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89

Methods to Reveal the


Structure of Lignin
Prof. Dr. Gsta Brunow
Department of Chemistry, Laboratory of Organic Chemistry, University of Helsinki,
P. O. Box 55, 00014 University of Finland, Finland; Tel: 358-919140361;
Fax: 358-919140366, E-mail: gosta.brunow@helsinki.fi
1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3
3.1
3.2

Lignin Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Milled Wood Lignin (MWL ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Determination of
Total Lignin in Lignocellulosics . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5
5.1
5.2
5.3
5.4
5.5

Methods to Determine the Molecular Mass of Lignin


Introduction . . . . . . . . . . . . . . . . . . . . . . .
Gel Permeation Chromatography . . . . . . . . . . .
Vapor Pressure Osmometry . . . . . . . . . . . . . .
Light Scattering . . . . . . . . . . . . . . . . . . . . .
Ultrafiltration . . . . . . . . . . . . . . . . . . . . . .

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6
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.7.1
6.7.2

Degradative Methods of Lignin Analysis . . . . .


Introduction . . . . . . . . . . . . . . . . . . . . .
Acidolysis and Thioacidolysis . . . . . . . . . . .
Permanganate Oxidation . . . . . . . . . . . . . .
Nitrobenzene and Cupric Oxide Oxidation . . .
Ozonolysis . . . . . . . . . . . . . . . . . . . . . .
Reductive Cleavage after Derivatization (DFRC )
Functional Group Analysis . . . . . . . . . . . . .
Methoxyl Group Analysis . . . . . . . . . . . . . .
Phenolic Hydroxyl Groups . . . . . . . . . . . . .

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3 Methods to Reveal the Structure of Lignin

6.7.3
6.7.4

Carbonyl Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quinoid Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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7
7.1
7.2
7.3

Nondegradative Methods of Lignin Analysis


Introduction . . . . . . . . . . . . . . . . . .
Functional Groups . . . . . . . . . . . . . .
Aromatic Nuclei and Side Chain Structures

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8
8.1
8.2
8.3
8.4

Structural Models . . . . . . . . .
Introduction . . . . . . . . . . . .
Softwood Lignin . . . . . . . . . .
Hardwood Lignin . . . . . . . . .
Non-Wood Lignin (Straw, Grass)

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Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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10

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

107

CEL
DHP
GPC
HPSEC
IR
MWL
NMR
SEC
UV/Vis
VPO

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cellulolytic enzyme lignin


dehydrogenation polymer
gel permeation chromatography
high-pressure size exclusion chromatography
infrared
milled wood lignin
nuclear magnetic resonance
size exclusion chromatography
ultraviolet/visible
vapor pressure osmometry

Introduction

Historical Outline

Lignin in the cell walls of vascular plants is


intimately mixed with the carbohydrate components. The structure of the polymer is
complex and irregular, and preparation of
pure samples of unchanged lignin is not easy.
This makes structure determination of lignin
more challenging than that of other biopolymers. In the following chapter, the most
useful methods for the isolation and structure
elucidation of lignin are described, with a
summary description of the structural pattern emerging from these studies.

Long after Anselme Payen (1839) first described the `encrusting material' in wood,
researchers were unclear about the nature of
this very abundant material. Although it had a
higher carbon content than the carbohydrates, its chemical nature remained obscure
for a long time; indeed, it was not until about
20 years later that the term `lignin' became
accepted for this material (Schulze, 1857).
Aromatic products formed on alkali fusion
(Bente, 1868 and references cited therein) led
to the conclusion that the noncellulosic
constituent of wood, or lignin, was aromatic
in nature. That the methoxyl group was

3 Lignin Preparations

typical of lignin and lacking in cellulose was


shown in 1890 (Benedikt and Bamberger,
1890). The methods to reveal the structure of
lignin evolved very slowly, and as recently as
1960 it might be read in a review that the
lignin building stone has a phenylpropane
structure that may be regarded as proven, but
how the stones are linked together in protolignin is still a mystery (Brauns, 1960). No
dimeric degradation product, that might have
revealed the nature of the bonding between
the units, had been isolated at that time. It was
in fact not degradation that led to a breakthrough in the understanding of lignin
chemistry, but an idea that usually is traced
back to P. Klason, who postulated that lignin
was an oxidation product of coniferyl alcohol
(Adler, 1977). The fruitfulness of this idea
was demonstrated by Freudenberg (1968)
when he succeeded in producing polymers
by oxidative dehydrogenation of coniferyl
alcohol. These dehydrogenation polymers
(DHPs) were found to contain the same
structural units as native lignin, and have
been used extensively as models for lignin. (It
was in fact the advent of new reducing agents
during the early 1950s that made it possible to
synthesize coniferyl alcohol in the large
amounts necessary for such a study.) To this
day, all analytical results in lignin chemistry
are interpreted in the light of the dehydrogenation hypothesis. Most structural units in
lignin can be regarded as oxidation products
of coniferyl alcohol, and the fact that there are
some structural units that do not fit into this
scheme, has not diminished its usefulness.
3

Lignin Preparations
3.1

Introduction

A critical review of methods for the isolation


of lignin was published by Lai and Sarkanen

(1971). The most commonly used method


involves thorough milling of the plant material, followed by extraction with dioxanwater
(Bjrkman, 1957; Lundquist, 1992a); this
material is referred to as milled wood lignin
(MWL ). The yields are usually low, and the
possibility of chemical changes occurring
during the isolation process must always be
taken into account.
3.2

Milled Wood Lignin (MWL)

When choosing a sample of plant tissue for


the isolation of MWL, consideration should
be taken into the possible variations in lignin
composition in different parts of the plant. To
prepare a lignin sample representative of a
certain species of wood, it is customary to
choose sapwood, which is free of reaction
wood. The milling is carried out either in a
nonswelling medium such as toluene, or in
the dry state. Treatment of the finely ground
wood meal with cellulolytic enzymes prior to
solvent extraction removes part of the polysaccharides and increases the yield of lignin.
Such preparations are referred to as cellulolytic enzyme lignins (CEL ) (Chang et al.,
1975). After extraction with dioxanewater,
the crude extract still contains carbohydrates.
In the original Bjrkman procedure, CEL is
purified by precipitation into water from a
solution in acetic acid. An alternative method
has been proposed that is based on liquid
liquid extraction (Lundquist, 1992a), this
yielding lignin preparations with less carbohydrate contamination. The remaining carbohydrate contaminants are difficult to remove, as some may be covalently bound to the
lignin. Hardwood MWLs are found to contain
more carbohydrates than softwood MWLs.
MWL usually constitutes about 25% of the
lignin in wood, and the question of its
morphological origin has been discussed
(Lai and Sarkanen, 1971; Lee et al., 1981;

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3 Methods to Reveal the Structure of Lignin

Whiting and Goring, 1981; Eom et al., 1987).


The milling probably causes chemical
changes (Itoh et al., 1995), but the extent of
these changes is largely unknown. In structural studies (see below) it has not been
possible to find any significant differences in
the chemical structure of MWL and lignin in
situ. CEL is probably more representative of
the total lignin, with higher yields and less
chemical change (Chang et al., 1975), but it
has other drawbacks, such as high carbohydrate content and its preparation procedure is
more tedious (Lapierre et al., 1985).

Determination of Total Lignin


in Lignocellulosics

There is no generally applicable method for


the quantitative determination of total lignin
in lignocellulosics. The oldest and most
common method is based on gravimetry. In
the Klason lignin determination which has
been standardized by the Technical Association of the Pulp and Paper Industry (Dence,
1992), the sample is first treated with 72%
sulfuric acid and subsequently heated with
dilute acid to hydrolyze the polysaccharides to
soluble fragments. The solid residue is
washed, dried and weighed. The method is
reliable provided that standard conditions are
strictly applied, but also has serious limitations that must be taken into consideration.
Correct values are obtained for softwoods, but
hardwoods contain variable amounts of `acidsoluble lignin' which must be estimated by
UV spectrophotometry. The method is also
not suitable for herbaceous and annual
plants. Such material tends to contain variable amounts of proteins and siliceous material that interfere with the determination of
Klason lignin. For such plant material there is
a modification of the Klason determination,
developed by Ellis (1949) and adapted as an

official method by the Association of Official


Agricultural Chemists (Dence, 1992). This
method involves pretreatment with a proteolytic enzyme, but since there is no assurance
that such pretreatment effects complete
removal of protein, it is often be necessary
to correct the values for acid-insoluble lignin
with calculated values based on nitrogen
analysis. In most cases, acid-soluble lignin
has also to be determined and the value
applied as a correction to arrive at an accurate
value for total lignin.
There are also methods that do not involve
isolation of the lignin. UV microspectrophotometry has been used to estimate lignin in
different morphological regions (Fergus and
Goring, 1970). Other spectrometric techniques include infrared spectroscopy (Schultz
et al., 1985) and solid-state NMR (13C CP/
MAS/NMR; Leary and Newman, 1992).
Some spectral techniques involve dissolving the whole plant material in a suitable
solvent and measuring the UV-absorbance at
a wavelength characteristic for the lignin in
question, usually 280 nm. Acetyl bromide in
acetic acid (Iiyama and Wallis, 1988; Hatfield
et al., 1999) seems to have earned widespread
acceptance as a rapid and simple method,
adaptable to samples of small size.
Special methods, which are used exclusively for the analysis of unbleached pulps, are
based on the consumption of oxidant. Lignin
concentration determined by such procedures is usually expressed as the amount of
oxidant per unit weight of pulp (for instance
the Roe chlorine number, or the kappa
number). These numbers may be converted
to Klason lignin or other lignin values by
application of empirically determined conversion factors. The two oxidants most commonly used are chlorine and potassium
permanganate. It is assumed that the oxidant
readily oxidizes lignin, whereas the carbohydrate constituents are relatively unreactive
(Dence, 1992).

5 Methods to Determine the Molecular Mass of Lignin

Methods to Determine the Molecular Mass


of Lignin
5.1

Introduction

An important limitation of the study of wood


polymers is that properties such as molecular
mass, molecular shape, crystallinity, density,
etc., have been investigated almost exclusively with isolated samples, and these characteristics of the wood polymers in situ can be
deduced only by inference (Goring, 1971).
Considering the large number of different
linkages between the phenylpropane units, it
has been assumed that lignin constitutes
a three-dimensional network polymer. To
break this down to soluble fragments, bonds
must be broken, and random scission of
bonds in such a network will lead to a wide
range of molecular sizes. It is typical of lignin
that isolated samples are very polydisperse,
and the measured molecular size range is
very much dependent on the isolation procedure. The polydispersity is accompanied by
rather small changes in chemical properties.
Soluble lignin preparations tend to be composed of molecules of similar constitution,
but differing widely in size. Most methods to
determine the molecular mass of lignins have
been developed for the study of industrial
spent liquors. For MWLs, the most important
methods are gel permeation chromatography
(GPC ), light scattering and vapor pressure
osmometry. There are also reports on the
application of ultrafiltration (Lin, 1992b) and
mass spectrometry (Metzger et al., 1992;
Evtuguin et al., 1999) to lignins.
5.2

Gel Permeation Chromatography

A commonly used method of measuring the


size of macromolecules is based on molecular

sieving in a chromatographic column. For


synthetic polymers in organic solvents the
techniques of GPC or `size exclusion chromatography' (SEC ) (Gellerstedt, 1992) have
been used; when applied to water-soluble
biopolymers, the technique is known as `gel
filtration'.
The procedure involves passing a solution
of macromolecules through a column of a
solvent-filled porous gel. Depending on their
size, the macromolecules can diffuse in
varying proportions into the porous gel.
Molecules with a small hydrodynamic radius
can penetrate into the gel, while large
molecules are excluded from the gel and pass
directly through the column. The elution
volume of any particular fraction is a function
of the dimensions of the macromolecules and
the sizes of the pores in the gel. In order to
convert the elution volumes into molecular
weight, the relationship between molecular
size and mass of the macromolecular solute
should be known. The simplest solution is to
run monodisperse fractions of known molecular weight through the column and thus
establish the relationship between molecular
mass and elution volume (Mnsson, 1981).
In earlier work, cross-linked polydextran gels
having varying pore sizes were employed as
stationary phases. The gels were swollen in
water and used for fractionating aqueous
solutions. Subsequently, modified polydextrans have become available that permit
fractionation in organic solvents (Connors,
1978). Recently, cross-linked rigid polystyrene gels have made it possible to perform GPC in high-pressure SEC systems
(HPSEC ), where the time of analysis can be
greatly reduced and the resolution enhanced
considerably when compared to normal GPC
(Yau et al., 1979). For milled wood lignins,
elution with tetrahydrofuran has been found
to give reliable results (Mnsson, 1981;
Chum et al., 1987). For added solubility, the
lignin samples are often derivatized (by

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methylation or acetylation) before analysis.


For calibration, polystyrene fractions or polypeptides (Brunow et al., 1993) of known
molecular weight and lignin model compounds have been used.

5.3

Vapor Pressure Osmometry

Vapor pressure osmometry ( VPO ) is commonly used to determine numberaverage


molecular masses in the range of 100 to
10,000. Beyond the upper limit the sensitivity
becomes unsatisfactory. In spite of that, VPO
now appears to be the preferred method
for determining number-average molecular
weights of lignins and lignin products. All
colligative methods are uncertain in the range
of 10,000 to 25,000 and average molecular
masses within this range are difficult to
determine accurately (Pla, 1992a). The principle of the method is based on the measurement (at a given temperature) of the vapor
pressure depression of the solvent for dilute
polymer solutions. The instrument must be
calibrated with a substance of known molecular mass.

5.4

Light Scattering

For measuring the number-average molecular mass of soluble lignins, low-angle laser
light-scattering photometers have been used
(Pla, 1992b), and are reported to overcome
some of the sources of error associated with
obtaining reproducible results from lightscattering measurements. The presence of
aggregates in the solution, and the absorbance or fluorescence of lignin in solution,
may influence the measurements.

5.5

Ultrafiltration

Membrane separation is a relatively new


technology that can be used for separating
lignins on the basis of molecular size.
Although not a standard method, it can be
used for the determination of molecular size
distribution of lignin samples. Membranes
are available for a wide range of molecular
sizes, from 1000 to 300,000. The solution of
lignin is filtered through a succession of
membranes and the yields of fractions are
determined by weighing the lignin fraction,
or by ultraviolet absorption (Lin, 1992a). The
chief advantage of the method is the insensitivity to impurities. Lignin permeates
through ultrafiltration membranes almost
unhindered by sugars or inorganic salts. Such
impurities render classical physical techniques, such as VPO and light scattering,
unusable.

Degradative Methods of Lignin Analysis


6.1

Introduction

It is not possible to degrade lignin into


monomeric fragments like most other biopolymers. In addition to the uncertainty
caused by the difficulty of preparing representative lignin samples that have not undergone any chemical change, all known methods of chemical degradation yield identifiable
products of small molecular weight in modest
yields only. Under such circumstances the
task of devising a satisfactory structural
picture of lignin macromolecules has been
likened (Sarkanen and Ludwig, 1971) to an
attempt to compose a picture-puzzle with an
incomplete number of pieces. What is contained in the missing pieces can only be

6 Degradative Methods of Lignin Analysis

guessed at, and there may be more than one


way to assemble the available pieces. The
degradative methods described below are all
aimed at some particular aspect of the
structure of lignin, and an extensive structural analysis implies the use of more than
one method and a combination with a
spectroscopic method.

6.2

Acidolysis and Thioacidolysis

Acid-catalyzed degradation aims at cleaving


the most important ether bonds in lignins,
the arylglycerol-b-aryl ethers. Acidolysis is the
term used for the method where the sample is
heated at 100 8C in 0.2 M HCl in dioxane
water (9:1, v/v; Lundquist, 1976, 1992b). This
causes selective cleavage of arylglycerol-b-

Scheme 1

Acidolysis (1) and thioacidolysis (2).

ethers and some other types of labile ether


linkages.
Monomeric and dimeric acidolysis products that contain a phenylpropane skeleton,
can be analyzed by gas chromatography after
silylation. The results are then interpreted
with the aid of low molecular weight `model'
compounds, that have undergone the same
treatment. The structural elements detected
with the aid of acidolysis studies include b-O4, b-5, b-b, b-1, glyceraldehyde-2-aryl ether,
2-aryloxypropiophenone, cinnamaldehyde,
cinnamic acid, benzaldehyde, benzoic acid
and quinoid types (see Figure 1). Some of
these elements have also been estimated
quantitatively. The same structural units are
detected by thioacidolysis, where the sample
is treated with boron trifluoride in dioxan
ethanethiol solution (Rolando et al., 1992).
Monomeric products substituted with thio-

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ethyl groups are formed, and these can be


analyzed by gas chromatography after silylation. Dimeric products can be analyzed after
removal of the sulfur substituents by reduction with Raney-nickel (Lapierre et al., 1991).
The yields of thioacidolysis products are
typically higher than in acidolysis, and there
are some noteworthy differences in the
structural information obtained. One such
difference is the occurrence of 5,5 or 5-O-4
bonded dimers (Figure 2); these are found in
thioacidolysis but are absent in acidolysis
mixtures. Also, the ratios of units of type 1 and
2 (Figure 3) are different in the product
mixtures from the two methods (Lapierre
et al., 1985). An important limitation, as far
as the structure of lignin is concerned, is that
both acidolysis and thioacidolysis only can
detect structural units bound by arylglycerolb-ether bonds (Scheme 1).
6.3

Permanganate Oxidation

In permanganate oxidation, the side chains in


the lignin are degraded to carboxyl groups
attached to the aromatic ring. The structures
of the products thus reveal the pattern of
substitution on the aromatic rings. Prior to
the oxidation, the sample is alkylated, usually
with dimethyl or diethyl sulfate (Freudenberg, 1968; Erickson et al., 1973a; Bose et al.,
1998). This protects the phenolic aromatic
rings from degradation, while all other
aromatic rings are degraded in the permanganate oxidation. Some biphenyls and diphenyl ethers also survive the oxidative
degradation. The mixture of acids is esterified
and analyzed by gas chromatography. The
structures provide information about the
substitution pattern of the phenolic structural units in the lignin. Monocarboxylic acids
are formed from uncondensed phenolic
groups, and dicarboxylic acids are formed
from condensed units with an additional

carbon substituent in the 5 or 6 position. The


presence of catechol units can be detected if
the alkylation is carried out with diethyl
sulfate. The structural information obtained
with permanganate oxidation is mostly qualitative. The generally low yields of degradation acids make it difficult to estimate the
quantitative distribution of structural units
from permanganate oxidation data (cf. Erickson et al., 1973b; Bose et al., 1998) (Scheme 2).
6.4

Nitrobenzene and Cupric Oxide Oxidation

Nitrobenzene oxidation and cupric oxide


oxidation give very similar degradation products, and are also more simple to perform
than permanganate oxidations. They are
primarily carried out in order to classify the
lignin in terms of the proportions of phenylpropane units of types 13 (Figure 3). It is
thus possible to characterize lignins of different botanical origin and in different morphological region of a plant. The reactions are
carried out in alkaline solutions at high temperature, though the reaction mechanisms
are still not well understood. The structural
information from these oxidations is limited
and they are usually combined with spectroscopic studies (Chen, 1992a) (Scheme 3).
6.5

Ozonolysis

Degradation of lignin with ozone achieves the


opposite of permanganate oxidation. While
permanganate degrades the side chains,
ozone destroys double bonds and the aromatic rings, leaving the side chains intact in
the form of carboxylic acids. The main
interest in ozonolysis lies in the fact that the
stereochemical configuration of the side
chain carbons in lignin is retained in the
oxidation products. Thus, the erythro form of
guaiacylglycerol-b-aryl ethers yield erythron-

6 Degradative Methods of Lignin Analysis

Scheme 2

Permanganate oxidation.

Scheme 3

Nitrobenzene/cupric oxide.

Scheme 4

Ozonolysis.

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ic acid and threonic acid is formed from the


corresponding threo isomer (Sarkanen et al.,
1992) (Scheme 4).

in unchanged lignins (Ralph et al., 1998)


(Scheme 5).
6.7

Functional Group Analysis

6.6

Reductive Cleavage after Derivatization (DFRC)

Acidolysis and thioacidolysis are specific


reactions that cleave the b-aryl ether bonds
in lignins. The strongly acidic treatments
involved cause extensive acid-catalyzed reactions that complicate the analysis of the
products of degradation. In an effort to
develop a simpler method with cleaner
cleavage reactions of b-aryl ether bonds, a
procedure has been introduced that involves
derivatization of lignin with acetyl bromide
and reductive cleavage of the resulting benzyl
bromides with zinc dust. This sequence of
reactions cleaves the b-aryl ether bonds, and
the liberated phenylpropane units can be
analyzed as cinnamyl alcohol derivatives. The
product mixtures in the derivatization followed by reductive cleavage (DFRC ) (Lu and
Ralph, 1997) tend to be simpler than those
obtained in acidolysis and thioacidolysis.
Apart from monomeric products, a number
of dimers and trimers have been identified.
These include representatives of all the common interunit linkages in softwood lignin
except the b-O-4 which is efficiently cleaved in
the degradation reaction (b-1, b-b, 5 5, b-5,
and 5-O-4) (Peng et al., 1998). Among the
trimers, a novel isochroman structure has
been found (Peng et al., 1999), and it is
suggested that such structures may exist

Scheme 5

6.7.1

Methoxyl Group Analysis


The methoxyl content is a widely used basis
for characterization of lignins. Methoxyl
groups are present in guaiacylpropane units
(1) and syringylpropane units (2). The methoxyl content of a lignin sample reflects the
distribution of units 1 3 (Figure 3). The
methoxyl content may give a measure of the
purity and the plant origin of lignin samples.
The method most widely used is a modification of one originally introduced by Zeisel
(1885), and is based on the formation of
methyl iodide when the sample is treated with
hydriodic acid at reflux temperature (Chen,
1992b). The methyl iodide is then treated
with bromine, which liberates the iodine and
converts it to iodic acid. The iodic acid is
treated with potassium iodide, and the liberated iodine is titrated with standard thiosulfate solution. In this sequence of reactions six
atoms of iodine are produced for each
methoxyl group, which allows determinations with a high degree of accuracy, even on a
micro scale (Scheme 6).
6.7.2

Phenolic Hydroxyl Groups


Common instrumental methods are potentiometric and conductometric titration, ionization difference UV spectroscopy (see be-

Reductive cleavage after derivatization (DFRC).

6 Degradative Methods of Lignin Analysis

Scheme 6

low), and NMR spectroscopy (Lai, 1992).


Oxidation of guaiacyl and syringyl structures
with aqueous sodium periodate to o-quinones, with the release of 1 mol of methanol
from each phenolic units, has been used to
estimate phenolic hydroxyl groups in lignins.
The amount of methanol is measured by gas
chromatography. The good reproducibility
and comparative simplicity makes it useful
for routine purposes (Francis et al., 1991).
The aminolysis method is based on the

Methoxyl group analysis.

finding that phenolic acetates are cleaved


with pyrrolidine much faster than aliphatic
acetates (Mnsson, 1983). It is a more
laborious procedure, and the accuracy is
critically dependent on the extent of acetylation and the selectivity of the deacetylation.
The inherent difficulties of obtaining reliable
data for phenolic hydroxyl contents in lignins
is reflected in the large variation in values
(from 18 to 33%) obtained for spruce MWL
with different methods (Lai, 1992).

Analysis of phenolic hydroxyl


groups; (1) oxidation with periodate, (2)
Aminolysis, (3) quinoid structures.

Scheme 7

99

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