structural analysis for lignin characteristics
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Review
Structural analysis for lignin characteristicsin biomass straw
Seyed Hamidreza Ghaffar, Mizi Fan*
School of Engineering and Design, Brunel University, Uxbridge, Middlesex, UB8 3PH, United Kingdom
a r t i c l e i n f o
Article history:
Received 31 March 2013
Received in revised form
15 July 2013
Accepted 19 July 2013
Available online 20 August 2013
Keywords:
Straw lignin
Quantitative and qualitative anal-
ysis
NMR
FT-IR
Structural characteristics
Thermal analyses
* Corresponding author. Department of CivilE-mail address: [email protected] (M
0961-9534/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.biombioe.2013.07.0
a b s t r a c t
Agricultural by-products are the most promising feedstock for the generation of renewable,
carbon neutral substitutes for synthetic materials (e.g. biofuel, building materials). The
demand for efficient utilisation of lignin biomass has induced detailed analyses of its
fundamental chemical structures and development of analysing technologies. This paper
reviews the structural analysis techniques for straw lignin together with the morphology of
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
techniques reviewed, the magnetic resonance techniques have proved to be efficient
analytical tools for the structural elucidation of these complex biopolymers. Quantitative
and qualitative structural analysis of lignin indicated a great potential for industrial crops
optimisation due to in-depth microstructure interpretation, and detailed and accurate
chemical composition although the composition and structure of straw lignin have been
discovered highly complex and varied considerably within and among plants. The struc-
ture of lignin has remained one of the most difficult biopolymers to characterise, however
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 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
Engineering, Brunel Univ. Fan).ier Ltd. All rights reserve15
research workers e.g. Refs. [3e5]. A number of chemical and
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
ersity, London UB8 3PH, United Kingdom.
d.
b i om a s s a n d b i o e n e r g y 5 7 ( 2 0 1 3 ) 2 6 4e2 7 9 265
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 agreedmodel 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 ligninmust 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].
with multiple chemical and biophysical functionalities, it is
largely underutilised due to its image as low quality and low
value addedmaterial. 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 variousmorphological 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 strawdetermined 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
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.
b i om a s s a n d b i o e n e r g y 5 7 ( 2 0 1 3 ) 2 6 4e2 7 9266
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 structuralanalysis 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
Fig. 2 e Micrograph of cross sections of (A) wheat straw under
epidermis, (b) parenchyma, (c) lumen, (d) vascular bundles [24];
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
light microscope [23]; (B) wheat straw internode SEM (a)
(C) rice straw internode SEM [25].
Table 3 e Assignments of FT-IR absorption bands (cmL1)[7].
Absorptionbands
Assignment
3429 OH stretching
2945 CH stretching of methyl, methylene
or methane group
1732, 1726 C¼O stretch in unconjugated ketone
and carboxyl group
1660, 1653 C¼O stretch in conjugated ketone
1606 Aromatic skeletal vibration
1507 Aromatic skeletal vibration
1460 Aromatic methyl group vibrations
1434 Aromatic skeletal vibration
1374 Aliphatic CeH stretch in CH3
1328 Syringyl ring breathing with CeO stretching
1242 Aromatic CeO stretching
1165 CeO stretch in ester groups
1135 Aromatic CeH in-plane deformation
for syringyl type
1043 Aromatic CeH in-plane deformation
for guaiacyl type
855, 844 Aromatic CeH out of plane bending
b i om a s s a n d b i o e n e r g y 5 7 ( 2 0 1 3 ) 2 6 4e2 7 9 267
characterisation into two groups of destructive and non-
destructive methods. Thioacidolysis (TA) [26], nitrobenzene
oxidation (NBO) [27] and also derivatization followed by
reductive cleavage (DFRC) [28] are amongst the most
destructive methods used. The non-destructive methods
include various spectroscopic methods (e.g. UV and Fourier
transform infrared spectroscopy (FT-IR spectra)) and Nuclear
magnetic resonance (NMR) techniques.
The chemical composition of agricultural biomass can also
be analysed with near infrared (NIR) spectroscopy, e.g. Kelley
et al. [29] tested the effectiveness of NIR for measuring the
chemical composition of biomass that has been subjected to a
wide variety of extraction and chemical treatments; Sander-
son et al. used NIR for directly measuring the chemical
composition of biomass [30].
Another technique to analyse lignin in the cell wall is based
onmercurization which was firstly used in the early history of
lignin chemistry to characterise its structure [31]. Mercuriza-
tion is a reaction occurring under mild conditions; it seems to
be less sensitive to the substitution pattern of the aromatic
ring. The determination of lignin, thought apparently an easy
matter, is one of the least satisfactory of the analysis
commonly carried out on plant materials and woods. Almost
all current methods contain the solution and hydrolysis of all
other plant constituents and assume that the residue after
such treatment is lignin. Various investigators have pointed
out that this is not the case, but in spite of this vital objection,
considerable reliance has been placed on figures so obtained.
3.1. Quantitative analysis
Quantitative measurement of lignin structures is an impor-
tant aspect when it comes to investigating lignin; the mea-
surement of various structures of lignin is possible when
appropriate standards or pulse sequences are applied [32e34].
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
chlorite lignin or permanganate lignin) [37]. The spectroscopic
techniques, such as infrared (IR) and 13C nuclear magnetic
resonance (13C NMR) spectroscopy, are complementary to the
above procedures since they provide information on the
whole structure of the polymer and avoid the possibility of
degradation artefacts [38]. This information is presented with
quantitative values.
3.1.1. FT-IRAmong all the quantitative analysis techniques, FT-IR spec-
troscopy shows interesting characteristics, including high
sensitivity and selectivity, high signal-to-noise ratio, accu-
racy, data handling facility, mechanical simplicity and short
time and small amount of sample required for the analysis
[39]. In addition, the spectrum of a lignin sample gives an
overall view of its chemical structure [40]. FT-IR spectroscopy
is non-destructive method of lignin investigation, which
opens perspective to find structural discrepancies in lignin
isolated by different methods.
Infrared radiation is electromagnetic radiation which
covers the wavenumbers between 13,300 cm�1 and 3.3 cm�1.
The infrared region is usually divided into 3 regions: near
infrared (13,300e4000 cm�1), middle infrared (4000e200 cm�1)
and far infrared (200e3.3 cm�1) regions. Organic compounds
have fundamental vibration bands in the mid infrared region,
which is why the region is widely used in infrared spectros-
copy. An infrared spectrum is unique to each substance and
can therefore, in principle, be used as an impartial charac-
teristic to identify the sample. Every lignin IR spectrum has a
strong wide band between 3500 and 3100 cm�1 assigned to OH
stretching vibrations. This band is caused by the presence of
alcoholic and phenolic hydroxyl groups involved in hydrogen
bonds. The intensity of the band increases during demethy-
lation and decreases during methylation since during deme-
thylation the OeCH3 bonds in methoxyl groups bonded to 3rd
or 5th carbon atom of the aromatic ring are split and CH3 is
replaced by a hydrogen atom producing a new OH group.
During methylation the OeH bonds are split and H is replaced
by CH3 group and the amount of OH group decreases hence
the intensity of the band decreases [41]. Acetylation of the
lignin causes a partial or full band loss since almost all of OH
groups are replaced by CH3COO [42].
The assignments of FT-IR absorption bands for the lignin
normally include the aromatic skeleton vibrations at 1606,
1507 and 1434 cm�1, in which the aromatic semicircle vibra-
tion (a vibration involving both CeC stretching and a change
of the HeCeC bond angle) is assigned at 1507 cm�1 [43], the
carbonyl and unconjugated ketone and carboxyl group
stretching at 1732 cm�1 and the small band of the conjugated
carbonyl stretching such as detected in organosolv lignin at
1653 cm�1. More detailed assignment are summarised in
Table 3 [7,44,45].
3.1.2. Thermal analysisThermal analysis has recently become prominent, especially
for fibres, plastics and other polymeric materials. Thermal
b i om a s s a n d b i o e n e r g y 5 7 ( 2 0 1 3 ) 2 6 4e2 7 9268
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 tomonitor 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 �Cand 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 Muller-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 mol�1 and from
107.3 to 107.9 min�1 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 mol�1 for the first and the second reaction zones,
Table 4 e NMR techniques for lignin analysis.
NMR techniques Quantitative data
13C NMR Aliphatic/phenolic OH groups and methoxy gro
HSQC Inter-unit bonding patterns31P NMR Aliphatic/phenolic OH groups, guaiacyl, siringy
condensed OH groups, carboxylic acids1H NMR Hydrocarbon contaminant, aliphatic and aroma
protons in methoxyl groups, benzylic protons in
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 kJmol�1 respectivelywith themethods 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. NMRNuclear 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
ups Inter-unit bonding patterns
Inter-unit bonding patterns
l, Assignments of guaiacyl, siringyl, condensed
OH groups, carboxylic acids
tic acetate,
b-b structures
Improved spectral resolution of key
lignin functionality
b i om a s s a n d b i o e n e r g y 5 7 ( 2 0 1 3 ) 2 6 4e2 7 9 269
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 NMRhave 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 NMRThe interest to study the mixture of increasing complexity of
lignin has created the demand to employ high magnetic field
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)
Table 5 e H/S/G ratios for types of biomass resources.
Type Lignin (%) H S G Reference
Wheat straw 16e21 9 46 45 [109]
Rice straw 6 15 40 45 [109]
Rye straw 18 1 53 43 [110]
Hemp 8e13 9 40 51 [111]
Jute 15e26 2 62 36 [112]
Flax 21e34 4 29 67 [109]
b i om a s s a n d b i o e n e r g y 5 7 ( 2 0 1 3 ) 2 6 4e2 7 9270
delivered very well resolved spectra that can be compared to
that of MWL [93]. Based on the results obtained, it was found
that in situ characterisation of pre-treated biomass by HSQC
NMR analysis is a beneficial structural analysis methodology
in the emerging biomass research field for the characterisa-
tion of EHR.
Heteronuclear multiple quantum coherence (HMQC) or
HSQCspectraof ligninhavebeenreviewed [80] andwell reported
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
structural type. 2D HSQC NMR technique is also useful in
determination of the structural characteristics of oxygenated
aliphatic region of lignin polymeric framework [32,99].
3.1.5. DFRC methodThe derivatization followed by reductive cleavage method
(DFRC)was developed by Lu and Ralph in 1997 as an alternative
to thioacidolysis [28,100]. Because of the mild conditions used
and its unique selectivity, the DFRCmethodhas becomewidely
used for characterising lignin from various origins [101e103].
One unique feature of the DFRCmethod is that esters on lignin
remain fully intact during the DFRC procedure [104]; therefore,
p-coumarates on lignin from grass plant materials can be
detected and measured [105]. With a few modifications to the
standard DFRC procedure by replacing the acetyl-containing
reagents (acetic acid and AcBr) with the propionyl analogues,
the modified DFRCmethod was applied to detect and quantify
the naturally occurring acetates on lignin from some plants
such as kenaf, aspen or other non-woody plants [106].
The DFRC procedure, as a basic component, has been
modified or combined with other techniques to provide more
precise and specific quantitative data about lignin structures
[107]. The combination of DFRCwith quantitative 31P NMRwas
shown to have significant potential for the determination of
arylglycerol-a-aryl ether and other linkage. Coupled with the
spread of advanced NMR techniques, during the past decade,
lignin structural enquiries have been greatly facilitated by the
development of various degradative protocols, such as DFRC
which efficiently cleaves the b-aryl ethers in lignin. DFRC is a
flexible method due to its three distinctly separated steps,
allowing modifications designed to the quantification of
different monomeric units and structures. On the basis of this
flexibility, Tohmura and Argyropoulos [108] proposed the
combination of DFRC with quantitative 31P NMR data.
3.1.6. H/S/G ratioQualitative or semi-qualitative indication of H/S/G ratio units
are not too informative and on the other hand they are very
laborious, lengthy and also prone to errors. Table 5 shows the
p-hydroxyphenylesyringyleguaiacyl (H/S/G) ratio for
different types of biomass which vary quite considerably
amongst each other.
Thioacidolysis is the most used analytical method for
lignin analysis to obtain information about uncondensed
structures (H/S/G ratios) [12]. Thioacidolysis was developed by
Lapierre, as an extension of acidolysis, to cleave b-aryl ethers
in lignin so that the basic units linked by b-aryl ethers are
released as monomers to be quantified by gas chromatog-
raphy [26]. Therefore, the yields of monomers released by
thioacidolysis reflect the proportion of uncondensed units in
lignin. When thioacidolysis is used for grass lignin, esters on
grass ligninmay decrease the efficiency of b-ether cleavage by
thioacidolysis [113,114]; hence this should be taken into
consideration when interpreting thioacidolysis results for
grasses.
Permanganate oxidation, nitrobenzene oxidation, GCeMS
pyrolysis, thioacidolysis and DFRC are degradative methods
that reveal theH/S/G composition of the lignin polymer. Itmust
be mentioned that degradative analytical techniques are
lengthy and complicated. All these methods release only a
fractionof the lignin for analysis, theyarebasedon thecleavage
of lignin backbone and analyses of the fragments obtained, and
provide partial data due to the specificity of the treatment.
The quantification of lignin H/S/G ratio has been reported
by 13C NMR and HSQC NMR analysis [115,116].
3.2. Qualitative analysis
Qualitative lignin analysis relies rather much on studies of
lignin degradation products; these methods only provide
preliminary information which could then be useful for the
quantification of lignin within straw.
3.2.1. SEM-EDXScanning electron microscopy (SEM) is used to examine the
microstructure, morphology, crystalline structure and size of
the straw. The microscope could be equipped with energy-
dispersive X-ray (EDX) analyser, which provides useful
element compositions and also information about the chem-
ical composition of the straw.
Despite its importance as imaging technique, SEM is not
capable of quantitatively evaluating the purity of typical lignin
sample. Moreover, there is no published algorithm to convert
such images into numerical data. SEM analysis also provides
information on the particle size and the surface properties of
straw. When the straw is being treated by means of chemical
ormechanical treatments, the particle sizesmight change and
the surface of straw might become smoother or rougher
depending on each step of the treatment.
Field emission scanning electron microscopy (FE-SEM) was
used by Koo et al. [117] in order to observe the change in lignin
distribution during pre-treatment; (Fig. 3), which is one of the
key applications of SEM technique used for qualitative anal-
ysis of biomass.
3.2.2. TEM and optical microscopyTransmission electron microscopy (TEM) has been proved to
be an effective tool for determining cell wall ultrastructure.
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].
b i om a s s a n d b i o e n e r g y 5 7 ( 2 0 1 3 ) 2 6 4e2 7 9 271
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
Fig. 4 e TEM micrograph of an ultrathin transverse section
of the cell wall of Forsythia suspensa fibre: S1 [ outer
secondary wall, S2 [ middle secondary wall and
S3 [ inner secondary wall [118].
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
Lignin monolignols are polymerised by a radical coupling
process that links them by carbonecarbon or ether bonds. A
linkagemay occur at any of several different locations on each
phenolic unit, causing many different linkage types to be
Fig. 5 e Light micrograph of cross section of grass stem,
showing four basic parts [23].
b i om a s s a n d b i o e n e r g y 5 7 ( 2 0 1 3 ) 2 6 4e2 7 9272
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
[119,120]. The ether type linkages dominate in native lignin,
estimated to make up one half to two thirds of the total
number of native plant lignin linkages. Monolignols form
branch points within the polymer giving a network-like
structure. Given the variety of linkages that occur, lignin
molecules cannot be depicted as a series of regular, defined
repeating units, as traditional polymers are. In contrast, lignin
is a highly irregular complex polymer [119,120].
Lignin is one of major cell wall component of herbaceous
crops. The composition and structure of lignin vary consid-
erably within and among plants; Lignin has been classified
into three groups: Gymnosperm (softwood), Angiosperm
(hardwood) and Grass lignin. However, it was recently found
that these categories are inadequate because of ignoringmost
of the herbaceous angiosperm lignin and some conifer fam-
ilies containing guaiacyl and syringyl types of lignin. It was
suggested by Gibbes that lignin should be categorised into two
groups: guaiacyl lignin and guaiacylesyringyl lignin according
to their overall chemical constitutions [12]. Each type also
generally contains a small proportion of p-coumaryl units,
though grass lignin generally contains a higher amount of p-
coumaryl units than woody lignins [121]. Other phenolic
monolignols have been identified, but they generally make up
a much smaller portion of the lignin molecule [122]. Other
phenolic compounds (alcohols, aldehydes, acids, esters and
amides) have been reported to act as lignin precursors and
illustrate the structural ‘‘plasticity” of the polymer and the
adaptability of the lignification mechanisms in plants
[123,124], but several of these compounds (such as p-hydrox-
ycinnamaldehydes, ferulic acid or 5-hydroxyconiferyl alcohol)
only provide a significant contribution to lignin in transgenic
plants with modified monolignol biosynthesis [124] and are
minor lignin precursors in normal plants [125]. On the other
hand, some phenolic compounds with saturated or no side-
chains, e.g. dihydrocinnamyl alcohol, sporadically incorpo-
rate to the lignin polymer as terminal units as confirmed by 2D
NMR [126,127].
The solubility of straw lignin in alkali has been attributed
mainly to the presence of significant amounts of p-hydrox-
yphenyl residues, which are bound to lignin as p-coumarate
units [128]. Ferulic and p-coumaric acids in grasses are known
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-
acidolysis confirmed the presence of g-acylation, although
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].
4.1. LCC linkages
Lignin is always associated with carbohydrates (in particular
with hemicelluloses) via covalent bonds at two sites: ⍺-carbon
and C-4 in the benzene ring, and this association is called
lignin carbohydrate complexes (LCC). The major inter-unit
linkages within the lignin monomer (H, S, and G) are b-O-4,
b-b, b-5, b-1 arrangements. The chemical linkages limit the
efficient separation of lignin from plant cell wall. Thus, it is
important to understand the LCC linkages of lignin samples.
Four types of linkages between lignin and carbohydrates
have been suggested: glycosidic linkages, ester linkages,
benzyl ether linkages and hemiacetal or acetal linkages [12].
Among these linkages, the possibilities of glycosidic, benzyl
ether and ester linkages have been demonstrated with model
compounds [13]. The association of lignin and carbohydrates
in grass cell walls is largely through free radical coupling of
ferulates or diferulates with monolignols or growing lignin
oligomers. Due to the complexity of LCCs, the isolation of
homogeneous preparation is the most important step for the
following compositional analysis and structural characteri-
sation. Many isolation methods for LCC have been tried, but
the finely divided plant meal [131,132] and MWL [133] were
preferred sources for LCC isolation because any chemical or
biochemical treatment before LCC isolation would break
possible linkages between lignin and carbohydrates.
The cross-linking of lignin and carbohydrates via ferulates/
diferulates in grass cell walls has been demonstrated [12]; it is
generally accepted that the cross-linking of carbohydrates
and lignin by ferulates presents the greatest barrier to efficient
utilisation of grass cell wall [113].
4.2. Physical properties
The physicochemical properties of straw lignin are known to
be different from those of softwoods or hardwoods, with
straw lignin possessing characteristic alkali solubility. The
physicochemical state of lignin dictates how and where it can
be utilised in the production of various products. The source
from which lignin is obtained and the method of extraction
has a strong bearing on its properties [16]. As lignin from
different crops or treatment can be extremely diverse in
b i om a s s a n d b i o e n e r g y 5 7 ( 2 0 1 3 ) 2 6 4e2 7 9 273
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’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 thementionedmethods 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 compoundmiddle lamella
(CML) have been carried out by using UVmicroscopy [141]. UV
absorption is a tool used for lignin identification because
Table 6 e Molecular weight and functional groups oflignin [1].
Lignin type Mn (g mol-1) COOH(%)
OHphenolic
(%)
Methoxy(%)
Soda
(bagasse)
2160 13.6 5.1 10.0
Organosolv
(bagasse)
2000 7.7 3.4 15.1
Soda (wheat
straw)
1700 7.2 2.6 16
Organosolv
(hardwood)
800 3.6 3.7 19
Kraft
(softwood)
3000 4.1 2.6 14
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 interferencemicroscopy 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.
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 7 e Lignin concentration and distribution in wheatstraw [22].
Cells S CML CC
Br-L X-ray counts
Thick wall fibre 1620 2156 2992
Thin wall fibre 1340 2004 3336
Parenchyma cell 2005 2599 e
Epidermis cell 1022 1690 1743
Lignin concentration (g g�1)
Thick wall fibre 0.168 0.412 0.571
Thin wall fibre 0.154 0.339 0.664
Lignin distribution (%)
Thick wall fibre 67.5 18.0 14.5
Thin wall fibre 57.5 20.3 22.22
b i om a s s a n d b i o e n e r g y 5 7 ( 2 0 1 3 ) 2 6 4e2 7 9274
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 reactionwith formaldehyde for
the production of lignin adhesive [150]. Lai described a pro-
cedure to determine free phenolic hydroxyl groups in lignin
[151]. Commonphysicalmethod 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
Fig. 6 e 31P NMR of wheat straw lignin phosphitylated with 2
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 lignincharacteristics
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 and13C 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
-chloro-4, 4, 5, 5-tetramethyl-1, 3, 2 dioxaphospholane.
b i om a s s a n d b i o e n e r g y 5 7 ( 2 0 1 3 ) 2 6 4e2 7 9 275
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 quantitative31P 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 lownitrogen 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.
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 structurewasmore
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 ligninwere 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 themost accurate way to study the
straw lignin could be the combination of several techniques,
each providing partial but complimentary information.
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