c© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim10.1002/14356007.a15 305.pub3

Lignin 1

Lignin

Bodo Saake, Institut fur Holzchemie, Bundesforschungsanstalt fur Forst- und Holzwirtschaft, Hamburg,Germany

Ralph Lehnen, Institut fur Holzchemie, Bundesforschungsanstalt fur Forst- und Holzwirtschaft, Hamburg,Germany

1. Occurrence and Functions . . . . . . . 12. Structure and Biosynthesis . . . . . . . 23. Physical Properties . . . . . . . . . . . . 64. Chemical Properties . . . . . . . . . . . 74.1. Kraft Pulping . . . . . . . . . . . . . . . . 74.2. Sulfite Pulping . . . . . . . . . . . . . . . 74.3. Other Pulping Processes . . . . . . . . . 84.4. Pulp Bleaching . . . . . . . . . . . . . . . 84.5. Yellowing Reactions . . . . . . . . . . . . 95. Commercial Lignins . . . . . . . . . . . 9

6. Analysis . . . . . . . . . . . . . . . . . . . . 106.1. Detection and Quantification . . . . . . 116.2. Isolation . . . . . . . . . . . . . . . . . . . 116.3. Spectroscopic Methods . . . . . . . . . . 116.4. Degradation Techniques . . . . . . . . . 126.5. Molar Mass Determination . . . . . . . 137. Uses . . . . . . . . . . . . . . . . . . . . . . 138. Toxicology . . . . . . . . . . . . . . . . . . 149. References . . . . . . . . . . . . . . . . . . 14

1. Occurrence and Functions

Lignin is one of the three major constituents ofvascular plants, the other two being celluloseand hemicelluloses. The name lignin is derivedfrom the Latin word lignummeaning wood. Af-ter cellulose, lignin is the most abundant natu-ral (terrestrial) organic polymer. Its content ishigher in softwoods (27–33%) than in hard-woods (18–25%) and grasses (17–24%). Thehighest amounts of lignin (35–40%) occur incompression wood on the lower part of branchesand leaning stems of conifers [1, 2]. Lignindoes not occur in algae, lichens, or mosses [3],whereas the “lignins” of bark differ in their struc-ture from typical wood lignins [4].

Lignin is a randomly branched polyphenol,made up of phenylpropane (C9) units, renderingit discernible from the other two major woodcomponents by its UV absorption maximum at280 nm. Figure 1 shows a typical UV micro-scopic imaging profile on a microtome cut ofan individual spruce wood fiber [5]. Novel in-struments can as well provide color-coded 2Dor 3D plots, showing the lignin distribution overthe cross-section of fibers [6]. The highest ligninconcentration (≈ 70%) is found between adja-cent cell walls (middle lamella) and at the cell

corners, while it is much lower (≈ 20%) acrossthe secondary wall. However, due to the muchlarger volume of the secondary wall, most lignin(≈ 80%) is located in the secondary wall of thewood cells.

Figure 1. A) Black spruce earlywoodCross section of tracheids of black spruce early-wood photographed in ultraviolet light (λ= 240 nm);B) Densitometer tracing across the tracheid wall showingthe variation of lignin concentration along the dotted line[5]

In accordancewith its distribution, lignin per-forms three important functions in the xylem tis-sue that are essential to the life of plants: Due

2 Lignin

to its lipophilic character, lignin decreases thepermeation of water across the cell walls, thatconsist of cellulose fibers and amorphous hemi-celluloses, thus enabling the transport of aque-ous solutions of nutrients and metabolites in theconducting xylem tissue. Secondly, lignin im-parts rigidity to the cell walls and, in woodyparts, together with hemicelluloses, functions asa binder between the cells generating a com-posite structure with outstanding strength andelasticity. Finally, lignified materials effectivelyresist attacks by microorganisms by impedingpenetration of destructive enzymes into the cellwalls.

The above mentioned functions of lignin areimpressively demonstrated by the strength of thetrunks of giant redwoods (Sequoia) in Califor-nia, growing up to more than 100 m, supportingcrown structures of several tons in weight forthousands of years, unparalleled by human con-structions.

2. Structure and Biosynthesis

After cell growth has ceased, lignin is formed bya dehydrogenative polymerization of three p-hy-droxycinnamyl alcohols (monolignols): i.e., p-coumaryl (1), coniferyl (2), and sinapyl alcohol(3) (Fig. 2), which are formed from d-glucosevia shikimic acid [7]. It has to be mentioned thatin lignin chemistry the designation of the carbonatoms does not follow the IUPAC nomenclature.The aromatic ring is numbered assigning thephenolic OH group to position 4. The propaneside chain is labeled with Greek letters, start-ing from the benzylic carbon, termed α carbon.The phenylpropanoid monomers are oxidized tophenoxy radicals by hydrogen peroxide in thepresence of peroxidase. The unpaired electronof the phenoxy radical is delocalized over the

aromatic ring and the conjugated olefinic doublebond of the side chain, the highest density of theunpaired electron being at the β-carbon atom,C-5, C-1, and O-4, as illustrated for coniferylalcohol in Figure 3. Random recombination oftwo monomeric phenoxy radicals leads to dilig-nols, which are further oxidized to oligolignolsand finally to the polymeric lignin. Themost im-portant structural units and their designation aredepicted in Figure 4.

Figure 2.Monomeric lignin precursors

In order to prove this pathway Freudenbergand his group carried out tracer experimentswith carbon-14 labeled d-glucose, phenylala-nine, coniferyl alcohol, and d-coniferin [7]. Inaddition they performed in vitro model studiesby dehydrogenation of coniferyl alcohol withperoxidase and hydrogen peroxide, obtainingpolymers which were not identical but rathersimilar to lignin [7]. The resulting products werecalled dehydrogenation polymers (DHP) andhave been intensively used as a lignin polymermodel by many research groups.

The random polymerization by recombina-tion of phenoxy radicals explains why lignin—in contrast to most other natural polymers suchas proteins, polysaccharides, nucleic acids, andnatural rubber—has an irregular structure andis optically inactive, though carbon atoms α and

Figure 3. Dehydrogenation of coniferyl alcohol (2) yielding phenoxy radicals

Lignin 3

Figure 4.Most important structural units of lignin

β in the side chains of the phenylpropane struc-tural units are asymmetric. However, it has to bekept in mind that the last polymerization steptakes place within the secondary cell wall ofdifferentiated wood cells in close contact withpartly crystalline cellulose fibers, so that the aro-matic nuclei in lignin may partly be aligned tan-gentially to the secondary wall [8]. Recently (inthe early 2000s) a new hypothesis has evolved,postulating that the synthesis of lignin might beat least partly controlled by dirigent proteins.Such proteins are involved in the polymeriza-tion of lignans, a group of oligomeric productswhich belong to the extractives of lignocellu-

losic plants. This hypothesis is discussed ratherintensely and summaries of the pros and conshave been published by the main protagonists[9, 10].

The random coupling reaction, which is stillthe most widely accepted concept, results in athree-dimensional, amorphous polymer withouta regular structure or repeating unit. Accord-ingly, no definite lignin structure can be deter-mined although several schemes have been pro-posed as a statistical structural model for var-ious plant species. A first concept for sprucelignin was developed by Freudenberg [7] basedon 18 C9 units, of which 2.5 (units 2, 3, 5)

4 Lignin

are coumaryl, one a syringyl (3,5-dimethoxy-4-hydroxyphenyl) (unit 16), and 14.5 guaiacyl(3-methoxy-4-hydroxyphenyl) units (Fig. 5). Astructural scheme of beech lignin was proposedby Nimz consisting of 25 structural units, ofwhich six (5/6, 9/10, 24/25) are partly replacedby the dilignol units enclosed in brackets (Fig.6). The concentrations of the latter units are be-low 4%. The scheme shows a representativesection of a ten to twenty times larger beechlignin molecule, in which ten different interunitbond types are randomly distributed. The rela-tive amounts of interunit linkages for the twolignin structures are listed in Table 1 [11]. Thedata show as well that the calculated C9 for-mulas are in good correlation with experimentaldata for isolated lignins. The most abundant in-terunit linkages in both schemes are the β-O-4linkages. It is the concentration of these link-ages in which lignins typically differ from other

polyphenols occurring also in plant materials.During the lignification process the quinoneme-thide can also react with hydroxyl or carbox-yl groups of polysaccharides forming covalentether and ester bonds in the α-C position. Thesecovalent bonds make it difficult to obtain ligninwithout carbohydrate impurities for both analyt-ical and technical purposes. For this close associ-ation of lignin and carbohydrates the term lignincarbohydrate complex (LCC) has been coined[12].

Although the structures in Figures 5 and 6do not include some new features such as thedibenzodioxocin structure (Fig. 3, 12) disco-vered in the 1990s [15], they are still good mod-els to describe the fundamental differences ofsoftwood and hardwood lignins. According tothe high proportion of guaiacyl units softwoodlignins are called guaiacyl lignins (G-lignins),while hardwood lignins are termed guaiacyl–

Figure 5. Constitution of spruce lignin after Freudenberg [7]

Lignin 5

Table 1. Proportions (%) of interunit linkages, functional groups and elementary composition per C9 unit of spruce and beech ligninaccording to structural schemes in Figures 5 and 6

Bond types, functional groups and elementary composition of C9unit

Spruce lignin Beech lignin

β-O-4 39–48 32–37α-O-4 11–16 28–32β-5 6–10 8β-β 7–10 6.45–5 7–9 24-O-5 6–7 2β-1 2 16α-5 7.2α-β 4Aliphatic OH 92–98 88Benzylic OH (α-OH) 18 4Phenolic OH 29.4 16Ketone groups 13.8 16Aldehyde groups 2.8 4Methoxy groups 91.7 136C9 formulae of structural schemes C9H7.82O2.4(OCH3)0.92 [7] C9H7.16O2.44(OCH3)1.36 [11]C9 formulae of MWLs* C9H7.95O2.4(OCH3)0.92 [13] C9H7.10O2.41(OCH3)1.36 [14]

* MWL (milled wood lignin), see Section 6.2

Figure 6. Constitutional scheme of beech lignin after Nimz [11]

6 Lignin

syringyl lignins (GS-lignins). Grass lignins ad-ditionally contain p-hydroxyphenyl (coumaryl)units (GSH-lignins).

Accordingly, hardwood lignins have thehighest methoxy values with 1.21–1.52 perC9 unit, followed by softwood lignins with0.80–1.01 and grass lignins with about 0.6–0.9 [16]. However, the low methoxy values ofgrass lignins may also be caused by substan-tial amounts of p-coumaric acid, linked to theγ-carbon atoms of grass lignins via ester bonds[17]. The methoxy group content of compres-sion wood MWL from larch (Larix leptolepis)was found to be 0.2 per C9 unit lower than thatof normal wood MWL [18]. From the compari-son of the carbon-13 NMR spectra of acetylatedcompression wood MWL with those of acety-lated GSH- and H-DHPs (p-hydroxyphenyl de-hydrogenation polymers), compression woodlignin was classified as a guaiacyl-p-hydroxy-phenyl (GH)-lignin [19].

Variations in the methoxy group contentscause changes in the constitution of the lignins.As shown in Table 1 the proportions of α-O-4 and β-1 interunit linkages in beech ligninare significantly higher than those in sprucelignin, while the content of 5–5 and 4–O–5 link-ages is lower, meaning that softwood ligninsare more condensed than hardwood lignins andhardwoods may be more easily delignified thansoftwoods, agreeing with the differences inthe chemical properties of hardwood and soft-wood lignins. Consequently, compression woodlignins have the highest level of condensationwith the lowest solubility and degradability. Areviewwith almost 700 references on the history,chemistry, and technology of lignin is given in[20].

3. Physical Properties

Solubility. As a branched polymer, nativelignin is insoluble in all neutral solvents at roomtemperature.At temperatures above 100 ◦Cor inalkaline solvents native lignin is partly dissolvedas a result of degradation reactions. For isolatedor technical lignin the solution properties varydepending on the isolation or production pro-cess and sample purity. Milled wood lignins aresoluble in dioxane and acetone, containing 5–10% water, as well as in dimethylformamide

and pyridine. Lignosulfonates can be dissolvedin water, while all isolated lignins are soluble inalkali.Most lignins can be solubilized in organicsolvents such as dimethyl sulfoxide, dimethyl-formamide, dioxane, and pyridine at least foranalytical purposes.

Molar Mass. The molar mass of lignin insitu is unknown and that of isolated lignins de-pends on the conditions of isolation. In generalthe molar mass determination of lignins withvarious methods show a large deviation (seeSection 6.5). For spruce milled wood lignin anaverage molar mass (M) of 11 000 g/mol hasbeen estimated by ultracentrifugation [21], cor-responding to a weight-average degree of poly-merization of approximately 60 C9 units. Forthe most important commercial lignins from thekraft, soda, and sulfite pulping process molarmasses in a range between 3 000 and 20 000g/mol and polydispersities between 2 and 12have been reported, while the intrinsic viscosi-ties were in a range from 0.04 to 0.08 dL/g. Theexponent a from the Mark–Houwink–Sakuradaequation varies between 0.1 and 0.5, which is inthe range between an Einstein sphere (a = 0) anda compact coil (a = 0.5) [22, 23].

Thermal Properties. Lignin is an amor-phous thermoplastic polymer. The glass tran-sition temperature Tg of lignin is strongly af-fected by moisture content and hydrogen bond-ing, a phenomenon of great importance for thewood processing industry. By dynamic mechan-ical thermal analysis the Tgs of native lignins inwood were calculated resulting in lower valuesin hardwoods (65-85 ◦C) than in softwoods (90-105 ◦C). For isolated lignins the Tg values arehigher and show deviations according to the rawmaterial, production process, and measurementprocedure: MWL lignins: 110 to 160 ◦C, kraftlignins: 124–174 ◦C, steam explosion lignin:113–139 ◦C [24]. The effect of moisture wasdemonstrated for an isolated periodate ligninwhich had a Tg of 195 ◦C when dry and a Tg of90 ◦C when containing 27% water [25]. Ther-mal properties are as well depending on molarmass. On fractionated kraft lignins the fractionof lowest molar mass (M 620 g/mol) had a Tgof only 32 ◦C while at highest molar mass (M180 000 g/mol) Tg amounted to 173 ◦C. A simi-lar tendencywas observed for the temperature of

Lignin 7

start of decompositionwhich increasedwithmo-lar mass from 181 to 238 ◦C [26]. The calorificvalue of lignin depends on the purity of the sam-ple. The dry matter of the black liquor from thekraft process has a value around 23.4 MJ/kg [1].

4. Chemical Properties

Besides ketone and aldehyde groups, the mostreactive functional groups in lignin are the phe-nolic groups, the benzyl alcohol (α-OH) andnoncyclic benzyl ether groups (α-O-4), whichunder acidic and alkaline conditions are proneto condensation reactions. From an industrialstandpoint the reactions of lignin in pulping pro-cesses are most important since these reactionsinfluence not only the pulping process itself butthe structural features of technical lignins ob-tained as by products.

4.1. Kraft Pulping

The leading technical pulping process withsodium hydroxide and sodium sulfide is calledkraft pulping. The word “kraft” originates fromthe German word “Kraft” (strength), meaningthe strength of the obtained kraft pulps in com-parison to that of sulfite pulps.

As a main reaction of the kraft process moi-eties with free phenolic groups are convertedinto quinonemethide groups (15), which add hy-drogen sulfide ions at the α-carbon atoms (Fig.7). The thus formed benzylthiolate anion (16)loses its β-phenolate anion in a neighboring dis-placement reaction. The units with free phenolicgroups thus createdmay again form quinoneme-thides and add hydrogensulfide ions, if their α-carbon atoms are bonded to hydroxyl or non-cyclic ether groups. At the high reaction tem-perature the sulfur from compound (17) is partlysplit off. Especially in the last stage of the kraftprocess carbon-carbon bonds can be formed bet-ween lignin units, a reaction which is referredto as “condensation”. This results in structureswhich are very difficult to cleave in further pro-cess stages. Furthermore, the hydrogensulfideions lead to demethylation reactions and sub-sequently to the formation of methyl mercaptanwhich resulted in former times in odor problemsin themills. An extensive reviewon the reactions

and technology of pulping processes is given in[27].

Figure 7.Mechanism of kraft cooking

4.2. Sulfite Pulping

In the technical sulfite pulping processeswood isusually reacted with calcium (pH≈1-2) or mag-nesium sulfite (pH ≈3-5) at ca. 125 to 150 ◦Cfor 3 to 7 h. The three major reactions occurringsimultaneously are sulfonation, hydrolysis, andcondensation. The dissolution of lignin in aque-ous solution is mainly caused by the introduc-tion of hydrophilic sulfonic acid groups at theα-carbon atoms (Fig. 8). In the sulfurous acidprocess at pH 1–1.6 and in the acid sulfite pro-cess at pH 1.8–3.1, benzyl alcohol and benzylether groups in lignin form benzylium ions (20),that preferably add nucleophilic sulfite ions, butto some extent may also lead to condensationproducts (21) [28, 29]. The degree of conden-sation may be less in the bisulfite process at pH

8 Lignin

4.5 and the bisulfite-sulfite process at pH 7. Theβ-O-4 bond is rather stable under acidic condi-tions. Some sulfite processes operate under neu-tral or alkaline conditions using sodium or am-monia as base. In these processes β-O-4 bondscan be cleaved as well depending on the reac-tion conditions. The spent sulfite liquor, besidesdegraded hemicelluloses contains lignosulfonicacids (22).

Figure 8.Mechanism of sulfite cooking

4.3. Other Pulping Processes

Especially in countries with small wood re-sources the soda process is used for the pro-duction of pulp from annual plants applyingonly sodium hydroxide as a reaction chemi-cal. In this process only α-aryl ether bondsin phenolic lignin units are effectively cleaved[29]. The addition of the redox catalyst sys-tem anthrahydroquinone/anthraquinone can im-prove the delignification when raw materials

with higher lignin content are processed. An-thrahydroquinone can catalyze the cleavage oflignin in a similar manner to sulfide, improvingespecially the cleavage of β-O-4 linkages [30].It can be applied in all alkaline processes suchas kraft or alkaline sulfite pulping.

A large number of pulping processes basedon organic solvents have been investigated overthe last decades and were summarized underthe term “organosolv” processes. Some werebased on organic solvents (especially methanoland ethanol) with and without addition of cata-lysts while others were applying organic acids(mainly formic and acetic acid) [31, 32]. Up tonow none of these processes has been imple-mented in industry. Nevertheless, in many pa-pers on new applications of lignin still organo-solv lignins from former pilot plant trials areused as a rawmaterial, especially when a sulfur-free lignin is required.Steam explosion or steam refining is a pro-

cess route for the production of fibers fromwoodor straw. These processes are often catalyzed au-tohydrolytically by organic acids liberated fromthe hemicelluloses of hardwoods or straw. Dur-ing steam explosion a part of the lignin (≈10–15%) is rendered water-soluble while the restcan be extracted with dilute sodium hydroxidesolution. During the steam treatment an acid-catalyzed hydrolysis of aryl ether linkages takesplace while on the other hand homolytic cleav-ages occur. At high reaction severity a recon-densation of lignin can occur as well. Accord-ingly steam explosion lignins have higher molarmasses than sulfite, kraft, or organosolv lignins.Steam explosion lignins might become interest-ing in the future, because this process is the firststep for saccharification of lignocelluloses forglucose or ethanol production, a process routewhich is currently under discussion as a sourcefor bioenergy and fuel [31, 33] (the whole issueof [33] deals with steam explosion.)

4.4. Pulp Bleaching

Unbleached pulps still contains between 1.5 and6% of lignin. Since the residual lignin is inten-sively colored it has to be removed in order toproduce pulps with high brightness and bright-ness stability. Previously chlorine and chlorinedioxide were major bleaching chemicals lead-

Lignin 9

ing to toxic chlorinated phenols in the bleachingeffluents (measured as adsorbable organic halo-gen (AOX). In order to reduce these effluents,chlorine (C)was almost completely and chlorinedioxide (D) partly replaced by alkaline oxygen(O), hydrogen peroxide (P), and ozone (Z), lead-ing to totally chlorine free (TCF) or elementalchlorine free (ECF) bleached pulps. In the pro-duction of ECF pulps chlorine dioxide is still ap-plied, but no gaseous chlorine. In the beginningof these developments it was often assumed thatTCF bleaching should be the long-term goal foran environmental-friendly industry. However, ithas been shown that a modern ECF sequencecan be operated with very low AOX levels andaccordingly ECF is now the dominating technol-ogy for modern pulp mills [34].

4.5. Yellowing Reactions

In the presence of alkali and oxygen or in day-light lignin shows an intense discoloring, whichis observed by the yellowing reaction of pa-per, paper products, and wood. Amajor reactionpathway is the absorption of ultraviolet light bythe α carbonyl group leading to a phenoxy rad-ical which reacts subsequently with oxygen toform quinoid chromophores (Fig. 9).

Figure 9.Main reaction pathway of photo yellowing

The photoyellowing is a rather complex pro-cesswhichwas recently summarized in [35]. Forapplications of lignin the sensitivity to UV lighthas to be considered being a potential problem

for product stability or a positive feature whenUV absorbing properties are required.

5. Commercial Lignins

Up to now commercial lignins are exclusivelyobtained as byproducts from the chemical pulp-ing industry. The kraft process is the dominat-ing technology with about 89% of the total pro-duction capacity while in 2000 the share of thesulfite process had declined to only 3.7%. Nev-ertheless, lignosulfonates from sulfite processesare still of major importance for the applicationof lignin as industrial products. About 10% ofthe total pulp production is based on nonwoodplant materials like bast and leaf fibers, straw,sugar cane bagasse, or bamboo. These materi-als are easier to delignify and are mainly pro-duced by the soda process. They are especiallyimportant raw materials in countries with smallwood resources like India andChina [36]. In for-mer times these lignins were not commerciallyavailable. Recently a plant for lignin recoveryfrom nonwood soda pulping has been installedwhich claims an annual production capacity of10 000 t sulfur-free lignin [37] and accordinglysoda lignins might become an alternative in thefuture. In 1998 some 50× 106 t of lignin wereproduced in thewestern hemisphere as a byprod-uct of pulp, less than 2% of this amount wasisolated and sold. Kraft lignin, making up some95% of the totally produced lignin, is predomi-nantly burnt to cover the energy demands of kraftmills and the recovery of caustic soda in themill.Most of the commercially used kraft lignin issulfonated to water-soluble lignosulfonates. Ex-cluding the former Soviet Union, lignosulfonateproduction capacity in 1998 was estimated tobe about 975 000 t/a with some new capacitiesunder construction in South Africa [38].

The lignins are contained in the pulpingliquor as a mixture with other wood degradationproducts with strongly fluctuating compositiondepending on the process and the raw material.Typical compositions of pulping liquors fromhardwoods and softwoods obtained by the sul-fite and kraft process are summarized in Tables 2and 3 [39]. Kraft lignins are preferably obtainedby precipitation from “black liquors” with min-eral acids or carbon dioxide, whereas crude lig-nosulfonates are partly used directly as “spent

10 Lignin

sulfite liquors”. The isolation of lignosulfonatescan be achieved by addition of excess lime, bytreatment with a long-chain alkylamine and ex-traction, or by ultrafiltration. In order to producehigh-purity lignosulfonates a chemical destruc-tion of sugars, or fermentation treatments canbe included into the refinement procedure [38].The sulfur contents of lignosulfonates (4–8%)are higher than those of kraft lignins (1–1.5%).Due to the sulfonate groups the lignosulfonatesshow good solubility in water over the entirepH range but are insoluble in many organic sol-vents. Kraft lignins are not soluble in water butin alkali at pH > 10.5. They are as well solu-ble in many organic solvents. The molar massesof lignins are difficult to determine in a reliableway and often results in controversial discus-sion of data. Furthermore molar masses dependstrongly on the pulping conditions. Inmost casesthe molar masses of lignosulfonates are highercompared to kraft lignin and even values up to60 000 g/mol have been reported [40], althoughlower data are found for most samples [22, 23].For Kraft lignins the data are mostly below 10000 g/mol [22, 23, 39].

Table 2. Example for the composition of spent sulfite liquors fromsoftwood and hardwood [39]

Component Percentage of total solids

Softwood Hardwood

Lignosulfonate 55 42Hexose sugars 14 5Pentose sugars 6 20Acetic and formicacid

4 9

Resin and extractives 2 1Ash 10 10

Table 3. Example for the composition of kraft black liquors fromsoftwood and hardwood [39]

Component Percentage of total solids

Softwood HardwoodKraft lignin 45 38Xyloisosaccharinicacids

1 5

Glucoisosaccharinicacid

14 4

Hydroxy acid 7 15Formic acid 6 6Acetic acid 4 14Resin and fatty acids 7 6Turpentine 1Others 15 12

Kraft lignins contain larger amounts of phe-nolic hydroxyl, carboxyl, and catechol groupsthan lignosulfonates and are more likely to pos-sess some unsaturated side chain structures. Forcertain application lignins can be further mod-ified in order to improve the dispersing, com-plexing, or binding properties. These treatmentscan be sulfonation, sulfoalkylation, desulfona-tion, oxidation, carboxylation, amination, cross-linking, graft polymerization, and various com-binations of the previous methods. Most of thekraft lignins are subjected to sulfonation eitherwith sodium sulfite at 150–200 ◦C or oxida-tive with oxygen and sulfite. They can also besulfomethylated with sulfide and formaldehydeat temperatures around 100 ◦C. Lignosulfonatesaremostly obtained asmagnesium, calcium, am-monium, or sodium salts [38 – 41].Trade Names: A large variety of trade names

and products exists. However, most of theseproducts aremarketed by two companies,Mead-Westvaco Speciality Chemicals andBorregaard-LignoTech, where the first company is dominat-ing the kraft lignin and the latter the lignosul-fonate market. Some trade names are listed be-low:

Unsulfonated kraft lignin IndulinSulfonated kraft lignin Polyfon, ReaxLignosulfonates Ameri-Bond, Borresperse,

Borresol, Collex, Diwatex,Dustex, Dynasperse, Kelig,LignoBond, LignoSol,Marasperse, Norlig, Ufoxane,Ultrazine, Wafex, Wanin, Zewa

Soda lignin Protobind, Biosurfact

6. Analysis

Due to its complicated structure the analysis oflignin is very difficult. For the pulp industry theanalysis of lignin contents in pulps and raw ma-terials is specified, while for technical lignins nowell-defined industrial standards exist. For thesereasons it is difficult to obtain reliable, detailedinformation on technical products. Due to thissituation the following information can provideonly a small insight into the field of lignin anal-ysis. A review of most classical techniques forlignin analysis is given in [42].

Lignin 11

6.1. Detection and Quantification

The easiest methods to detect lignin in plant ma-terials are color reactions. However, those testscan be perturbed by some polyphenolic plantextractives. In the Wiesner reaction the mate-rial is treated with phloroglucinol in hydrochlo-ric acid, leading to a purple conjugated quinone[43], formed between phloroglucinol and cin-namyl aldehyde groups in lignin. The reactionis positive with all lignins, but is weak withhardwood lignins with high contents of syringylunits.

For the Maule test [44] lignified material istreated with a 1% potassium permanganate so-lution, followed by washing, a treatment with12% hydrochloric acid, and a final moisteningwith aqueous ammonia. With hardwoods an in-tensive purple-red color is produced, while withsoftwood lignins the color is an indefinite brown-ish shade. The chromophores are probably chlo-rinated quinones.

The quantitative determination of lignin inplants most commonly used, the Klason proce-dure, is based on the observation that celluloseand hemicelluloses are hydrolyzed by concen-trated sulfuric acid to soluble sugars while ligninis condensed to an insoluble cross-linked poly-mer which is recovered by filtration on a filtercrucible and can be gravimetrically determined.This procedure is a standard test for raw materi-als in the pulp industry [45].

Another method for the quantitative determi-nation of lignins in wood uses the solubility ofwood in a mixture of acetyl bromide and aceticacid [46]. After removal of the reagent, the ab-sorbance of the resulting solution is measuredat 280 nm. A detailed overview on all analyticalmethods for the determination of lignin in rawmaterials as well as in pulps is given in [47]. Acritical comparisonof severalmethods includingnot only wood and straw but various agriculturalresidues was presented in [48].

6.2. Isolation

The complete isolation of unchanged ligninfrom wood is not possible, due to its intimatemerging with cellulose and hemicelluloses inthe secondary walls of wood cells. Lignin has

to be degraded before extraction and accord-ingly the structure depends on the conditionsof isolation. The most common procedure forstructural and analytical purposes is produc-tion of milled wood lignin (MWL), also calledBjorkman lignin [49]. In the isolation procedurepreextracted, dry wood meal is milled for, e.g.,30 days in a vibratory ball mill under a non-swelling solvent (toluene). In the subsequent ex-traction step with dioxane –water (9:1) up to50% of the lignin can be isolated from coniferwood which can be further purified in order toreduce carbohydrate impurities. Further meth-ods for the isolation of lignins from pulps andpulping liquors are summarized in [42].

Recently it has been proposed to dissolveball-milled cell wall material in dimethyl sulf-oxide and tetrabutylammonium fluoride or N-methylimidazole. The product mixture of ligninand polysaccharides can be directly acetylated inthe solution and allows further analyses, e.g., by2D NMR techniques. Although this is no isola-tion procedure in the strict sense it can serve thesame purposes for analytical techniques. A ma-jor advantage is that a higher lignin yield than intraditional isolation procedures is obtained [50].

For pulp fibers a method has been developedwhich is based on the enzymatic saccharificationof polysaccharides and allows recovering of theundissolved lignin residue from the aqueous so-lution [51].

6.3. Spectroscopic Methods

UV Spectroscopy. The aromatic structure oflignin gives rise to a strong absorptionmaximumat 280 nm, while wood carbohydrates are trans-parent in the near-ultraviolet range. As, accord-ing to the Lambert – Beer law, the absorption ofUV light by dissolved lignin is proportional toits concentration, UV absorption can be used forquantitative determination of lignin in solutionor as a detection method in liquid chromatogra-phy [52]. In alkaline solution, the UV spectrumof lignin shows a bathochromic shift and hyper-chromic effect, due to the formation of phenolateanions. An alkaline ionization difference spec-trum (∆εi) is obtained by subtracting the UVspectrum of the neutral solution from that of thealkaline solution. ∆εi -Spectra are often used toobtain information on the amount of phenolic

12 Lignin

hydroxyl groups. These spectra show as wellspecific absorption peaks for conjugated (car-bonyl and olefinic) groups in lignin, which thuscan be determined semiquantitatively [53, 54].The UV absorption can be used also in the solidstate forUVmicroscopyonmicrotomecuts (Fig.1).

FTIR spectra of lignins are usually mea-sured on finely ground solid samples dispersedin potassium bromide pellets. In solution thelignin spectra are influenced by strong interfer-ing absorption bands of the solvents, i.e., semi-quantitative work can be carried out better withKBr pellets using controlled pressing conditionsand weight ratio of lignin to potassium bromide.The assignment of infrared absorption bands oflignins has been reviewed in [55] and [56].

NMR Spectroscopy. Of all physical meth-ods, NMRspectroscopy provides by far themostcomplete information on the chemical structureof lignins. The assignment of some 40 signals inthe 1H broad-band decoupled carbon-13 NMRspectra of spruce and beech MWLs and DHPsas well as their acetates has been achieved inthe early 1970s by comparison with the spec-tra of more than 50 lignin model compounds[57]. By now 1H and 13C shifts for a varietyof model compounds have been determined anda large database is provided by the U.S. DairyForage Research Center in Madison, Wiscon-sin. This database is continuously updated andavailable free of charge in the internet [58]. Al-though solid-state NMR (13C cross-polarizationmagic angle spinning (CP-MAS) NMR) spec-tra of lignin have been frequently recorded [59],the solution NMR techniques have by far thehigher importance due to their higher resolu-tion. One-dimensional high resolution 1Hor 13Cspectra of lignin can be obtained directly or af-ter acetylation. The acetylation can improve theresolution of spectra and provide as well infor-mation on the primary, secondary, and phenolicOH groups from the carbonyl signals of the cor-responding esters. For a good signal-to-noise ra-tio extreme longmeasurement times are requiredfor 13C spectra [60]. Here the short measure-ment times of 1H spectra are advantageously,although the information is more limited. Be-sides the methoxy signals, a differentiation ofphenolic and aliphatic hydroxyl groups, and in-

formation on the protons in aromatic and vari-ous aliphatic structures can be obtained. As analternative 31P and 19F NMR spectra can berecorded with good sensitivity and short mea-surement times after labeling reactions with Pand F containing reagents. The resulting spectracannot provide information on the entire ligninstructure, but on different hydroxyl, aldehyde,keto and quinone groups [61, 62].

The large number of signals in the carbon andproton spectra results in an intense overlappingof signals. This can be resolved by heteronuclearsingle- andmultiple-bond shift correlation spec-trawhich provide 2Dplots for proton and carbonshifts. Under normal operation conditions 2Dspectra are not suitable for quantification. How-ever, various approaches have been developed inthe early 2000s in order to enable a quantitativedetermination of lignin either by a calibration ofthe 2D spectra [63] or by a remodeling of thepulse sequences [64]. Also 3D spectra of ligninhave been recorded [65, 66].

6.4. Degradation Techniques

Many methods for the determination of inter-monomer bonds of lignin are based on analyti-cal degradation procedures. The degradation byalkaline solutions of potassium permanganatewas used intensively by Freudenberg’s groupin the 1950s and 1960s and further modified byMiksche and coworkers. Accordingly differentvariations of the method exist and are reviewedin [61]. The protocol consists of several reactionsteps and result in mono- and dimeric aromaticcarboxylic methyl esters, which can be analyzedby gas chromatography. This shows not only theproportion of hydroxyphenyl, guaiacyl, and sy-ringyl units but allow calculation of the abun-dance of different lignin substructures on a mo-lar basis. The procedure is applicable to all kindof lignins but has the disadvantage of a rathercomplicated multi-step protocol which requiresan elaborated data treatment in order to concludeon the original lignin structure [68].

The thioacidolysis of lignin encompassesmainly the cleavage of β-O-4 linkages withboron trifluoride etherate and ethanethiol inanhydrous media. The monomeric degradationproducts can be analyzed by gas chromatogra-phy and normally between 1000 to 3000 µmol/g

Lignin 13

of mono- and dimeric degradation products areobtained. The protocol is accordingly simplerand allows an evaluation of the amount of alkyl-aryl ethers in lignin samples. A disadvantageis the strong odor of the reagents and the factthat the method is less informative on degradedsamples, which is sometimes the case with sometechnical lignins [68, 69].

Derivatization followed by reductive cleav-age (DFRC) was introduced as a new degrada-tion method for lignin analysis [70]. Lignin isderivatized with acetyl bromide, followed by areductive cleavage with zinc dust in a mixtureof dioxane, acetic acid, and water (5:4:1). Theresulting products are then acetylated and ana-lyzed by gas chromatography. In the DFRC pro-cedure hydroxyphenyl, guaiacyl, and syringylunits linked by α- and β-aryl ethers are cleavedand transferred into 4-acetocinnamyl acetate,coniferyl diacetate, and sinapyl diacetate.

The pyrolysis of lignin or lignocellulosic ma-terials, i.e., the thermal degradation in the ab-sence of oxygen, leads to the formation of alarge variety of volatile components, which con-tain information on the original structure. In an-alytical pyrolysis this degradation technique iscombined online or offline with other analyti-cal techniques and most importantly with massspectrometry (Py-MS) or gas chromatographyandmass spectrometry Py-GC/MS. In the GC orGC/MS analysis of pyrolysis degradation prod-ucts more than 100 peaks are found. Accord-ingly, the evaluation requires a large databaseand is based on statistical evaluation methods.Information on various pyrolysis techniques isfound, e.g., in [71, 72]. A general review onMS techniques in combination with all relevantlignin degradation techniques can be found in[73].

6.5. Molar Mass Determination

Size-exclusion chromatography (SEC) is themost common method to determine molarmasses of lignins. Frequently, lignins wereacetylated followed by SEC in THF usingcolumns based on polystyrene – divinylbenzene[74]. However, in various round robin testscarried out by the World Energy Associationand the European network Eurolignin this ap-proach resulted in a tremendous variation of

the calculated molar masses [75, 76]. Thisproblem might be partly due to aggregationphenomena and partly due to interaction withthe column material. Furthermore, the acetyla-tion is not suitable for lignins containing car-bohydrate impurities and for lignosulfonates,the most important technical lignins. Ligno-sulfonate samples have been recently investi-gated using a complexmixture of water, DMSO,sodium phosphate buffer, and sodium dodecylsulfate [40]. A universal eluent system applica-ble to all technical lignins is sodium hydrox-ide, however, the handling is not so easy be-cause most commercial prepacked columns forHPSEC cannot withstand the high alkalinity[74]. RecentlyDMSO–water – lithiumbromideand dimethylacetamide – lithium chloride wereapplied as eluents, showing a good agreementwith alkaline SEC [23]. The determination ofabsolutemolarmasses by light scattering is com-plicated by the fluorescence of lignin, while vis-cosimetric methods suffer from the low overallviscosity of the samples. All methods are af-fected by the impurities of technical samplesespecially by carbohydrates and ash. Recentlymatrix-assisted laser desorption ionization/timeof flight (MALDI/TOF) was applied to ligninsafter a fractionation of the samples on SECcolumns in order to obtain fractions with nar-row polydispersity [77].

7. Uses

Most applications of lignin and lignosulfonatesare based on their dispersing, binding, complex-ing, and emulsion-stabilizing properties. About50% of all lignosulfonates produced worldwideare used for concrete mixtures, usually in theformof calciumor sodium salts. Addition of 0.1-0.3% lignosulfonates to cement retards the set-ting or hydration of concrete. The second mostimportant application is as a binder for animalfeed pellets, where mainly calcium and ammo-nium salts are applied in order to improve pel-let durability and abrasive resistance. A maxi-mum dosage of 4% is possible in finished pel-lets. Primarily chrome and ferrochrome salts oflignosulfonates function in oilwell drillingmudsas mud thinners, clay conditioners, viscosity-control agents, and fluid-loss additives. Mud

14 Lignin

Table 4. Data on the toxicity of different lignosulfonates [38]

Property Calcium lignosulfonate Sodium lignosulfonate Ammonium lignosulfonateAcute toxicity LD50, mg/kg > 2 000 > 10 000 > 10 000Eye irritant no no noSkin irritant no no noFish toxicityLC50, mg/L > 1 000 > 1 000 > 1 000Bacteria toxicity EC10, mg/L 5 000 5 000 343

systems conditioned with 0.2–0.5% lignosul-fonates, applied in the crude oil industry, per-form well at high pressures and at temperaturesof up to 175 ◦C. Another major market, espe-cially for crude lignosulfonate spent liquors, isthe dust control application for stabilizing in-surfaced roads. In addition to these bulk applica-tions a variety of specialitymarkets exist. Ligno-sulfonates are used for the granulation, complex-ation, or encapsulation of pesticides and as addi-tives for gypsum boards in order to disperse thestucco. Lignosulfonates are used as dispersantin water-based paints and inks. Lignosulfonatesand spent sulfite liquors are used for water treat-ment plants and paper machines to reduce de-posits and slime formation or to complex metalssuch as zinc in cooling-water cycles. They areused in industrial cleaner formulations, for thecomplexation of nutrients in soil stabilization,for dyes or as additives in the brick industry.Modified lignosulfonates are additives in lead –acid batteries, which extend the service life ofthe product significantly. As further future usesthe stabilization of enzyme formulations, bio-cide neutralization, and applications exploitingthe antiviral and the chelating properties havebeen targeted [38].

Borregaard LignoTech still produces vanillinfrom softwood lignosulfonates, while all otherproducers closed down their production due tolower costs for vanillin production from petro-chemical feedstocks. This was also the reasonfor stopping the production of dimethyl sulf-oxide (DMSO) from kraft lignin by heatingblack liquor with sulfur to above 200 ◦C to ob-tain dimethyl sulfide (DMS), which upon oxi-dation with dinitrogen tetroxide (N2O4) yieldedDMSO [78].

Up to now,muchwork has been carried out onnew utilizations of lignin (see for instance [79]).Only few applications are economically feasi-ble, taking into account that lignin is currentlyburnt, covering not only the energy demand ofthe pulp mills but providing a significant surplus

of energy. Sometimes the argument occurs thatthe utilization of lignin as a chemical feedstockwill increase at higher oil prices. However, onehas to keep in mind that the economic burningvalue of lignin is increasing as well when theoverall energy costs are rising. Accordingly, thechanges for implementing new applications arehigher when lignin is used not as a bulk chem-ical but as a specialty product using its specificproperties.

8. Toxicology

Lignosulfonates are considered as nontoxic atthe typical use levels and they are approved,e.g., by the U.S. Food and Drug Administra-tion for a variety of applications in food packag-ing and food production. Examples are the useas a boiler-water additive for the production ofsteam, which comes into contact with food (21CFR 173.310), or the approval of calcium lig-nosulfonates as a “specific usage additive” forthe regulations on food additives for direct ad-dition to food for human consumption (21 CFR172.172). Some data for various lignosulfonatesare listed in Table 4 [38]. However, one shouldkeep in mind that the properties of a specificproduct are not only influenced by its lignosul-fonate component but by its impurities.

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