallière, was
more amused than if everything had gone on in its proper way.
The reservoir was not very deep, but the Gascon had lost all self-
possession, and he floundered about like a water-god, to the great
detriment of so much of his finery as yet remained, until he got near
enough to the edge of the basin for Maître Picard to hook him out
with his halberd, and drag him half-drowned and half-roasted to dry
ground.
 CHAPTER XVIII.
THE RUE DE L’HIRONDELLE

On the southern bank of the Seine, touching the water-boundary of

the Quartier Latin, and running parallel with the river from the Place
du Pont St. Michel, which is situated at the foot of the bridge from
which it takes its name, there is a dark and noisome street, bordered
by tall gloomy houses, and so narrow in its thoroughfare that the
inhabitants on either side of the way can all but shake hands with
each other across the footway—for carriages could not pass. It is
called—for it exists in all its pristine squalor and wretchedness at the
present day—the Rue de l’Hirondelle. The pure air can scarcely
penetrate to its reeking precincts, the way is choked up with offal and
things flung from the houses to decay in the streets. The houses are
tenanted by the lowest orders, and the dirt of ages has been suffered
to accumulate on the walls and passages: in fact, it bears some
resemblance to the miserable portion of the ‘Rookery’ still left in
London, with the exception, that this Rue de l’Hirondelle is narrower
and darker. Gloomy at all times, at night the thinly scattered lamps
scarcely illuminate its entrance; and he would be a bold man indeed
who chose to pass along it alone. And in the seventeenth century,
before the introduction of street-lights, when the poverty of its
inhabitants would not allow them to place lanterns before their doors,
it was always in total darkness, even when bright moonlight fell upon
the quays and open places.
It was the evening of the funeral of M. d’Aubray, the father. The
night was stormy, and the wind howled over the city as if bearing on
its wings spirits wailing for the dead and crying for retribution. Few
cared to be abroad: the few lamps had been extinguished after
struggling against the blast and were not relighted: and one window
only in the Rue de l’Hirondelle gave token that the houses were
inhabited.
In a miserable room of one of the worst-conditioned houses—so
ruinous in its appearance that large black beams crossed the street
from its front to the opposite side of the narrow street, to prop it up
from falling and crushing those who might be below—there were two
persons seated at a small fire. In one of them any person who had
once seen him could have recognised the Italian Exili, although his
imprisonment had left traces of its privations upon his face. His
features were more wan, his hair was grizzled, and his eyes had
sunk yet deeper, glaring from the bottom of the orbits with riveting
intensity. His companion was dressed in a fantastic costume of old
black velvet, with a capuchin cowl which, when worn over his head,
nearly concealed his face, and his head was now buried in it,—less,
however, for privacy than to shield himself from the cold draughts of
air that poured in through the broken, ill-fitted windows. On a rough
table before him were pieces of money, of all degrees of value: and
these he was counting, as he put them away in a box heavily
clasped with iron.
‘Sorcery is still thriving,’ said the latter personage, ‘and we have
had a good day. Here are twelve pistoles from the Demoiselle La
Varenne, who came to-day suspicious of her new patron, M.
Chanralon, the Archbishop of Paris. He has taken up with the
Marchioness of Gourville.’
‘The sister of the maréchal?’ asked Exili.
‘The same. Ho! ho! ours is a brave court!’ continued the other with
a derisive laugh. ‘Better be magician than superintendent at the
Gobelins. Here is a piece of gold from the same clique. Pierre-Pont,
the lieutenant of the Gardes-du-corps, is crazy with jealousy for La
Varenne. He came to-day for a philtre: he will come for poison next.’
‘Hush!’ exclaimed Exili; ‘the very echoes linger about these walls
to repeat themselves to the next comers. I find liberty too sweet to
run the chance of another sojourn in the Bastille, where Sainte-Croix
would too gladly see me—curses wither him!’
‘He will be here to-night,’ replied Lachaussée—for such was Exili’s
companion—‘to have his wound dressed. M. François d’Aubray is an
expert swordsman, and the Captain found his match on the terrain
last night.’
The ex-superintendent alluded to a duel which had been fought on
the preceding night on a lonely piece of waste-ground behind Notre
Dame, frequently chosen for such engagements from the facility of