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Structural analysis for lignin characteristics in

biomass straw

ARTICLE in BIOMASS AND BIOENERGY · AUGUST 2013

Impact Factor: 3.39 · DOI: 10.1016/j.biombioe.2013.07.015

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Review

Structural analysis for lignin characteristics

in biomass straw

Seyed Hamidreza Ghaffar, Mizi Fan*

School of Engineering and Design, Brunel University, Uxbridge, Middlesex, UB8 3PH, United Kingdom

article info abstract

Article history: Agricultural by-products are the most promising feedstock for the generation of renewable,
Received 31 March 2013 carbon neutral substitutes for synthetic materials (e.g. biofuel, building materials). The
Received in revised form demand for efficient utilisation of lignin biomass has induced detailed analyses of its
15 July 2013 fundamental chemical structures and development of analysing technologies. This paper
Accepted 19 July 2013 reviews the structural analysis techniques for straw lignin together with the morphology of
Available online xxx the lignin biomass and the study of the form and structure of organisms and their specific
structural features. The review showed that the studies on lignin could be divided into the
qualitative and quantitative analyses; different analytical methods could provide signifi-
cantly different results that are even sometimes not directly comparable. Among many
Keywords:
techniques reviewed, the magnetic resonance techniques have proved to be efficient
Straw lignin
analytical tools for the structural elucidation of these complex biopolymers. Quantitative
Quantitative and qualitative anal-
and qualitative structural analysis of lignin indicated a great potential for industrial crops
ysis
optimisation due to in-depth microstructure interpretation, and detailed and accurate
NMR
chemical composition although the composition and structure of straw lignin have been
FT-IR
discovered highly complex and varied considerably within and among plants. The struc-
Structural characteristics
ture of lignin has remained one of the most difficult biopolymers to characterise, however
Thermal analyses
recent advances in analytical chemistry and spectroscopy have dramatically improved the
understanding of this natural resource, and further value added utilisations are being
expected for the lignin and its related biomass.
ª 2013 Elsevier Ltd. All rights reserved.

1. Introduction research workers e.g. Refs. [3e5]. A number of chemical and

Research concerning lignin has significantly increased due to
its renewability and availability and the value added produc-
tion of lignin based products, such as biofuel and sustainable
construction materials for replacing petrochemicals based
products [1,2]. The structure of lignin is probably the single
most important parameter for understanding and hence uti-
lising lignin materials, and has been analysed by many
physical methods used for characterising lignin’s structure
are destructive. However there are non-destructive methods
too, such as FT-IR which is believed to be one of the most
informative methods of lignin investigation, ultraviolet (UV),
carbon-13 nuclear magnetic resonance spectroscopies (13C
NMR) and gas permeation chromatography (GPC) [6].
Lignin is an extremely complex three-dimensional poly-
mer (typically found in vascular plants) formed by radical

* Corresponding author. Department of Civil Engineering, Brunel University, London UB8 3PH, United Kingdom.
E-mail address: mizi.fan@brunel.ac.uk (M. Fan).
0961-9534/$ e see front matter ª 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biombioe.2013.07.015

Please cite this article in press as: Ghaffar SH, Fan M, Structural analysis for lignin characteristics in biomass straw, Biomass
and Bioenergy (2013), http://dx.doi.org/10.1016/j.biombioe.2013.07.015
2 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6
coupling polymerisation of p-hydroxycinnamyl, coniferyl and
sinapyl alcohols; these three lignin precursors monolignols
give rise to the so-called p-hydroxyphenyl (H), guaiacyl (G) and
syringyl (S) phenylpropanoid units, which show different
abundances in lignin from different groups of vascular plants,
as well as in different plant tissues and cell-wall layers [7].
These aromatic building units are linked with a variety of
ether and carbonecarbon bonds. The predominant linkage is
the so called b-O-4 linkage. About 40e60% of all inter-unit
linkages in lignin are via this ether bond. Different types of
linkage between phenyl propane units form three-dimen-
sional net structures and make it difficult to completely
degrade non-regular lignin macromolecules. Due to lignin’s
structural complexity it is hard to come to an agreed model for
their chemical and physical structure. Lignin is primarily a
structural material to add strength and rigidity to cell walls
and constitutes between 15 and 40% mass fraction of the dry
matter of plants, whether wood, straw or other natural woody
plants. Lignin is more resistant to most forms of biological
attack than cellulose and other structural polysaccharides
[8e10]. Lignin acts as a matrix together with hemicelluloses
for the cellulose microfibrials which are formed by ordered
polymer chains that contain tightly packed, crystalline re-
gions (Fig. 1). Covalent bonds between lignin and the carbo-
hydrates have been suggested to consist of benzyl esters,
benzyl ethers and phenyl glycosides [11e13].
There is a little study of lignin native (in situ) which does not
change the structure of lignin, such as, chemically unmodified
lignin by using ionic liquid 1-ethyl-3-methylimidazolium ace-
tate [Emim] (CH3COO) as a pre-treatment solvent [14]. How-
ever, there has been much interest for industrial lignin due to
the existence of large scale manufacturing processes, i.e. wood
pulping, one of the largest industries in the world in which the
main objective is to separate individual fibres from wood and
to do so lignin must firstly be removed, and lignin from various
resources and industries were studied through elemental
analysis in order to understand the fundamentals of lignin and
their properties for value added applications [15]. Huge quan-
tities of lignin are extracted by wood pulping and other in-
dustries annually, though the bulk of which is still either
burned to recover energy or otherwise considered waste. Only
about 1e2% of this lignin is used to make other products [16].
Despite lignin’s unique characteristics as a natural product
Fig. 1 e Cellulose strands surrounded by hemicellulose and
lignin [1].
Please cite this article in press as: Ghaffar SH, Fan M, Structural analysis for lignin characteristics in biomass straw, Biomass
and Bioenergy (2013), http://dx.doi.org/10.1016/j.biombioe.2013.07.015
with multiple chemical and biophysical functionalities, it is
largely underutilised due to its image as low quality and low
value added material. Current work of researchers has focused
on expanding the frontiers of application of NMR to straw
lignin analysis in order to explore lignin’s potential [4,17,18].
The main goal of this paper is to study in details the
structure of straw lignin and to review the analytical tech-
niques up to date for the analysis of lignin; this will then
contribute to a better understanding of lignin and hence could
lead to the optimisation of lignin for specific and value added
industrial utilisations.
2. Composition and morphology of straw
Straw consists mainly of three groups of organic compounds:
cellulose, hemicellulose and lignin which account for more
than 80% of the dry matter of cereal straws such as oats,
barley and wheat [19]. Straw also contains various other
organic compounds including protein, small quantities of
waxes which protect the epidermis of the straw, sugars, salts
and insoluble ash including silica. Different fractions across
the straw vary in chemical composition which is also affected
by soil type and fertiliser treatment. Overall, there is a higher
concentration of cellulose in internode, of ash (in which silica
is the main constituent) in leaf and leaf base and of lignin in
the node cored compared to the other parts of straw (Table 1)
[20,21].
Similar to wood, straw is considered a natural composite
material because of its composition of polysaccharides (cel-
lulose and hemicelluloses) and lignin. The former two com-
ponents are hydrophilic and the latter is hydrophobic.
However, they are practically insoluble in water due to
hydrogen bonding and covalent bonding with lignins [6]. The
ultrastructure of straw fibres, like wood tracheids, consists of
middle lamella (ML), primary wall (P) and secondary walls (the
outer S1, the middle S2, the inner S3). The percentage volume
fraction (PVF) and thickness of various morphological layers of
wheat straw can be different from those of wood (e.g. spruce)
(Table 2) [22], especially the S1 layer of wheat straw fibre is
thicker than that of the spruce tracheid and PVF of ML and cell
corner (CC) in wheat straw fibre is greater than that of spruce
tracheid.
When it comes to anatomical structure of straw, normally
the sclerenchyma cells in the bundle itself have a small
diameter and a thick fibre wall while the extra vascular fibres
often have a much larger diameter and a very variable wall
Table 1 e Chemical components of wheat straw
determined by gravimetric analysis [20].
Mass fraction of dry material (%)
Leaf Internode Leaf base Node core
Hot-water soluble 14.6 7.2 18.9 13.2
Lignin 15.3 14.2 14.1 16.7
Hemicellulose 32.4 33.8 34.2 32.7
Cellulose 37.7 44.8 32.7 37.5
Ash 11.5 4.7 12.4 6.3
 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6 3

Table 2 e The thickness and PVF of wheat straw [22].

Morphological layers Wheat straw Spruce tracheid

Thick fibre Thin fibre

Thickness (mm) PVF (%) Thickness (mm) PVF (%) Thickness (mm) PVF (%)

ML þ P 0.1e0.2 9.3 0.06e0.12 12.3 0.05e0.1 10.2a

S1 0.1e0.3 0.2e0.3 0.15e0.2 9.9
S2 1.8e2.5 83.5 0.5e0.8 80.7 0.7e2.0 75.9
S3 0.15e0.3 0.1e0.2 0.1 4.0
CC e 5.4 e 7.0 e e

a Containing the CC region.

thickness. In wheat straw these fibres occur as coarse bast
fibres just inside the epidermal layer (Fig. 2A). Unlike wheat,
the cross-section of rice straw reveals a different type of ul-
trastructure, beside the tubular concentric ring structure, the
centre of the rice straw internodes contains a core (Fig. 2B and
C). Moreover, rice is an aquatic plant and has a different type
of protecting layer, including substances composed of lignin,
amorphous silica, and other inorganic substances.
3. Analytical techniques for structural
analysis of straw lignin
The need of properly characterising and classifying lignin for
the promotion of the new applications is clear. At the product
development level, appropriate analysis will facilitate the
definition of specifications for commercial purposes and also
allow the reliable monitoring of physical and chemical mod-
ifications which eventually will promote the understanding of
the relationship between properties and performance. The
utilisation of lignin for a range of natural and industrial pur-
poses is dependent on the analysis of lignin and the charac-
teristics of this polymer. Also a comprehensive understanding
of lignin is needed when considering processes such as
various pre-treatment techniques, including physicochemical
and biological pre-treatments. The complicated and hetero-
geneous structure of lignin has been traditionally revealed by
a series of chemical analysis, such as thioacidolysis (TA) [26],
nitrobenzene oxidation (NBO) [27] and derivatization followed
by reductive cleavage (DFRC) [28]. Nevertheless recent de-
velopments in nuclear magnetic resonance (NMR) technology
have made this technique the most widely used technique in
the structural characterisation of lignin, the reason being its
versatility in indicating structural features and structural
transformation of lignin.
The studies on lignin are divided into two different sec-
tions: qualitative and quantitative analyses. Some researchers
(e.g. Ref. [3]) divided the analytical methods of lignin

Fig. 2 e Micrograph of cross sections of (A) wheat straw under light microscope [23]; (B) wheat straw internode SEM (a)
epidermis, (b) parenchyma, (c) lumen, (d) vascular bundles [24]; (C) rice straw internode SEM [25].

Please cite this article in press as: Ghaffar SH, Fan M, Structural analysis for lignin characteristics in biomass straw, Biomass
and Bioenergy (2013), http://dx.doi.org/10.1016/j.biombioe.2013.07.015
4 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6

characterisation into two groups of destructive and non- The infrared region is usually divided into 3 regions: near
destructive methods. Thioacidolysis (TA) [26], nitrobenzene infrared (13,300e4000 cm1), middle infrared (4000e200 cm1)
oxidation (NBO) [27] and also derivatization followed by and far infrared (200e3.3 cm1) regions. Organic compounds
reductive cleavage (DFRC) [28] are amongst the most have fundamental vibration bands in the mid infrared region,
destructive methods used. The non-destructive methods which is why the region is widely used in infrared spectros-
include various spectroscopic methods (e.g. UV and Fourier copy. An infrared spectrum is unique to each substance and
transform infrared spectroscopy (FT-IR spectra)) and Nuclear can therefore, in principle, be used as an impartial charac-
magnetic resonance (NMR) techniques. teristic to identify the sample. Every lignin IR spectrum has a
The chemical composition of agricultural biomass can also strong wide band between 3500 and 3100 cm1 assigned to OH
be analysed with near infrared (NIR) spectroscopy, e.g. Kelley stretching vibrations. This band is caused by the presence of
et al. [29] tested the effectiveness of NIR for measuring the alcoholic and phenolic hydroxyl groups involved in hydrogen
chemical composition of biomass that has been subjected to a bonds. The intensity of the band increases during demethy-
wide variety of extraction and chemical treatments; Sander- lation and decreases during methylation since during deme-
son et al. used NIR for directly measuring the chemical thylation the OeCH3 bonds in methoxyl groups bonded to 3rd
composition of biomass [30]. or 5th carbon atom of the aromatic ring are split and CH3 is
Another technique to analyse lignin in the cell wall is based replaced by a hydrogen atom producing a new OH group.
on mercurization which was firstly used in the early history of During methylation the OeH bonds are split and H is replaced
lignin chemistry to characterise its structure [31]. Mercuriza- by CH3 group and the amount of OH group decreases hence
tion is a reaction occurring under mild conditions; it seems to the intensity of the band decreases [41]. Acetylation of the
be less sensitive to the substitution pattern of the aromatic lignin causes a partial or full band loss since almost all of OH
ring. The determination of lignin, thought apparently an easy groups are replaced by CH3COO [42].
matter, is one of the least satisfactory of the analysis The assignments of FT-IR absorption bands for the lignin
commonly carried out on plant materials and woods. Almost normally include the aromatic skeleton vibrations at 1606,
all current methods contain the solution and hydrolysis of all 1507 and 1434 cm1, in which the aromatic semicircle vibra-
other plant constituents and assume that the residue after tion (a vibration involving both CeC stretching and a change
such treatment is lignin. Various investigators have pointed of the HeCeC bond angle) is assigned at 1507 cm1 [43], the
out that this is not the case, but in spite of this vital objection, carbonyl and unconjugated ketone and carboxyl group
considerable reliance has been placed on figures so obtained. stretching at 1732 cm1 and the small band of the conjugated
carbonyl stretching such as detected in organosolv lignin at
3.1. Quantitative analysis 1653 cm1. More detailed assignment are summarised in
Table 3 [7,44,45].
Quantitative measurement of lignin structures is an impor-
tant aspect when it comes to investigating lignin; the mea- 3.1.2. Thermal analysis
surement of various structures of lignin is possible when Thermal analysis has recently become prominent, especially
appropriate standards or pulse sequences are applied [32e34]. for fibres, plastics and other polymeric materials. Thermal
Quantitative analysis is based on gravimetry or UV-absorption
[35] either to estimate lignin as an insoluble residue after
strong sulphuric acid treatment (Klason lignin) [36] or to oxi-
dise away the lignin from a holocellulose preparation (acidic
Table 3 e Assignments of FT-IR absorption bands (cmL1)
chlorite lignin or permanganate lignin) [37]. The spectroscopic [7].
techniques, such as infrared (IR) and 13C nuclear magnetic
Absorption Assignment
resonance (13C NMR) spectroscopy, are complementary to the bands
above procedures since they provide information on the
3429 OH stretching
whole structure of the polymer and avoid the possibility of
2945 CH stretching of methyl, methylene
degradation artefacts [38]. This information is presented with
or methane group
quantitative values. 1732, 1726 C¼O stretch in unconjugated ketone
and carboxyl group
3.1.1. FT-IR 1660, 1653 C¼O stretch in conjugated ketone
Among all the quantitative analysis techniques, FT-IR spec- 1606 Aromatic skeletal vibration
troscopy shows interesting characteristics, including high 1507 Aromatic skeletal vibration
1460 Aromatic methyl group vibrations
sensitivity and selectivity, high signal-to-noise ratio, accu-
1434 Aromatic skeletal vibration
racy, data handling facility, mechanical simplicity and short
1374 Aliphatic CeH stretch in CH3
time and small amount of sample required for the analysis 1328 Syringyl ring breathing with CeO stretching
[39]. In addition, the spectrum of a lignin sample gives an 1242 Aromatic CeO stretching
overall view of its chemical structure [40]. FT-IR spectroscopy 1165 CeO stretch in ester groups
is non-destructive method of lignin investigation, which 1135 Aromatic CeH in-plane deformation
opens perspective to find structural discrepancies in lignin for syringyl type
1043 Aromatic CeH in-plane deformation
isolated by different methods.
for guaiacyl type
Infrared radiation is electromagnetic radiation which 855, 844 Aromatic CeH out of plane bending
covers the wavenumbers between 13,300 cm1 and 3.3 cm1.

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and Bioenergy (2013), http://dx.doi.org/10.1016/j.biombioe.2013.07.015
 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6
analysis is a set of techniques used to describe the physical or
chemical changes associated with substances as a function of
temperature. The thermal stability of the isolated hemi-
celluloses, cellulose and lignin preparations is determined by
thermogravimetric analysis (TGA) and differential scanning
calorimetry (DSC). TGA can be used to monitor the weight loss
of the lignin as it is heated, cooled or held isothermally, while
DSC is performed to determine the melting temperature and
enthalpy of the lignin. DSC is the most widely accepted
method for determining glass transition temperatures (Tg) of
lignin or modified lignin samples [46]. Thermoanalytical
techniques, in particular TGA and derivative thermogravi-
metric (DTG), allow the information about chemical compo-
sition of straw to be obtained in a simple and straightforward
manner. Pyrolysisegas chromatography/mass spectrometry
(PyeGC/MS) has also been applied to examine reaction prod-
ucts of thermal degradation [47] and an effective tool to
investigate fast pyrolysis of biomass and on-line analysis of
the pyrolysis vapours [48,49].
PyeGC/MS provides a rapid and easy alternative to tedious
chemical degradation procedures for analysing the mono-
lignol composition of lignin samples [50]. It requires only a
small amount of lignin (<1 mg) and samples do not have to be
isolated from the associated plant polysaccharides to be
effective. Compounds separated on a GC column can easily be
identified from their mass spectra as being derived from p-
hydroxyphenyl (H), syringyl (S), or guaiacyl (G) propane units.
Integration of the chromatograph peaks can determine if the
sample in question comes from a G, S/G, or H/S/G type lignin.
For TGA and DSC the heating rate is usually between 5  C
and 10  C. The tests are undertaken in both nitrogen and air
atmosphere between room temperature of 25  Ce30  C up to
730  Ce800  C [51]. Due to the complexity of lignin and the
difficulty in extraction, the literature related to the pyrolysis
behaviour of lignin is scarce. However Müller-Hagedorn and
Bockhorn [52] obtained kinetic parameters for straw lignin in a
TGA based on the LevenbergeMarquardt and improved Run-
geeKutta laws. The activation energies and frequency factor
for barley straw lignin varied from 92 to 102 kJ mol1 and from
107.3 to 107.9 min1 respectively. Ghaly et al. [53] used TGA and
differential thermal analysis (DTA) to study the behaviour of
oat straw in an oxidising atmosphere (15% oxygen and 85%
nitrogen); two distinct reaction zones were observed on the
TGA and DTA curves. Higher thermal degradation rates were
observed in the first reaction zone due to the rapid release of
volatiles as compared to those in the second reaction zone.
The activation energies were in the range of 83e102 and
58e75 kJ mol1 for the first and the second reaction zones,
Table 4 e NMR techniques for lignin analysis.
NMR techniques Quantitative data
13
C NMR Aliphatic/phenolic OH groups and methoxy groups
HSQC Inter-unit bonding patterns
31
P NMR Aliphatic/phenolic OH groups, guaiacyl, siringyl,
condensed OH groups, carboxylic acids
1
H NMR Hydrocarbon contaminant, aliphatic and aromatic acetate,
protons in methoxyl groups, benzylic protons in b-b structures
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5
respectively. The thermogravimetric dynamics parameters of
wheat straw enzymatic acidolysis lignin (EMAL) were calcu-
lated by the methods of Kissinger and Ozawa, respectively;
the activation energy of wheat straw EMAL was 103.92 and
107.69 kJ mol1 respectively with the methods of Kissinger and
Ozawa [54].
Carrier et al. [55] have also investigated the suitability of
TGA as a new method to obtain lignin, hemicellulose and
⍺-cellulose content in biomass. The study indicated compa-
rable results to common methods used for ⍺-cellulose con-
tent; however, this method cannot be extended to the lignin
content because of important deviations in the correlation
curves. Through this alternative method, which is faster,
easier to implement and less cost effective than the existing
wet chemical techniques, it is possible to determine the
hemicelluloses and ⍺-cellulose contents of biomass samples.
Thermal degradation of lignin is reviewed by Brebu and
Vasile [56] where the information on the temperature range,
kinetics and mechanism of thermal degradation is presented.
Samples for the thermal analysis of straw such as TGA are
usually the same as those for FTIR spectroscopy test.
3.1.3. NMR
Nuclear magnetic resonance spectroscopy (NMR) has been
shown to be reliable and comprehensive among the various
physical and chemical methods for the characterisation of
lignin, because it not only gives quantitative data but also
provides some qualitative information (Table 4). An inclusive
review on NMR application for structural analysis of lignin
was published in 1971 [57].
In the past, proton NMR (1H NMR) was mainly used for
lignin characterisation and the 1H NMR spectrum of acety-
lated lignin is used to determine the quantity of different
hydroxyl groups. Lundquist [58e60] has published works
about NMR characterisation of lignin. With the development
of NMR techniques, 13C NMR became popular in lignin char-
acterisation as it is powerful and capable of revealing a large
amount of lignin structural information including the pres-
ence of aryl ethers, condensed and uncondensed aromatic
and aliphatic carbons. Since the 1980s, many studies on 13C
NMR spectra of lignin have been conducted [61e64]. Many
attempts have been made to increase the sensitivity and
signal-to-noise ratios of the quantitative 13C NMR spectra,
especially for understanding the structural changes of lignin
polymer in pulping processes and other isolation processes
[3]. 13C NMR has been used to aid in the elucidation of pulping
or delignification mechanism (soda pulping, kraft pulping and
oxygen/peracid treatments), as well as pre-treatments, which
Qualitative data
Inter-unit bonding patterns
Inter-unit bonding patterns
Assignments of guaiacyl, siringyl, condensed
OH groups, carboxylic acids
Improved spectral resolution of key
lignin functionality
6 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6
are discussed in detail in a recent book by Ralph and Landucci
[65].
Quantitative 13C NMR is commonly used for the estimation
of some specific component [65,66]. However, the most recent
practices in the use of quantitative 13C NMR spectroscopy for
the characterisation of lignin are confined to using the aro-
matic and methoxyl signals as internal standards in
expressing the various functional groups per C9 [65]. Such a
practice is applicable to native lignin but inapplicable for in-
dustrial lignin or modified lignin [66]. To overcome the above
defects, Xia et al. [66] suggested a novel protocol for acquiring
quantitative 13C NMR spectra of lignin by using the internal
reference compounds 1,3,5-trioxane and pentafluorobenzene.
Trioxane offers a convenient internal standard for collecting
inverse gated proton decoupled 13C NMR spectra for lignin.
The internal reference compounds provide single and un-
overlapped sharp signals in the middle of the spectral re-
gion, permitting superficial integration.
Detailed approaches for the quantification of different
lignin structures in milled wood lignin (MWL) have also been
reported by using quantitative 13C NMR techniques [67,68],
including the amount of different linkages, various phenolic/
etherified non-condensed/condensed guaiacyl and syringyl
moieties. This approach is comparable to that reported from
other wet chemistry techniques, but requiring only rather
short experimental times. Much progress has been achieved
on the 13C NMR spectra of lignin, but there still remain some
issues to be explained, such as precise signal assignments and
true quantification based on 13C NMR spectra of lignin, which
are difficult due to signal overlap and other factors. Both solid-
state and solution-state 13C NMR have been used for structural
characterisation of lignin [69,70], however the solution-state
NMR is more informative because of its better resolution,
although soluble lignin preparations may not be representa-
tive of the whole lignin fraction in plants [71,72].
It is worth mentioning that structural characterisation of
lignin could be directly investigated by NMR techniques via
non-derivatization solvent systems, such as DMSO-d6/NMI-d6
[73], DMSO-d6 [74] and DMSO-d6/pyridine-d5 (as gelling sol-
vent) [75]. The rapid NMR characterisation method provides
what appears to be the best tool for the detailed structural
study of the complex cell wall polymers. The DMSO-d6 (gel-
state) method is used for lignin analysis, not polysaccharides
analysis. Nevertheless, based on these non-acetylated solvent
systems, lignin composition (notably, the syringyl: guaiacyl: p-
hydroxyphenyl ratio) could be quantified without the need for
lignin isolation [3].
In summary, with NMR techniques, the true quantification
in lignin is difficult due to signal overlap and other factors. The
best quantification method remains the relatively tedious
inverse-gated technique, along with an internal standard
substance. Fortunately, most of the NMR techniques are
adequate when the researchers want to follow changes in
structure of straw biomass over time during a particular
treatment, provided that the desired precision is maintained
[65].
3.1.4. Two-dimensional HSQC NMR
The interest to study the mixture of increasing complexity of
lignin has created the demand to employ high magnetic field
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NMR spectrometers to improve resolution and sensitivity. The
strong self-association of lignin chains and harsh treatment
required for lignin-derived fragments solubilisation are major
factors that make studying lignin primary structures difficult
[76]. Quantification of complex samples with 1H NMR spec-
troscopy suffers from resonance overlap even at high mag-
netic fields, which can prevent an accurate quantification of
sample compounds. Protonecarbon correlated two-
dimensional (2D) NMR can exploit the wide chemical shift
range of carbon, thus offering a significantly improved reso-
lution. Therefore, the application of protonecarbon correlated
2D NMR in the determination of molar concentration of the
sample compounds has gained interest in studies of natural
products, biological samples and processes taking place in
living organisms [77] and 2D NMR of both homo-and hetero-
nuclear became popular and much powerful tools for lignin
characterisation [78e80].
The most used and valuable 2D NMR is heteronuclear-
single-quantum-coherence (HSQC) that provides the correla-
tion between directly bonded protons and carbons in two di-
mensions [67,81]. Overlapping protons that are attached to
carbons with different shifts are separated by their carbon
shift difference; while overlapping carbons may be separated
by their attachment to protons with differing chemical shifts.
Therefore, the apparent resolution of 2D spectra is much
better than anything that can be achieved in 1D spectrum [12].
Thus, quantitative measurement of various structures of
lignin is possible when appropriate standards or pulse se-
quences are applied [33,67]. The assignment for lignin corre-
lations comes from the extensive database of lignin model
compounds along with data from a long history of NMR of
both isolated and synthetic lignin [32,82e87]. The 2D-HSQC
experiments of non-acetylated lignin samples have been
valuable in assigning major structures (b-O-4, b-b, b-5, etc.) in
the lignin samples according to the previous studies and the
previously mentioned database of lignin model compounds
[80].
The HSQC approaches have also been valuable in assigning
major structures of non-acetylated lignin from different ori-
gins in recent years, such as some non-woody plants [32], Jute
fibres [82], bamboo [84,85,87], Triploid poplar [88e90] and
wheat straw [83]. HSQC spectra have been crucial in recog-
nising new and minor lignin structural units. The clear iden-
tification of dibenzodioxocins (5e5 linkages) as major new
structures in lignin has been a significant finding [91,92].
Evidence provided by 2D NMR is far more diagnostic than
1D data purely because of the simultaneous constraints that
are revealed in the data. Consequently, the observation that
there is a proton at 4.9 part per million (ppm) directly attached
to a carbon at 84.4 ppm and a proton at 4.1 ppm attached to a
carbon at 82.5 ppm is more revealing than just observing two
new carbons at 84.4 and 82.5 ppm in the 1D spectrum [92].
Zhang and Gellerstedt [33] presented a new analytical method
based on the 2D-HSQC NMR sequence and quantitative 13C
NMR, which can be applied for quantitative structural deter-
mination of complicated polymers, such as lignin and cellu-
lose derivatives. This method is a combination of HSQC and
quantitative 13C NMR techniques, which gives more reliable
data about the lignin linkages. A recent study showed that 2D-
HSQC NMR spectra of enzymatic hydrolysis residue (EHR)
 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6 7

delivered very well resolved spectra that can be compared to

Table 5 e H/S/G ratios for types of biomass resources.
that of MWL [93]. Based on the results obtained, it was found
that in situ characterisation of pre-treated biomass by HSQC Type Lignin (%) H S G Reference
NMR analysis is a beneficial structural analysis methodology Wheat straw 16e21 9 46 45 [109]
in the emerging biomass research field for the characterisa- Rice straw 6 15 40 45 [109]
tion of EHR. Rye straw 18 1 53 43 [110]
Heteronuclear multiple quantum coherence (HMQC) or Hemp 8e13 9 40 51 [111]
Jute 15e26 2 62 36 [112]
HSQC spectra of lignin have been reviewed [80] and well reported
Flax 21e34 4 29 67 [109]
by previous researchers [4,74,75,78,94e98]. The assignments in
HMQC/HSQC spectra should be made with comprehensive
knowledge of both carbon and proton chemical shifts for each
thioacidolysis reflect the proportion of uncondensed units in
structural type. 2D HSQC NMR technique is also useful in
lignin. When thioacidolysis is used for grass lignin, esters on
determination of the structural characteristics of oxygenated
grass lignin may decrease the efficiency of b-ether cleavage by
aliphatic region of lignin polymeric framework [32,99].
thioacidolysis [113,114]; hence this should be taken into
consideration when interpreting thioacidolysis results for
3.1.5. DFRC method
grasses.
The derivatization followed by reductive cleavage method
Permanganate oxidation, nitrobenzene oxidation, GCeMS
(DFRC) was developed by Lu and Ralph in 1997 as an alternative
pyrolysis, thioacidolysis and DFRC are degradative methods
to thioacidolysis [28,100]. Because of the mild conditions used
that reveal the H/S/G composition of the lignin polymer. It must
and its unique selectivity, the DFRC method has become widely
be mentioned that degradative analytical techniques are
used for characterising lignin from various origins [101e103].
lengthy and complicated. All these methods release only a
One unique feature of the DFRC method is that esters on lignin
fraction of the lignin for analysis, they are based on the cleavage
remain fully intact during the DFRC procedure [104]; therefore,
of lignin backbone and analyses of the fragments obtained, and
p-coumarates on lignin from grass plant materials can be
provide partial data due to the specificity of the treatment.
detected and measured [105]. With a few modifications to the
The quantification of lignin H/S/G ratio has been reported
standard DFRC procedure by replacing the acetyl-containing
by 13C NMR and HSQC NMR analysis [115,116].
reagents (acetic acid and AcBr) with the propionyl analogues,
the modified DFRC method was applied to detect and quantify
the naturally occurring acetates on lignin from some plants 3.2. Qualitative analysis
such as kenaf, aspen or other non-woody plants [106].
The DFRC procedure, as a basic component, has been Qualitative lignin analysis relies rather much on studies of
modified or combined with other techniques to provide more lignin degradation products; these methods only provide
precise and specific quantitative data about lignin structures preliminary information which could then be useful for the
[107]. The combination of DFRC with quantitative 31P NMR was quantification of lignin within straw.
shown to have significant potential for the determination of
arylglycerol-a-aryl ether and other linkage. Coupled with the 3.2.1. SEM-EDX
spread of advanced NMR techniques, during the past decade, Scanning electron microscopy (SEM) is used to examine the
lignin structural enquiries have been greatly facilitated by the microstructure, morphology, crystalline structure and size of
development of various degradative protocols, such as DFRC the straw. The microscope could be equipped with energy-
which efficiently cleaves the b-aryl ethers in lignin. DFRC is a dispersive X-ray (EDX) analyser, which provides useful
flexible method due to its three distinctly separated steps, element compositions and also information about the chem-
allowing modifications designed to the quantification of ical composition of the straw.
different monomeric units and structures. On the basis of this Despite its importance as imaging technique, SEM is not
flexibility, Tohmura and Argyropoulos [108] proposed the capable of quantitatively evaluating the purity of typical lignin
combination of DFRC with quantitative 31P NMR data. sample. Moreover, there is no published algorithm to convert
such images into numerical data. SEM analysis also provides
3.1.6. H/S/G ratio information on the particle size and the surface properties of
Qualitative or semi-qualitative indication of H/S/G ratio units straw. When the straw is being treated by means of chemical
are not too informative and on the other hand they are very or mechanical treatments, the particle sizes might change and
laborious, lengthy and also prone to errors. Table 5 shows the the surface of straw might become smoother or rougher
p-hydroxyphenylesyringyleguaiacyl (H/S/G) ratio for depending on each step of the treatment.
different types of biomass which vary quite considerably Field emission scanning electron microscopy (FE-SEM) was
amongst each other. used by Koo et al. [117] in order to observe the change in lignin
Thioacidolysis is the most used analytical method for distribution during pre-treatment; (Fig. 3), which is one of the
lignin analysis to obtain information about uncondensed key applications of SEM technique used for qualitative anal-
structures (H/S/G ratios) [12]. Thioacidolysis was developed by ysis of biomass.
Lapierre, as an extension of acidolysis, to cleave b-aryl ethers
in lignin so that the basic units linked by b-aryl ethers are 3.2.2. TEM and optical microscopy
released as monomers to be quantified by gas chromatog- Transmission electron microscopy (TEM) has been proved to
raphy [26]. Therefore, the yields of monomers released by be an effective tool for determining cell wall ultrastructure.

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8 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6

Fig. 3 e Droplets on surface of pre-treated biomass due to the isolation and migration of lignin (a: untreated biomass; b: pre-
treated at 120  C; c: pre-treated at 130  C; d: pre-treated at 140  C) [117].

TEM observations deliver further details of lignification and
the distribution of lignin. Based on the intensity of the stain-
ing, the concentration of lignin in different morphological
regions of straw could also be qualitatively examined by TEM
micrograph, for example, Fig. 4 shows the distribution and
concentration of lignin under TEM. The dark staining of the
cell corner middle lamella (CCML) and compound middle
lamella (CML) indicates that both of them are strongly ligni-
fied. Like SEM, TEM analysis will also be helpful in finding
particle size and surface properties of straw.
Optical microscopy is unable to provide the detailed fea-
tures of the materials under examination but is a good tool to
observe the lignin distribution within the straw. It is also a
quick and easy method to observe the overall structure of
straw and hence to observe the changes that may occur after
any treatments used on straw. The evaluation of straw
morphology for preliminary research can usually be carried
out using optical microscopy or light microscopy. Basic parts,
including epidermis, cortex, vascular bundle and pith of the
straw, can be observed, with the epidermis cells arranging in
parallel rows and being closely packed to protect the internal
parts of the plant, the cortex being the zone between
epidermis and the vascular bundles and containing collen-
chyma and parenchyma cells, the pith being the central part
of the stem which is composed of thin walled parenchyma
cells, and the vascular bundles in the stem of the grass plants
varying in number and size but having the same basic struc-
ture composed of xylem and phloem (Fig. 5).

4. Structure of straw lignin

Fig. 4 e TEM micrograph of an ultrathin transverse section Lignin monolignols are polymerised by a radical coupling
of the cell wall of Forsythia suspensa fibre: S1 [ outer process that links them by carbonecarbon or ether bonds. A
secondary wall, S2 [ middle secondary wall and linkage may occur at any of several different locations on each
S3 [ inner secondary wall [118]. phenolic unit, causing many different linkage types to be

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 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6 9

to be implicated in the cross-linking of cell wall carbohydrates

with lignin [129]. Obviously such cross-linking can have a
dramatic influence on the mechanical properties and biode-
gradability of straw [4].
All grasses have lignin that is acylated by p-coumaric acid.
Early attempts at establishing the acylation regiochemistry
(the site on lignin where the acylation occurs) utilised UV and
concluded that p-coumarates were mainly at the g-position of
lignin side chains [130]. NMR work on corn lignin, subse-
quently on wheat [4] and other grasses showed that the
acylation was exclusively at the g-position, implicating
enzymatic processes in the formation of the ester. Thio-
Fig. 5 e Light micrograph of cross section of grass stem, acidolysis confirmed the presence of g-acylation, although
showing four basic parts [23]. esters are partially cleaved [113]. The DFRC method, which
leaves such g-esters intact, further established the g-acylation
and, as for acetates, indicated that p-coumarates were pre-
dominantly on syringyl units [105].
possible. The most common linkage types found in a lignin
molecule are b-O-4, a-O-4, b-5, 5e5, 4-O-5, b-1 and b-b
4.1. LCC linkages
[119,120]. The ether type linkages dominate in native lignin,
estimated to make up one half to two thirds of the total
Lignin is always associated with carbohydrates (in particular
number of native plant lignin linkages. Monolignols form
with hemicelluloses) via covalent bonds at two sites: ⍺-carbon
branch points within the polymer giving a network-like
and C-4 in the benzene ring, and this association is called
structure. Given the variety of linkages that occur, lignin
lignin carbohydrate complexes (LCC). The major inter-unit
molecules cannot be depicted as a series of regular, defined
linkages within the lignin monomer (H, S, and G) are b-O-4,
repeating units, as traditional polymers are. In contrast, lignin
b-b, b-5, b-1 arrangements. The chemical linkages limit the
is a highly irregular complex polymer [119,120].
efficient separation of lignin from plant cell wall. Thus, it is
Lignin is one of major cell wall component of herbaceous
important to understand the LCC linkages of lignin samples.
crops. The composition and structure of lignin vary consid-
Four types of linkages between lignin and carbohydrates
erably within and among plants; Lignin has been classified
have been suggested: glycosidic linkages, ester linkages,
into three groups: Gymnosperm (softwood), Angiosperm
benzyl ether linkages and hemiacetal or acetal linkages [12].
(hardwood) and Grass lignin. However, it was recently found
Among these linkages, the possibilities of glycosidic, benzyl
that these categories are inadequate because of ignoring most
ether and ester linkages have been demonstrated with model
of the herbaceous angiosperm lignin and some conifer fam-
compounds [13]. The association of lignin and carbohydrates
ilies containing guaiacyl and syringyl types of lignin. It was
in grass cell walls is largely through free radical coupling of
suggested by Gibbes that lignin should be categorised into two
ferulates or diferulates with monolignols or growing lignin
groups: guaiacyl lignin and guaiacylesyringyl lignin according
oligomers. Due to the complexity of LCCs, the isolation of
to their overall chemical constitutions [12]. Each type also
homogeneous preparation is the most important step for the
generally contains a small proportion of p-coumaryl units,
following compositional analysis and structural characteri-
though grass lignin generally contains a higher amount of p-
sation. Many isolation methods for LCC have been tried, but
coumaryl units than woody lignins [121]. Other phenolic
the finely divided plant meal [131,132] and MWL [133] were
monolignols have been identified, but they generally make up
preferred sources for LCC isolation because any chemical or
a much smaller portion of the lignin molecule [122]. Other
biochemical treatment before LCC isolation would break
phenolic compounds (alcohols, aldehydes, acids, esters and
possible linkages between lignin and carbohydrates.
amides) have been reported to act as lignin precursors and
The cross-linking of lignin and carbohydrates via ferulates/
illustrate the structural ‘‘plasticity” of the polymer and the
diferulates in grass cell walls has been demonstrated [12]; it is
adaptability of the lignification mechanisms in plants
generally accepted that the cross-linking of carbohydrates
[123,124], but several of these compounds (such as p-hydrox-
and lignin by ferulates presents the greatest barrier to efficient
ycinnamaldehydes, ferulic acid or 5-hydroxyconiferyl alcohol)
utilisation of grass cell wall [113].
only provide a significant contribution to lignin in transgenic
plants with modified monolignol biosynthesis [124] and are
4.2. Physical properties
minor lignin precursors in normal plants [125]. On the other
hand, some phenolic compounds with saturated or no side-
The physicochemical properties of straw lignin are known to
chains, e.g. dihydrocinnamyl alcohol, sporadically incorpo-
be different from those of softwoods or hardwoods, with
rate to the lignin polymer as terminal units as confirmed by 2D
straw lignin possessing characteristic alkali solubility. The
NMR [126,127].
physicochemical state of lignin dictates how and where it can
The solubility of straw lignin in alkali has been attributed
be utilised in the production of various products. The source
mainly to the presence of significant amounts of p-hydrox-
from which lignin is obtained and the method of extraction
yphenyl residues, which are bound to lignin as p-coumarate
has a strong bearing on its properties [16]. As lignin from
units [128]. Ferulic and p-coumaric acids in grasses are known
different crops or treatment can be extremely diverse in

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10 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6

structure and hence physical properties, it is necessary to
determine these differences through analytical methods. The
reactivity and physicochemical properties of lignin are
dependent to certain extent on their molecular weight distri-
bution (Table 6). The reactivity of lignin will impact on the
attributes of the end products. For example, Muller and
Glasser [134] found that kraft lignin-based phenol formalde-
hyde resins have superior properties to steam exploded
lignin-based phenol formaldehyde resins. Methods for lignin
molecular weight determinations are developed and
described by previous researchers [135,136]; gel permeation
chromatography (GPC) involves the chromatographic frac-
tionation of macromolecules according to molecular size.
The technique of fractionating macromolecules on Sephadex
gel has been applied to lignin materials since early 1960s
[137,138].
Another important parameter is the glass transition tem-
perature, Tg, which is an indirect measure of crystallinity and
degree of crosslinking and directly indicates the rubbery re-
gion of the material [139]. Lignin cross-linking degree will
depend on the quantity of water and polysaccharides, as well
as molecular weight and chemical functionalisation. Lignin
cross-linking degree generally increases with increasing mo-
lecular weight.
4.3. Lignin distribution and concentration
lignin absorption occurs at 280 nm. The weight concentration
of lignin was estimated to be 16% and 73% for the secondary
wall and CML of Norway spruce tracheids respectively [142].
Lange and Kjaer [143] proposed interference microscopy based
on the pathways of rays which is another method for quan-
titative analyses of lignin distribution within the cell wall
layers. With the development of electron dispersive X-ray
analysis (EDXA) combined with SEM and TEM, the interfer-
ence microscopy in conjunction with EDXA was applied to
determine lignin distribution in wood and straw, in differen-
tiating xylem and in chips during pulping [144,145]. The
technique is fully quantitative and precise. Donaldson [146]
used confocal laser scanning microscopy (CLSM) to study
lignin distribution in agricultural fibres.
Although many chemical studies of grass lignin have been
carried out, there is still little quantitative data on lignin dis-
tribution in grass species. Zhai and Lee [22] used SEM-EDXA
technique to determine the Br-L X-ray counts of the
different brominated wheat straw cell which are presented in
Table 7. In fibre and non-fibre cells of wheat straw, the lignin
concentration is the highest in the cell corners (CC) and the
lowest in the secondary wall. In the morphological regions of
all cells, the lignin concentration is the highest in parenchyma
cells, followed by fibres, and is the lowest in epidermis cell
[22]. Interference microscopy [144] and CLSM [147] were also
used by researchers to examine the lignin concentration of
wheat straw in the ML and in the secondary wall.

Lignin’s properties are significantly different with carbohy-

drates such as activity and solubility, according to which the
distribution of lignin in plant cell wall can be analysed. Many
techniques have been applied to the study of distribution of
lignin and organisation of cellulose components. One of the
oldest procedures is selective staining which follows by the
study under light microscope and another method to study
lignin distribution is electron microscopy of lignin skeleton
[140]. Although the mentioned methods are useful in studying
lignin distribution, only qualitative or semi-quantitative data
of lignin in various morphological regions can be provided.
The studies that focused on quantitative data of lignin dis-
tribution in the secondary wall and compound middle lamella
(CML) have been carried out by using UV microscopy [141]. UV
absorption is a tool used for lignin identification because
4.4. Lignin functional groups and their characteristics
The key chemical functional groups in lignin are the hydroxyl,
methoxyl, carbonyl and carboxylic groups. The quantity of
these groups depends on the genetic origin and isolation
processes adapted. Functional group characterisation can be
used to determine the lignin structure. Various analytical
methods for determining functional groups in technical lignin
have been studied, while the statistical comparison of the
various analytical methods for hydroxyl groups have been
found not fully comparable [148], the aminolysis and non-
aqueous potentiometry are assumed to be the most reliable
for phenolic hydroxyl and for determining the total carbonyl,
and the oximating method was found the most reliable
method which was first described by Faix et al. [149]. The

Table 6 e Molecular weight and functional groups of

lignin [1]. Table 7 e Lignin concentration and distribution in wheat
Lignin type Mn (g mol-1) COOH OH Methoxy straw [22].
(%) phenolic (%) Cells S CML CC
(%)
Br-L X-ray counts
Soda 2160 13.6 5.1 10.0 Thick wall fibre 1620 2156 2992
(bagasse) Thin wall fibre 1340 2004 3336
Organosolv 2000 7.7 3.4 15.1 Parenchyma cell 2005 2599 e
(bagasse) Epidermis cell 1022 1690 1743
Soda (wheat 1700 7.2 2.6 16 Lignin concentration (g g1)
straw) Thick wall fibre 0.168 0.412 0.571
Organosolv 800 3.6 3.7 19 Thin wall fibre 0.154 0.339 0.664
(hardwood) Lignin distribution (%)
Kraft 3000 4.1 2.6 14 Thick wall fibre 67.5 18.0 14.5
(softwood) Thin wall fibre 57.5 20.3 22.22

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 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6
phenolic hydroxyl group is a significant functionality affecting
the physical and chemical properties of lignin polymer.
Moreover, the chemical reactivity of lignin in various modifi-
cation processes is influenced by its phenolic hydroxyl con-
tent which is significant in the reaction with formaldehyde for
the production of lignin adhesive [150]. Lai described a pro-
cedure to determine free phenolic hydroxyl groups in lignin
[151]. Common physical method used to estimate the phenolic
hydroxyl content of lignin is UV spectroscopy, which is based
on difference in absorption of phenolic units in neutral and
alkaline solutions [152].
Methoxyl groups (eOCH3) are found in lignin from all
plants. The amount of the groups depends on plants species
and on the method of isolation. The amount of the methoxyl
groups is often used as a measure of the purity of lignin
preparation, since the isolated lignin sometimes is contami-
nated with hydrocarbons. The classical method for methoxyl
determination of lignin uses hydroiodic acid to promote
demethylation and gas chromatography to determine the
percentage methoxyl. Alternative method, which is less
tedious, involves the use of proton nuclear magnetic reso-
nance (1H NMR) [153].
There are the primary aliphatic hydroxyl groups bonded to
the g-C-atom, secondary aliphatic hydroxyl groups bonded to
the ⍺-C-atom and phenolic hydroxyl groups bonded to the 4-
C-atom of the aromatic ring in lignin. The accurate determi-
nation is difficult since they have very similar chemical
properties and reactivity. The amount of total hydroxyl group
in lignin was determined by potentiometry [154].
Natural lignin contains a low concentration of COOH-
groups. However, when native lignin is subjected to chemi-
cal or biological treatments, carboxyl groups are frequently
detected in significant quantities, hence quantitative mea-
surements of carboxyl groups may provide information
regarding the degree to which the lignin has been degraded or
modified as a result of treatment. Further carboxyl groups are
produced during delignification as a result of oxidation of
hydroxyl and carbonyl groups. Oxidation of the lignin struc-
ture will result in an increase of carboxyl absorption in the FT-
IR spectra as it was observed by Xu et al. [7]. In work on lignin
31
Fig. 6 e P NMR of wheat straw lignin phosphitylated with 2-chloro-4, 4, 5, 5-tetramethyl-1, 3, 2 dioxaphospholane.
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11
characterisation by Sahoo et al. [15], FT-IR analysis pointed
out the differences in the bonding pattern and the functional
groups. These different chemical functionalities are very
important components when it comes to utilising lignin in
biomass for composite applications, the final properties of the
composites.
5. Wheat and rice straw lignin
characteristics
Researchers have focused on the structural analysis of wheat
and rice straw by using a combination of qualitative and
quantitative techniques in order to either, optimise these
renewable raw materials for industrial applications or to
investigate the changes within the composition of straw
biomass after a certain treatments. Most of the researches in
this area are comparative studies between different types of
biomass to establish the structural differences between them.
Xu et al. [7] in a comparative study examined the chemical
characteristics of lignin in order to get more information on
chemical structures and relationship between physical prop-
erties and chemical structures. The analytical quantitative
techniques such as FT-IR and 1H and 13C NMR spectroscopy
were used to investigate the changes occurring in lignin
structure during the organosolv treatment processes. 1H and
13
C NMR spectra revealed that the lignin comprised guaiacyl,
syringyl and a small amount of p-hydroxyphenyl units. The
various covalent linkages and physicochemical interactions
between lignin, cellulose, hemicelluloses, phenolic acids and
other polyphenolic or proteinaceous or mineral extractives all
contribute structural integrity to the wheat straw cell wall
composite [155].
Recently, an in-situ quantitative 2D-HSQC NMR technique
(the ball-milled plant cell wall was dissolved in DMSO-d6) was
used for characterising the changes in the cell wall during the
hydrothermal pre-treatment (195  C for 6 min) process of
wheat straw for second-generation bioethanol production
[156]. This study provides an effective quantitative method to
reveal the structural changes of cell wall components based
12 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6
on in situ 2D-HSQC NMR techniques. The results revealed
substantial lignin b-aryl ether cleavage, deacetylation via
cleavage of the natural acetates at the 2-O- and 3-O-positions
of xylan, and uronic acid depletion via cleavage of the (1/2)-
linked 4-O-methyl-a-D-glucuronic acid of xylan after hydro-
thermal treatment.
By combining mild alkaline hydrolysis with quantitative
31
P NMR, Crestini and Argyropoulos [4] have been able to
arrive at a protocol for determining the various ester linkages
and their relative contributions to the overall structure of
wheat straw lignin; their technique has been applied on
samples of milled wheat straw (ML), dioxane acidolysis wheat
straw lignin (AL), and a milled wood lignin from black spruce
(BSL). A typical spectrum together with the detailed signal
assignment can be obtained (based on earlier efforts of
Granata and Argyropoulos [17] and Jiang et al. [18]) (Fig. 6).
Rice straw has high lignin and low nitrogen contents which
in combination are responsible for its low nutritive value.
Researchers in recent years have attempted to improve its
nutritive value by chemical treatments [157]. Rice straw is
highly heterogeneous and complex, and its fractionation and
utilisation should be conducted on different levels according
to structural properties. Dohnani et al. [158] evaluated the
variability in chemical and morphological variables of Euro-
pean rice straw in their work with the aim to relate these
variations to changes in “in sacco” degradation. In their work
fifteen rice straw varieties are analysed for their chemical and
morphological compositions, the results indicated that both
chemical and morphological characteristics have great vari-
ability. For instance the ash content of rice straw samples
ranged from 9.6 to 14.1% which are very low compared to the
reported ash content (averaged 14.1%) in Philippine varieties
[159], (16.6%) in Asian varieties [160], and (18.6%) in Californian
varieties [161]. NIR spectroscopy was used for determination
of rice straw analytical parameters such as total ash, mois-
ture, cellulose, hemicellulose and lignin; where the chemical
composition content varied among different rice straw sam-
ples (Table 8). This resulted from the different rice straw va-
rieties, the various tissue parts and the different origins [162].
Thermal gravimetric analysis and differential thermal
behaviour of rice straw were investigated [163], where the
degradation was found to be of first order reaction and hemi-
cellulose was found to be less stable than cellulose. The study
of structural features and physicochemical and thermal prop-
erties of the lignin, isolated with 1 M NaOH at 30  C for 18 h
from de-waxed rice straw, maize stems and rye straw using a
combination of several destructive and non-destructive
Table 8 e Chemical composition of rice straw [162].
Component Range (%) Average (%)a
Moisture 4.2e9.8 6.9
Hemicellulose 19.8e31.6 25.6
Cellulose 30.3e38.2 33.9
Klason lignin 7.2e12.8 10.2
Acid insoluble ash 3.8e13.2 7.8
Total ash 7.8e15.6 11.8
a Average of 34 samples.
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techniques, showed the treatment with 1 M NaOH at 30  C for
18 h can significantly cleave the ether linkages between lignin
and hemicelluloses from the cell walls of the straws and stems
[6]. The hemicelluloses of rice straw can substantially be
decomposed between 200 and 300  C and above 245  C the
decomposition of cellulose starts to take place reaching to a
maximum peak around 300  C and yielding mainly volatile
products [6]. The decomposition process of lignin covered a
larger temperature range, between 250 and 600  C, however at
temperatures lower than 400  C only 40% was decomposed,
rendering charcoal as the residue product. This result implied
that hemicelluloses degraded in first place, while lignin
showed less degradation, and therefore, its structure was more
stable. Cellulose, on the other hand, showed an important
degradation process, mainly between 250 and 330  C.
6. Conclusions
The analytical techniques up to date for the structural analysis
of straw lignin were reviewed and the resultant un-
derstandings of the structure of straw lignin were summarised.
The studies on lignin could be divided into the qualitative
and quantitative analyses, the former includes SEM-EDX,
TEM, optical microscopy and the latter includes FT-IR, TGA/
DSC, NMR and DFRC. Different analytical methods applied to
same lignin samples could provide significantly different re-
sults that are not directly comparable. This problem, coupled
with the extreme heterogeneity of lignin and its chemical
structure among different crop species, morphology and
maturation degree has made to date the quantitative struc-
tural characterisation of lignin an open debate. More sensitive
and reliable quantitative HSQC techniques are the most
diagnostic and accurate analytical tools to investigate lignin
structure, among them the magnetic resonance techniques
when applied to lignin have proved to be efficient analytical
tools for the structural elucidation of these complex bio-
polymers. These NMR techniques are capable of detecting and
quantitatively determining all functional groups in lignin
possessing labile hydroxyl groups, i.e. aliphatic OH, the
various forms of phenolic OH and carboxylic acids.
The composition and structure of straw lignin varies
considerably within and among plants. The nature of lignin
carbohydrate linkages and occurrence is not completely clear
and the isolation processes could affect the lignin structure.
Because of the complexity of the chemical composition of
lignin in straw, it is difficult to find a single technique to
analyse its structure, hence the most accurate way to study the
straw lignin could be the combination of several techniques,
each providing partial but complimentary information.
references
[1] Doherty WOS, Mousavioun P, Fellows CM. Value-adding to
cellulosic ethanol: lignin polymers. Ind Crop Prod
2011;33:259e76.
[2] Stewart D. Lignin as a base material for materials
applications: chemistry, application and economics. Ind
Crops Prod 2008;27:202e7.
 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6
[3] Wen J, Sun S, Xue B, Sun R. Recent advances in
characterization of lignin polymer by solution-state nuclear
magnetic resonance (NMR) methodology. Materials
2013;6:259e391.
[4] Crestini C, Argyropoulos D. Structural analysis of wheat
straw lignin by quantitative 31P and 2D NMR spectroscopy.
The occurrence of ester bonds and a O-4 substructures. J
Agric Food Chem 1997;45:1212e9.
[5] Derkacheva O, Sukhov D. Investigation of lignins by FTIR
spectroscopy. Macromol Symp 2008;265:61e8.
[6] Xiao B, Sun XF, Sun R. Chemical, structural, and thermal
characterizations of alkali-soluble lignins and
hemicelluloses, and cellulose from maize stems, rye straw,
and rice straw. Polym Degrad Stab 2001;74:307e19.
[7] Xu F, Sun J, Sun R, Fowler P, Baird MS. Comparative study of
organosolv lignins from wheat straw. Ind Crop Prod
2006;23:180e93.
[8] Akin DE, Benner R. Degradation of polysaccharides and
lignin by ruminal bacteria and fungi. Appl Environ Microbiol
1988;54:1117e25.
[9] Baurhoo B, Ruiz-Feria CA, Zhao X. Purified lignin: nutritional
and health impacts on farm animalsda review. Anim Feed
Sci Technol 2008;144:175e84.
[10] Kirk T. Effects of microorganisms on lignin. Annu Rev
Phytopathol 1971;9:185e210.
[11] Smook G. Handbook for pulp and paper technologies. 3rd
ed. Vancouver, BC: Angus Wilde Publications Inc; 2002.
[12] Sun R. Lignin. In: Lu F, Ralph J, editors. Cereal straw as a
resource for sustainable biomaterials and biofuels.
Amsterdam, the Netherlands: Elsevier; 2010. p. 169e207.
[13] Azuma J. In: Linskens H, Jackson J, editors. Analysis of
lignin-carbohydrate complexes of plant cell walls. Springer
Berlin Heidelberg; 1989. p. 100e26.
[14] Lee SH, Doherty TV, Linhardt RJ, Dordick JS. Ionic liquid-
mediated selective extraction of lignin from wood leading
to enhanced enzymatic cellulose hydrolysis. Biotechnol
Bioeng 2009;102:1368e76.
[15] Sahoo S, Seydibeyog  lu MÖ, Mohanty AK, Misra M.
Characterization of industrial lignins for their utilization in
future value added applications. Biomass Bioenergy
2011;35:4230e7.
[16] Lora J, Glasser W. Recent industrial applications of lignin: a
sustainable alternative to nonrenewable materials. J Polym
Environ 2002;10:39e48.
[17] Granata A, Argyropoulos D. 2-Chloro-4, 4, 5, 5-tetramethyl-
1, 3, 2-dioxaphospholane, a reagent for the accurate
determination of the uncondensed and condensed phenolic
moieties in lignins. J Agric Food Chem 1995;43:1538e44.
[18] Jiang Z, Argyropoulos D, Granata A. Correlation analysis of
31P-NMR chemical shifts with substituent effect of phenols.
Magn Reson Chem 1995;43:1538e44.
[19] Morrison I. The degradation and utilization of straw in the
rumen. In: Grossbard E, editor. Straw decay and its effects on
disposal and utilization. Chichester: Wiley; 1979. p. 237e45.
[20] Harper S, Lynch J. The chemical components and
decomposition of wheat straw leaves, intemodes and
nodes. J Sci Food Agic 1981;32:1057e62.
[21] Theander O. Review of straw carbohydrate research. In:
Hill R, Munck L, editors. New approaches to research on
cereal carbohydrates. Amsterdam: Elsevier Science
Publisher BV; 1985. p. 217e30.
[22] Zhai H, Lee Z. Ultrastructure and topochemistry of
delignification in alkaline pulping of wheat straw. J Wood
Chem Technol 1989;9:387e406.
[23] Sun R. Structure, ultrastructure, and chemical composition.
In: Xu F, editor. Cereal straw as a resource for sustainable
biomaterials and biofuels. Amsterdam, the Netherlands:
Elsevier; 2010. p. 9e47.
Please cite this article in press as: Ghaffar SH, Fan M, Structural analysis for lignin characteristics in biomass straw, Biomass
and Bioenergy (2013), http://dx.doi.org/10.1016/j.biombioe.2013.07.015
13
[24] Yu H, Liu R, Shen D, Wu Z, Huang Y. Arrangement of
cellulose microfibrils in the wheat straw cell wall.
Carbohydr Polym 2008;72:122e7.
[25] Reddy N, Yang Y. Properties of high-quality long natural
cellulose fibers from rice straw. J Agric Food Chem
2006;54:8077e81.
[26] Lapierre C, Monties B, Rolando C. Thioacidolysis of lignin:
comparison with acidolysis. J Wood Chem Technol
1985;5:277e92.
[27] Freudenberg K, Lautsch W, Engler K. Die Bildung von
Vanillin aus Fichtenlignin. Berichte der deutschen
chemischen Gesellschaft (A B Series) 1940;73:167e71.
[28] Lu F, Ralph J. Derivatization followed by reductive cleavage
(DFRC Method), a new method for lignin analysis: protocol
for analysis of DFRC monomers. J Agr Food Chem
1997;45:2590e2.
[29] Kelley SS, Rowell RM, Davis M, Jurich CK, Ibach R. Rapid
analysis of the chemical composition of agricultural fibers
using near infrared spectroscopy and pyrolysis molecular
beam mass spectrometry. Biomass Bioenergy
2004;27:77e88.
[30] Sanderson MA, Agblevor F, Collins M, Johnson DK.
Compositional analysis of biomass feedstocks by near
infrared reflectance spectroscopy. Biomass Bioenergy
1996;11:365e70.
[31] Freudenberg K, Sohns F, Dürr W, Niemann C. Über Lignin,
Coniferylalkohol und Saligenin. Cellulosechemie
1931;12:263e75.
[32] Martı́nez ÁT, Rencoret J, Marques G, Gutiérrez A, Ibarra D,
Jiménez-Barbero J, et al. Monolignol acylation and lignin
structure in some nonwoody plants: a 2D NMR study.
Phytochemistry 2008;69:2831e43.
[33] Zhang L, Gellerstedt G. Quantitative 2D HSQC NMR
determination of polymer structures by selecting suitable
internal standard references. Magn Reson Chem
2007;45:37e45.
[34] Sette M, Wechselberger R, Crestini C. Elucidation of lignin
structure by quantitative 2D NMR. Chemistry? A Eur J
2011;17:9529e35.
[35] Morrison I. A semi-micro method for the determination of
lignin and its use inpredicting the digestibility of forages. J
Sci Food Agric 1972;23:455e63.
[36] Theander O, Aman P. Studies on dietary fibres. Swedish J
Agric Res 1979;9:97e106.
[37] Van Soest P, Wine R. Determination of lignin and cellulose
in acid-detergent fibre with permanganate. J Assoc Off Anal
Chem 1968;51:780e5.
[38] Fidalgo M, Terron M, Martinez A, Gonzalez A, Gonzalez-
vila F, Galletti G. Comparative study of fractions from
alkaline extraction of wheat straw through chemical
degradation, analytical pyrolysis, and spectroscopic
techniques. J Agric Food Chem 1993;41:1621e6.
[39] Hortling B, Tarja T, Kentta E. Determination of carboxyl
and non-conjugated carbonyl groups in dissolved and
residual lignins by IR spectroscopy. Holzforschung
1997;51:405e10.
[40] Gilarranz M, Rodrı́guez F, Oliet M, Garcı́a J, Alonso V.
Phenolic OH group estimation by FTIP and UV spectroscopy.
Application to organosolv lignins. J Wood Chem Technol
2001;21:387e95.
[41] Durie R, Lynch B, Sternhell S. Comparative studies of brown
coal and lignin. I. Infra-red spectra. Austral J Chem
1960;13:156e68.
[42] Bolker HI, Somerville NG. Infrared spectroscopy of lignins.
Pulp Paper Can Mag 1963;64:187e94.
[43] Colthup N, Daly L, Wiberley S. Introduction to infrared and
Raman spectroscopy. London: Academic Press Limited;
1990.
14 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6
[44] Collier W, Kalasinsky V, Schulz T. Infrared study of lignin:
reexamination of aryealkyl ether CeO stretching peak
assignment. Holzforschung 1997;46:523e8.
[45] Faix O. Classification of lignins from different botanical
origins by FT-IRspectroscopy. Holzforschung 1991;45:21e7.
[46] Glasser W. Classification of lignin according to chemical
and molecular structure. In: Glasser W, Northey R,
Schultz T, editors. Lignin: historical, biological, and
materials perspectives. ACS Symposium Series No. 742.
Washington, DC: American Chemical Society; 2000.
p. 216e38.
[47] Nowakowski DJ, Jones JM. Uncatalysed and potassium-
catalysed pyrolysis of the cell-wall constituents of biomass
and their model compounds. J Anal Appl Pyrolysis
2008;83:12e25.
[48] Qiang L, Wen-zhi L, Dong Z, Xi-feng Z. Analytical
pyrolysisegas chromatography/mass spectrometry (PyeGC/
MS) of sawdust with Al/SBA-15 catalysts. J Anal Appl
Pyrolysis 2009;84:131e8.
[49] Fahmi R, Bridgwater AV, Thain SC, Donnison IS, Morris PM,
Yates N. Prediction of Klason lignin and lignin thermal
degradation products by PyeGC/MS in a collection of Lolium
and Festuca grasses. J Anal Appl Pyrolysis 2007;80:16e23.
[50] Del Rı́o J, Gutierrez A, Rodriguez I, Ibarra D, Martinez A.
Composition of non-woody plant lignins and cinnamic
acids by Py-GC/MS, Py/TMAH and FT-IR. J Anal Appl Pyrol
2007;79:39e46.
[51] Hornsby PR, Hinrichsen E, Tarverdi K. Preparation and
properties of polypropylene composites reinforced with
wheat and flux straw fibres part 1 fibre characterization. J
Mater Sci 1997;32:443e9.
[52] Müller-Hagedorn M, Bockhorn H. Pyrolytic behaviour of
different biomasses (angiosperms) (maize plants, straws,
and wood) in low temperature pyrolysis. J Anal Appl
Pyrolysis 2007;79:136e46.
[53] Ghaly AE, Ergüdenler A, Al Taweel AM. Determination of
the kinetic parameters of oat straw using
thermogravimetric analysis. Biomass Bioenergy
1993;5:457e65.
[54] Yang Q, Wu S, Lou R, Lv G. Analysis of wheat straw lignin by
thermogravimetry and pyrolysisegas chromatography/
mass spectrometry. J Anal Appl Pyrolysis 2010;87:65e9.
[55] Carrier M, Loppinet-Serani A, Denux D, Lasnier J, Ham-
Pichavant F, Cansell F, et al. Thermogravimetric analysis as
a new method to determine the lignocellulosic composition
of biomass. Biomass Bioenergy 2011;35:298e307.
[56] Brebu M, Vasile C. Thermal degradation of ligninea review.
Cellulose Chem Technol 2009;44:353e63.
[57] Ludwig C. Magnetic resonance spectra. In: Sarkanen K,
Ludwig C, editors. Lignin, occurrence, formation, structure
and reactions. New York: Wiler-Interscience; 1971.
p. 299e344.
[58] Lundquist K. NMR-studies of lignins. 4. Investigation of
spruce lignin by H-1-NMR spectroscopy. Acta Chem Scand B
1980;34:21e6.
[59] Lundquist K. NMR-Studies of Lignins. 5. Investigation of
non-derivatized spruce and birch lignin by H-1-NMR
spectroscopy. Acta Chem Scand B 1981;35:497e501.
[60] Lundquist K. Proton (1H) NMR spectroscopy. In: Lin S,
Dence C, editors. Methods in lignin chemistry. New York,
NY, USA: Springer-Verlag; 1992. p. 242e9.
[61] Gellerstedt G, Robert D. Quantitative 13C NMR analysis of
kraft lignins. Acta Chem Scand B 1987;41:541e6.
[62] Pan D, Tai D, Chen C. Comparative studies on chemical
composition of wood components in recent and ancient
woods of Bischofia polycarpa. Holzforschung 1990;44:7e16.
[63] Robert D. Carbon-13 nuclear magnetic resonance
spectrometry. In: Lin S, Dence C, editors. Methods in lignin
Please cite this article in press as: Ghaffar SH, Fan M, Structural analysis for lignin characteristics in biomass straw, Biomass
and Bioenergy (2013), http://dx.doi.org/10.1016/j.biombioe.2013.07.015
chemistry. New York, NY, USA: Springer Berlin Heidelberg;
1992. p. 250e73.
[64] Hawkes G, Smith C, Utley J, Vargas R, Viertler H. A
comparison of solution and solid state 13C-NMR spectra of
lignins and lignin model compounds. Holzforschung
1993;47:302e12.
[65] Ralph J, Landucci L. NMR of lignins. In: Heitner C, Dimmel D,
Schmidt J, editors. Lignin and lignans: advances in
chemistry. Boca Raton, FL, USA: CRC Press; 2010. p. 137e234.
[66] Xia Z, Akim L, Argyropulos D. Quantitative 13C NMR
analysis of lignins with internal standards. J Agric Food
Chem 2001;49:3573e8.
[67] Capanema E, Balakshin M, Kadla J. A comprehensive
approach for quantitative lignin characterization by NMR
spectroscopy. J Agric Food Chem 2004;52:1850e60.
[68] Capanema E, Balakshin M, Kadla J. Quantitative
characterization of a hardwood milled wood lignin by
nuclear magnetic resonance spectroscopy. J Agric Food
Chem 2005;53:9639e49.
[69] Terashima N, Atalla R, Vanderhart D. Solid state NMR
spectroscopy of specifically C-13-enriched lignin in wheat
straw from coniferin. Phytochemistry 1997;46:863e70.
[70] Gu R, Xie Y, Zeng S, Wu H, Yasuda S. Carbon-13enrichment
of rice stalk lignin traced by solid state C-13 NMR
spectroscopy. Chem J Chinese Univ 2002;23:1073e6.
[71] Landucci L. Quantitative C-13 NMR characterization of
lignin. 1. A methodology for high-precision. Holzforschung
1985;39:355e9.
[72] Landucci L. Application of modern liquid-state NMR to
lignin characterization.2. C-13 signal resolution and useful
techniques. Holzforschung 1991;45:425e32.
[73] Yelle D, Ralph J, Frihart C. Characterization of
nonderivatized plant cell walls using high-resolution
solution-state NMR spectroscopy. Magn Reson Chem
2008;46:508e17.
[74] Kim H, Ralph J, Akiyama T. Solution-state 2D NMR of ball-
milled plant cell wall gels in DMSO-d6. Bioenerg Res
2008;1:56e66.
[75] Kim H, Ralph J. Solution-state 2D NMR of ball-milled plant
cell wall gels in DMSO-d(6)/pyridine-d(5). Org Biomol Chem
2010;8:576e91.
[76] Davin LB, Lewis NG. Lignin primary structures and dirigent
sites. Curr Opin Biotechnol 2005;16:407e15.
[77] Koskela H. Quantitative 2D NMR studies. Ann Rep NMR
Spectrosc 2009;66:1e31.
[78] Ede R, Brunow G. Application of 2-dimensional homonuclear
and heteronuclear correlation NMR-spectroscopy to wood
lignin structure determination. J Org Chem 1992;57:1477e80.
[79] Ede R, Kilapelainen I. Homo-nuclear and hetero-nuclear 2d
NMR techniqueseumambiguous structural probes for
noncyclic benzyl aryl ethers in soluble lignin samples. Res
Chem Intermediat 1995;21:313e28.
[80] Ralph J, Marita J, Ralph S, Hatfield R, Lu F, Ede R, et al.
Solution-state NMR of lignins. In: Argyropoulos D, editor.
Advances in lignocellulosics characterization. Atlanta, GA,
USA: TAPPI Press; 1999. p. 55e108.
[81] Liitia T, Maunu S, Hortling B, Toikka M, Kilpelainen I.
Analysis of technical lignins by two- and three-dimensional
NMR spectroscopy. J Agr Food Chem 2003;51:2136e43.
[82] Del Rı́o J, Rencoret J, Marques G, Li J, Gellerstedt G, Jiménez-
Barbero J, et al. Structural characterization of the lignin
from jute (Corchorus capsularis) fibers. J Agric Food Chem
2009;57:10271e81.
[83] Del Rı́o J, Rencoret J, Prinsen P, Martı́nez A, Ralph J,
Gutiérrez A. Structural characterization of wheat straw
lignin as revealed by analytical pyrolysis, 2D-NMR, and
reductive cleavage methods. J Agric Food Chem
2012;60:5922e35.
 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6
[84] Wen J, Sun Z, Sun Y, Sun S, Xu F, Sun R. Structural
characterization of alkali-extractable lignin fractions from
bamboo. J Biobased Mater Bioenergy 2010;4:408e25.
[85] Wen J, Xue B, Xu F, Sun R, Pinkert A. Unmasking the
structural features and property of lignin from bamboo. Ind
Crop Prod 2013;42:332e43.
[86] Li M, Sun SN, Xu F, Sun R. Formic acid based organosolv
pulping of bamboo (Phyllostachys acuta): comparative
characterization of the dissolved lignins with milled wood
lignin. Chem Eng J 2012;179:80e9.
[87] Shi Z, Xiao L, Jia-Deng, Xu Feng, Sun R. Physicochemical
characterization of lignin fractions sequentially isolated
from bamboo (Dendrocalamus brandisii) with hot water
and alkaline ethanol solution. J Appl Polym Sci
2012;125:3290e301.
[88] Yuan T, Sun S, Xu F, Sun R. Isolation and physico-chemical
characterization of lignins from ultrasound irradiated fast-
growing poplar wood. BioResources 2011;6:414e33.
[89] Yuan T, Sun S, Xu F, Sun R. Characterization of lignin
structures and ligninecarbohydrate complex (LCC) linkages
by quantitative 13C and 2D HSQC NMR spectroscopy. J Agric
Food Chem 2011;59:10604e14.
[90] Yuan T, Sun S, Xu F, Sun R. Structural characterization of
lignin from triploid of Populus tomentosa Carr. J Agric Food
Chem 2011;59:6605e15.
[91] Karhunen P, Rummakko P, Sipilä J, Brunow G, Kilpelainen I.
The formation of dibenzodioxocin structures by oxidative
coupling. A model reaction for lignin biosynthesis.
Tetrahedron Lett 1995;36:4501e4.
[92] Zhang A, Lu F, Sun R, Ralph J. Isolation of cellulolytic
enzyme lignin from wood preswollen/dissolved in dimethyl
sulfoxide/N-methylimidazole. J Agric Food Chem
2010;58:3446e50.
[93] Wen J, Xue B, Xu F, Sun R. Unveiling the structural
heterogeneity of bamboo lignin by in situ HSQC NMR
technique. Bioenergy Res 2012;5:886e903.
[94] Robert D, Ammalahti E, Bardet M, Brunow G, Kilpelainen I,
Lundquist K, et al. Improvement in NMR structural studies
of lignin through two- and three-dimensional NMR
detection and isotopic enrichment. In: Lewis NG,
Sarkanen S, editors. Lignin and lignan biosynthesis. ACS
Symposium Series, vol. 697. Washington DC: American
Chemical Society; 1998. p. 237e54.
[95] Ralph J, Grabber JH, Hatfield RD. Lignin-ferulate cross-links
in grasses: active incorporation of ferulate polysaccharide
esters into ryegrass lignins. Carbohydr Res 1995;275:
167e78.
[96] Lu F, Ralph J. Non-degradative dissolution and acetylation
of ball-milled plant cell walls: high-resolution solution-
state NMR. Plant J 2003;35:535e44.
[97] mmalahti E, Brunow G, Bardet M, Robert D, Kilpelainen I.
Identification of side-Chain structures in a poplar lignin
using three-dimensional HMQC-HOHAHA NMR
spectroscopy. J Agric Food Chem 1998;46:5113e7.
[98] Balakshin MY, Capanema EA, Chen CL, Gracz HS.
Elucidation of the structures of residual and dissolved pine
kraft lignins using an HMQC NMR technique. J Agric Food
Chem 2003;51:6116e27.
[99] Gutiérrez A, Rencoret J, Cadena EM, Rico A, Barth D, del
Rı́o JC, et al. Demonstration of laccase-based removal of
lignin from wood and non-wood plant feedstocks. Bioresour
Technol 2012;119:114e22.
[100] Lu F, Ralph J. DFRC method for lignin analysis. 1. New
method for beta aryl ether cleavage: lignin model studies. J
Agr Food Chem 1997;45:4655e60.
[101] Hu Z, Yeh T, Chang H, Matsumoto Y, Kadla J. Elucidation of
the structure of cellulolytic enzyme lignin. Holzforschung
2006;60:389e97.
Please cite this article in press as: Ghaffar SH, Fan M, Structural analysis for lignin characteristics in biomass straw, Biomass
and Bioenergy (2013), http://dx.doi.org/10.1016/j.biombioe.2013.07.015
15
[102] Lu F, Ralph J. The DFRC method for lignin analysis. 2.
Monomers from isolated lignins. J Agr Food Chem
1998;46:547e52.
[103] Yelle DJ, Ralph J, Lu F, Hammel KE. Evidence for cleavage of
lignin by a brown rot basidiomycete. Environ Microbiol
2008;10:1844e9.
[104] Lu F, Ralph J. Efficient ether cleavage in lignins: the “DFRC”
method as abasis for new analytical methods. In: Lewis N,
Sarkanen S, editors. Lignin and lignin biosynthesis.
Washington DC: American Chemical Society; 1998.
p. 294e322.
[105] Lu F, Ralph J. Detection and determination of p-coumaroylated
units in lignins. J Agr Food Chem 1999;47:1988e92.
[106] Del Rı́o J, Marques G, Rencoret J, Martinez A, Gutierrez A.
Occurrence of naturally acetylated lignin units. J Agr Food
Chem 2007;55:5461e8.
[107] Guerra A, Norambuena M, Freer J, Argyropoulos D.
Determination of arylglycerol-beta-aryl ether linkages in
enzymatic mild acidolysis lignins (EMAL): comparison of
DFRC/P-31 NMR with thioacidolysis. J Nat Prod
2008;71:836e41.
[108] Tohmura S, Argyropoulos D. Determination of arylglycerol-
beta-aryl ethers and other linkages in lignins using DFRC/
(31)P NMR. J Agr Food Chem 2001;49:536e42.
[109] Buranov AU, Mazza G. Lignin in straw of herbaceous crops.
Ind Crop Prod 2008;28:237e59.
[110] Sun RC, Fang JM, Tomkinson J. Delignification of rye straw
using hydrogen peroxide. Ind Crops Prod 2000;12:71e83.
[111] del Rı́o JC, Gutiérrez A, Rodrı́guez IM, Ibarra D, Martı́nez ÁT.
Composition of non-woody plant lignins and cinnamic
acids by Py-GC/MS, Py/TMAH and FT-IR. J Anal Appl
Pyrolysis 2007;79:39e46.
[112] del Rı́o JC, Gutiérrez A, Martı́nez ÁT. Identifying acetylated
lignin units in non-wood fibers using pyrolysis-gas
chromatography/mass spectrometry. Rapid Commun Mass
Spectrom 2004;18:1181e5.
[113] Grabber J, Quideau S, Ralph J. P-coumaroylated syringyl
units in maize lignin: implications for beta-ether cleavage
by thioacidolysis. Phytochemistry 1996;43:1189e94.
[114] Durot N, Gaudard F, Kurek B. The unmasking of lignin
structures in wheat straw by alkali. Phytochemistry
2003;63:617e23.
[115] Samuel R, Pu Y, Raman B, Ragauskas A. Structural
characterization and comparison of switchgrass ball-milled
lignin before and after dilute acid pretreatment. Appl
Biochem Biotechnol 2010;162:62e74.
[116] Mansfield SD, Kim H, Lu F, Ralph J. Whole plant cell wall
characterization using solution-state 2D NMR. Nat Protocols
2012;7:1579e89.
[117] Koo B, Min B, Gwak K, Lee S, Choi J, Yeo H, et al. Structural
changes in lignin during organosolv pretreatment of
Liriodendron tulipifera and the effect on enzymatic
hydrolysis. Biomass Bioenergy 2012;42:24e32.
[118] Ma JF, Yang GH, Mao JZ, Xu F. Characterization of anatomy,
ultrastructure and lignin microdistribution in Forsythia
suspensa. Ind Crop Prod 2011;33:358e63.
[119] Dence C, Lin S. Introduction. In: Lin S, Dence C, editors.
Methods in lignin chemistry. Berlin: Springer-Verlag; 1992.
p. 3e19.
[120] Sjöström E. Wood chemistry fundamentals and
applications. 2nd ed. San Diego: Academic Press, Inc; 1993.
[121] Lora J. Characteristics, industrial sources, and utilization of
lignins from nonwood plants. In: Hu T, editor. Chemical,
modification, properties, and usage of lignin. New York:
Kluwer Academic/Plenum Publishers; 2002. p. 267e82.
[122] Whetten R, MacKay J, Sederoff R. Recent advances in
understanding lignin biosynthesis. Annu Rev Plant Physiol
1998;49:585e609.
16 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e1 6
[123] Boerjan W, Ralph J, Baucher M. Lignin biosynthesis. Annu
Rev Plant Biol 2003;54:519e46.
[124] Ralph J. Perturbing lignification. In: Entwistle K, Harris P,
Walker J, editors. The compromised wood workshop.
Canterbury, New Zealand: University of Canterbury; 2007.
p. 85e112.
[125] Del Rı́o J, Martı́nez A, Gutiérrez A. Presence of 5-
hydroxyguaiacyl units as native lignin constituents in
plants as seen by Py-GC/MS. J Anal Appl Pyrolysis
2007;79:33e8.
[126] Balakshin MY, Capanema EA, Barry G, John F, Kadla JF. NMR
studies on Fraser fir Abies fraseri (Pursh) Poir. lignins.
Holzforschung 2005;59:488.
[127] Zhang L, Henriksson G, Gellerstedt G. The formation of beb
structures in lignin biosynthesis e are there two different
pathways? Org Biomol Chem 2003;1:3621e4.
[128] Scalbert A, Monties B, Rolando C, Sierra-Escudero A.
Formation of ether linkage between phenolic acids and
gramineae lignin: a possible mechanism involving quinone
methides. Holzforschung 1986;40:191e5.
[129] Higuchi T, Ito Y, Shimada M, Kawamura I. Chemical
properties of milled wood lignin of grasses. Phytochem
1967;6:1551e6.
[130] Shimada M, Fukuzuka T, Higuchi T. Ester linkages of p-
coumaric acid in bamboo and grass lignins. TAPPI
1971;54:72e8.
[131] Brownell H. Stability of lignin-carbohydrate complexes.
TAPPI 1971;54:66e71.
[132] Ford C. Comparative structural studies of lignin
carbohydrate complexes from Digitaria-Decumbens
(Pangola Grass) before and after chlorite delignification.
Carbohyd Res 1986;147:101e17.
[133] Lundquist K, Simonson R, Tingsvik K. On the occurrence of
carbohydrates in milled wood lignin preparations. Sven
Papperstidn 1979;82:272e5.
[134] Muller P, Glasser W. Engineering plastics from lignin. VIII.
Phenolic resin prepolymer synthesis and analysis. J Adhes
1984;17:157e73.
[135] Milne TA, Chum HL, Agblevor F, Johnson DK. Standardized
analytical methods. Biomass Bioenergy 1992;2:341e66.
[136] Cathala B, Saake B, Faix O, Monties B. Association behaviour
of lignins and lignin model compounds studied by
multidetector size-exclusion chromatography. J
Chromatogr A 2003;1020:229e39.
[137] Gellerstedt G. In: Lin S, Dence C, editors. Gel permeation
chromatography. Springer Berlin Heidelberg; 1992. p. 487e97.
[138] Kaj F, Raimo K, Sagfors P-E. Determination of the molar
mass distribution of lignins by gel permeation
chromatography. In: Glasser WG, Sarkanen A, editors.
Lignin properties and materials. ACS Symposium Series,
vol. 397. American Chemical Society; 1989. p. 124e33.
[139] Gargulak JD, Lebo SE. Commercial use of lignin-based
materials. In: Glasser WG, Northey RA, Schultz TP, editors.
Lignin: historical, biological, and materials perspectives.
ACS Symposium Series, vol. 742. American Chemical
Society; 1999. p. 304e20.
[140] Timell T. Studies on opposite wood in conifers part III:
distribution of lignin. Wood Sci Technol 1973;12:1e15.
[141] Lange P. The distribution of lignin in the cell wall of normal
and reaction wood from spruce and a few hardwoods. Sven
Papperstidn 1954;57:525e32.
[142] Wood J, Goring D. The distribution of lignin in stem wood
and branch wood of Douglas fir. Pulp Pap Can Mag
1971;72:61e8.
Please cite this article in press as: Ghaffar SH, Fan M, Structural analysis for lignin characteristics in biomass straw, Biomass
and Bioenergy (2013), http://dx.doi.org/10.1016/j.biombioe.2013.07.015
[143] Lange P, Kjaer A. Quantitative chemical analysis of the
different parts of the cell wall in wood and cellulose fibres
by interference microscopy. Norsk Skogind 1957;11:425e32.
[144] Donaldson L, Ryan K. A comparison of relative lignin
concentration as determined by interference microscopy
and bromination/EDXA. Wood Sci Technol 1987;21:303e9.
[145] Saka S, Goring D. Distribution of lignin in white birch wood
as determined by bromination with TEM-EDXA.
Holzforschung 1988;42:149e53.
[146] Donaldson L. Determination of lignin distribution in
agricultural fibres, vol. 4418. Wood Processing Division,
New Zealand Forest Research Institute; 1996. p. 1e25.
[147] Donaldson L, Hague J, Snell R. Lignin distribution in coppice
poplar, linseed and wheat straw. Holzforschung
2001;55:379e85.
[148] El Mansouri N, Salvadó J. Analytical methods for
determining functional groups in various technical lignins.
Ind Crop Prod 2007;26:116e24.
[149] Faix O, Andersons B, Zakis G. Determination of carbonyl
groups of six round robin lignins by modified oximation and
FTIR spectroscopy. Holzforschung 1998;52:268e74.
[150] Olivares M, Guzman JA, Natho A, Saavedra A. Kraft lignin
utilization in adhesives. Wood Sci Technol 1988;22:157e65.
[151] Lai Y. In: Lin S, Dence C, editors. Determination of phenolic
hydroxyl groups. Springer Berlin Heidelberg; 1992.
p. 423e34.
[152] Aulin-Erdtman G. Studies on ultraviolet absorption changes
caused by modifications of chromophores with special
reference to lignin chemistry. Stockholm; 1958.
[153] Aberu H, Freire M. Methoxyl content determination of
lignins by 1H NMR. An Bras Ci 1995;67:379e82.
[154] Gosselink RJA, Abächerli A, Semke H, Malherbe R, Käuper P,
Nadif A, et al. Analytical protocols for characterisation of
sulphur-free lignin. Ind Crop Prod 2004;19:271e81.
[155] Iiyama K, Lam T, Stone B. Covalent cross links in the cell
wall. Plant Physiol 1994;104:315e20.
[156] Yelle D, Kaparaju P, Hunt C, Hirth K, Kim H, Ralph J, et al.
Two-dimensional NMR evidence for cleavage of lignin and
xylan substituents in wheat straw through hydrothermal
pretreatment and enzymatic hydrolysis. Bioenergy Res
2013;6:211e21.
[157] Rahal A, Singh A, Singh M. Effect of urea treatment and diet
composition on, and prediction of nutritive value of rice
straw of different cultivars. Anim Feed Sco Technol
1997;68:165e82.
[158] Agbagla-Dohnani A, Nozière P, Clément G, Doreau M. In
sacco degradability, chemical and morphological
composition of 15 varieties of European rice straw. Anim
Feed Sci Technol 2001;94:15e27.
[159] Bainton S, Plumb V, Capper B, Juliano B. Botanical
composition, chemical analysis and cellulose solubility of
rice straw from different varieties. Anim Prod 1987;44:481.
[160] Walli TK, Ørskov ER, Bhargava PK. Rumen degradation of
straw 3. Botanical fractions of two rice straw varieties and
effects of ammonia treatment. Anim Sci 1988;46:347.
[161] Abou-El-Enin OH, Fadel JG, Mackill DJ. Differences in
chemical composition and fibre digestion of rice straw with,
and without, anhydrous ammonia from 53 rice varieties.
Anim Feed Sci Technol 1999;79:129e36.
[162] Jin S, Chen H. Near-infrared analysis of the chemical
composition of rice straw. Ind Crops Prod 2007;26:207e11.
[163] Sefain MZ, El-Kalyoubi SF, Shukry N. Thermal behavior of
holo- and hemicellulose obtained from rice straw and
bagasse. J Polym Sci Polym Chem Edition 1985;23:1569e77.