Basic Lignin Chemistry

David Wang’s Wood Chemistry Class

Lignin
n Lignin is the second abundant and important organic
substance in the plant world.
n The incorporation of lignin into the cell walls of plants
gave them the chance to conquer the Earth’s land
surface.
n Lignin increased the mechanical strength properties to
such an extent that huge plants such as trees with
heights of even more than 100 m can remain upright.
 Lignin
n Lignin is derived from the Latin word for wood (lignum). de Candolle
firstly introduced this term in 1819. (Cellulose was named by Anselme
Payer in 1838)
n In 1897, Peter Klason studied the composition of lignosulfonates and
put forward the idea that lignin was chemically related to coniferyl
alcohol.
n In 1907, Klason proposed that lignin is a macromolecular substance.
After ten year (1917) Klason further purposed that coniferyl alcohol units
are joined together by ether linkages.

Lignin
n Definition
q A very irregular, randomly cross-linked polymer of phenylpropane
units joined by many different linkages.
q A polymer derived from the phenylpropanoid compound, coniferyl
alcohol and related alcohols.
q Amorphous, cross-linked polymers that occur uniquely in vascular
plants.
q Lignin may be defined as an amorphous, polyphenolic material
arising from an enzyme-mediated dehydrogenative polymerization of
three phenylpropanoid monomers, conniferyl, synapyl and p-
coumaryl alcohols.
Lignin
n Lignin precursors (three cinnamyl alcohol)
q p-Coumaryl alcohol
q Coniferyl alcohol
q Sinapyl alcohol

Lignin Precursors and Aromatic Constituents

Lignin
o Lignification

n Formation lignin in wood

o Lignin function

n Supportive agent → mechanical strength

n Antioxidant → protection

n Sealant and reinforcing agents → bonding cellulose

and hemicellulose together
n Cross linker → cross link carbohydrates

Lignin Isolation
oGeneral
n Lignin can be isolated from extractives-free
wood as an insoluble after hydrolytic removal
of polysaccharides.

n Lignin can be hydrolyzed and extracted from

wood or converted to a soluble derivative.
 Lignin Isolation — Removing the polysaccharides

n Klason lignin
q Klason lignin is obtained after removing the polysaccharides from
extractives-free wood by hydrolysis with 72% sulfuric acid.
n Cellulolytic enzyme lignin (CEL)
q The polysaccharide may be removed by enzymes from finely divided
wood meal. This method is tedious, but the resulting CEL retains its
original structure essentially unchanged.
n Dioxane containing water and HCl
q Considerable changes in its structure occur.

Lignin Isolation — Removing the polysaccharides

n Milled wood lignin (MWL)

q Björkman lignin
q It is the best preparation known as far and it has been widely used for
structure studies.
q Wood meal is ground in a ball mill either dry or in the presence of non-
swelling solvents (ex. toluene), the cell structure of the wood is destroyed
and a portion of lignin (usually less than 50%) can be obtained from the
suspension by extraction with a dioxane-water mixture.
q MWL preparation always contain some carbohydrate materials.
 Lignin Isolation — Removing the polysaccharides

n Lignin preparation by artificial

q Dehydrogenation polymer (DHP)

Coniferyl alcohol was treated with H2O2 in the presence of

peroxidase enzyme yield synthetic lignin named DHP.

q Released suspension culture lignin (RSCL)

RSCL was isolated suspension cultures of spruce wood cells as a

secretion products (RSCL represents a carbohydrate-free
coniferous lignin)

Lignin Isolation — Removing the lignin

o Lignosulfonates
n Soluble lignin derivates, lignosulfonates, are formed by treating wood at
elevated temperatures with solutions containing sulfur dioxide and
hydrogen sulfite ions.
o Sulfate lignin or Kraft lignin
n Lignin is dissolved as alkali lignin when wood is treated at elevated
temperature (170 °C) with NaOH, or better, with a mixture of NaOH and
Na2SO4.
n Lignin is further converted to an alkali soluble derivative a solution of
hydrochloric acid and thioglycolic acid at 100 °C.
 Measurement on Lignin Content
n Softwood
¨ Softwood lignin can be determined gravimetrically by klason
method.
¨ Normal
softwood contains 26-32% lignin while the lignin content of
compression wood is 35-40%.
n Hardwood
¨ The lignin present in hardwood is partly dissolved during the acid
hydrolysis and hence the gravimetric values must be corrected for
the “acid-soluble lignin” using UV spectrophotometer.
¨ Normal hardwood contains 20-25% lignin. Tropical hardwood can
have a lignin content exceeding 30%. Tension wood contains only
20-25% lignin.

Biosynthesis and structure of Lignin

Lignin are polymers of phenylpropane units.

Many aspects in the chemistry of lignin still remain
unclear.
The ability to synthesize lignin has been essential in
the evolutionary adaptation of plants from an aquatic
environment to land.
Although researchers have studied lignin for more than
a century, many aspects of its biosynthesis remain
unresolved. The monolignol biosynthetic pathway has
been redrawn many times and remains a matter of
debate.
n Ethnolysis

¨The hydrolytic treatment of wood or lignin with dilute

alcoholic hydrochloric acid under pressure was the
original method for obtaining defined phenylpropenoid
ketones (Hibbert ketones) by splitting β-aryl ether
linkages.
n Lange (1954) who applied UV microscopy at
various wavelength directly on thin wood sections,
obtaining spectrum typical of aromatic compounds.

Biosynthesis of Lignin Precursor

o Precursors of lignin

n Gymnosperms: coniferyl alcohol (guaiacyl unit)

n Angiosperms: coniferyl alcohol + sinapyl alcohol

(guaiacyl unit + syringyl unit)

n Grasses: p-coumaryl alcohol (p-hydroxyphenyl unit)

o Lignin precursors are generated from D-glucose through

reaction of enzyme.
Outline of the
Biosynthetic
Pathway of
Phenylpropanoids

1. Shikimate pathway
phosphoenol pyruvic acid

3-dehydroquinic acid
3-deoxy-D-arabino-heptulosonic
D-erythrose 4-phosphate acid 7-phosphate

NADP-linked shikimate
dehydrogenase

3-dehydroshikimic acid
phosphochorismic acid shikimic acid
(1)
phosphorylated

phenylpyruvic acid
苯丙胺酸

arogenic acid
prephenic acid
chorismic acid

p-hydroxyphenylpyruvic acid 酪氨酸

2. Cinnamate pathway

PAL
cinnamic acid
phenylalanine ammonia-lyase

TAL
p-hydroxy cinnamic acid or
tyrosine ammonia-lyase p-coumaric acid

hydroylase

caffiec acid

O-methyl transferase
(OMT)
ferulic acid

5-hydroxyferulic acid

sinapic acid

— Reduction of Ferulic Acid to Coniferyl Alcohol

hydroxycinnamate-CoA
ligase

hydroxycinnamoyl-CoA
reductase
feruloyl adenylate
feruloyl CoA thio ester

• These enzyme in angiosperms reduce both

coniferyl and sinapyl aldehyde almost
equally, but the gymnosperm enzymes are
remarkably specific for coniferyl aldehyde.
• O-methyltransferase, p-hydroxycinnamyl
oxidoreductase alcohol oxidoreductase is obviously one of
the key enzymes controlling the specificity of
coniferyl aldehyde the lignin precursors.
 Pathways to monolignols. The
complete metabolic grid of
reactions is shown. All enzyme
reactions shown with a solid
arrow have been
demonstrated to occur in vitro.
Reactions shown in smaller type
may not occur in vivo. The
reactions shown in green seem
the most likely route to
G lignin in vivo. The reactions in
red represent those reactions
consistent with both in vivo and in
vitro evidence for being involved
speci.cally in S
lignin biosynthesis. The
intermediate in orange is common
to both G and S lignin pathways.

Major Segments of Phenylpropanoid Metabolism in Vascular

Plants as Currently Understood
 A Metabolic Channel Model for Independent
Pathways to G and S Monolignols

A Representation of the Random Model for Lignin Formation

Polymerization of Lignin

n Erdman (1930) studied the oxidative dimerization of various phenols in

the biogenesis of natural products and reached the conclusion that

lignin must be formed α,β-unsaturated C6C3 precursors of the coniferyl

alcohol type via enzymatic dehydrogenation.

n Freudenberg and co-worker (1940-1970) had demonstrated the

polymerization of precursors to lignin in nature does indeed occur as

Erdman described.

n One-electron transfer from coniferyl alcohol by enzymatic

dehydrogenation yield resonance-stabilized phenoxy
radicals.
 n Oligomeric products formed through coupling of coniferyl
alcohol radicals

n Endwise β-O-4 coupling of

a coniferyl alcohol radical
with a growing lignin group
radical to an intermediate
quinone methide (3), which
is stabilized to a
quaiacylglycerol-β-aryl
ether (4) structure through
addition of water.
 Type of Linkages and Dimeric Structure
l Common linkages between the phenylpropane units

Lignin Structure
n Methods based on classical organic chemistry led to the
conclusion, already by 1940, that lignin is build up
phenylpropane units.
¨ Permanganate oxidation (methylation softwood lignin)

10% veratric acid

1. KOH, 170℃
2. Methylation 藜蘆酸
3. KMnO4
Lignin
minor isohemipinic acid

異半派酸

The formation of isohemipinic acid

support the occurrence of condensed
structure (β-5 or γ-5) dehyrodiveratric acid
— Nitrobenzene oxidation

Softwood

Vanillin (25% of lignin)

Hardwood

Nitrobenzene oxidation
Lignin
syringaldehyde
Grass

p-hydroxybenzaldehyde

— Hydrolysis

H2/CAT
Lignin

— Ethnolysis Hibert ketons

2% HCl in EtOH
Lignin
n Acid hydrolysis (Acidolysis)
q The term acidolysis refers specifically to the refluxing of lignin or
lignocellulose with 0.2 M HCl in dioxane-water (9:1, v/v).
q Acidolysis has a close relationship to ethanolysis (heating of
lignin in HCl in ethanol). Both treatment result in the
degradation of lignin with formation of substantial amounts of
arylpropanes (but, dioxane-water is a better solvent for lignins
than is ethanol).
q The majority of the acidolysis monomers originate from
arylglycerol β-aryl ether structure.

q Formation of monomeric phenols from β-O-4 structures in lignin

during acidolysis.
n Thioacidolysis
q Thioacidolysis is solvolysis in dioxane-ethanethiol with boron
trifluoride etherate.
乙醚合三氟化硼

q It is an acid-catalyzed reaction with results in the

depolymerization of lignins.
q As in acidolysis, thioacidolysis proceeds mainly by cleavage of
arylglycerol-β-aryl ether linkages.
q In contrast to acidolysis, thioacidolysis is preformed in
anhydrous media, ether-cleaving reagent combines a hard
Lewis acid (Et2O-BF3, boron trifluoride etherate), and a soft
nucleophile (EtSH, ethanethiol)

n Thioacetolysis

q Thioacetolysis-lignin samples are subjected to a treatment with

thioacetic acid and boron trifluoride.

硫代醋酸

q The cleavage of the ether bonds by using thioacidolysis is

equally specific, but more complete than by thioacetolysis.

q The reaction products are separated as TMS (trimethylsilyl

ethers by GC.
n Permanganate oxidation
¨Much of our current knowledge about structure of lignin
in wood and pulp is based on results obtained from
permanganate oxidation.
¨This method involved the selective degradation of all
aliphatic side chains attached to aromatic moieties
structures henceforth referred to as “acids”.
¨The original permanganate oxidation method of
methylated lignin has also been considerably improved
when it is performed at alkaline instead of neutral
conditions.

n Permanganate oxidation
¨The methylated fragments are seperated by GC and
identified MS
¨Reaction sequence for the oxidative degradation of
lignin with potassium permanganate
l Major carboxylic acid methyl esters formed in the
oxidation of lignin with potassium permanganate

Type of Linkages and Dimeric Structure

n It is clear that phenylpropane units are joined together both with C-O-C
(ether) and C-C linkages.
¨ C-O-C linkages is dominant; approximately 2/3 or more.
¨ The rest are the C-C type.

n Proportions of different type of linkages connecting the phenylpropane

units in lignin
Functional Groups
n Lignin polymer contains characteristic methoxyl groups, phenolic
hydroxyl groups, and some terminal aldehyde groups in the side chain.
n Only relatively few of the phenolic hydroxyls are free; most of them are
occupied through linkages to the neighboring phenylpropane units.
n The syringly units in hardwood lignin are extensively etherified.
n Alcoholic hydroxyl groups and carbonyl groups are introduced into the
final lignin polymer during the dehydrogenative polymerization
process.

Functional Groups
n In some wood species substantial amounts of the
alcoholic hydroxyl groups are esterified with p-
hydroxybenzoic acid or p-hydroxycinnamic acid.
¨ Ester of p-hydroxybenzoic acid are typical in aspen lignin.
¨ p-Hydroxycinnamic acid are abundant in bamboo and grass lignin.
 Lignin Formula

o The formula of lignin presented in its final shape in 1968 by

Freudenburg for softwood lignin (spruce) has attained
general acceptance.
n This scheme for spruce lignin represents 18
phenylpropane units as a section of the total molecule
which was assumed to consist of more than 100 units in
the native state.
o Adler (1977) gave a structural scheme for spruce lignin
comprising 16 prominent C9-units, mainly derived from the
results of oxidative degradation experiment.

A Structure Segment of Softwood lignin Proposed by Adler

β-6 glyceraldehyde-2-aryl ether

pinoresinol

only low amount in softwood

Spruce lignin
Softwood Lignin Model designed by Computerized Evaluation
Loblolly pine lignin

(Glasser, 1981)

Lignin Formula

n In addition, formulas
for hardwood lignins
have been suggested
as well.
n The structure concept
of beech lignin (Nimz,
1974).
 Lignin-Carbohydrate Bonds
n The possible existence of covalent bonds between lignin and
polysaccharides has been a subject of much debate and intensive
studies.
n It is obviously and now generally accepted that such chemical bonds
must exist, and the term “lignin-carbohydrate complex (LCC)” is used
for the covalently bonded aggregates of this type.
n Chemical bonds have been reported between lignin and practically all
the hemicellulose constituents (even between lignin and cellulose).
These linkages can be either of ester or ether type and even glycosidic
bonds are possible.

Examples of suggested LCC bonds

An ester linkage to
xylan through 4-O-
methyl glucuronic
acid as a bridge
group.

An ether linkage to
An ether linkage to galactoglucomannan
xylan through an through a galactopyranose
arabinofuranose unit. unit.
 Lignin-Carbohydrate Bonds
o Ether linkages are more stable and common between lignin and
carbohydrates.
n The α-position is even in this case the most possible connection
point between lignin and hemicellulose.

o LCC bridge groups

n Arabinose unit (HO-2 or HO-3)

n Galactoglucomannans, the galactose unit (HO-3)

n In middle lamella and primary wall (pectic polysaccharides)

galactan → HO-6 in galactose

arabinan → HO-5 in arabinose

Lignin-Carbohydrate Bonds

o Glycosidic linkages for LCC

n Benzylic alcohol group, which is the most probable

connection point, the phenolic group may also be partly

occupied through glycosidation.

n The glycosidic linkages are easily cleaved with acid.

 Classification and Distribution of Lignin
l Lignin can be divided into several classes according to their structure
elements
l Guaiacyl lignin
It is a largely polymerization product of coniferyl alcohol. Guaiacyl
lignin occurs in almost all softwoods.
l Guaiacyl-syringyl lignin
The typical lignin of hardwood, this type of lignin is a copolymer of
coniferyl and sinapyl alcohol. The ratio varing from 4:1 to 1:2 for
the two monomeric units.
l p-Hydroxyphenyl lignin
Compression wood, which has a high proportion of phenylpropane
units of the p-hydroxyphenyl type in addition to the normal
quaiacyl units.

Transverse Section of a Spruce Tracheid Photographed in

UV Light (240 nm)
 Classification and Distribution of Lignin
n In hardwood, there are still many uncertainties involved, because of

the more heterogeneous nature of the wood and the presence of both

quaiacyl and syringyl units in the lignin.

q The lignin located in the secondary wall of hardwood fibers has a

high content of syringly units whereas larger amounts of guaiacyl

units are present in the middle lamella lignin.

q The vessel in birch seem to contain only guaiacyl lignin, whereas

syringyl lignin predominates in parenchyma cell.

Polymer Properties of Lignin

n The limitation for studying the macromolecular properties for lignin :
q Low solubility in most solvents
q It will cause degradation during the isolation process.
q The polymer properties of lignin is depend on its location in the cell
wall.
n The method for characterizing the polymer properties of lignin:
q Vapor pressure osmometry
q Light scattering
q Ultracentrifugation
Polymer Properties of Lignin
n The Mw of softwood lignin around 20000, the lower values have been
reported for hardwood lignin.
n Compared with cellulose, the polydiversity of lignin is relatively high,
roughly 2.0 -3.0 for softwood MWL.