M. Lawoko et al.

: Quantitative Preparation of Lignin-Carbohydrate Complex from Kraft Pulp 69

Holzforschung
57 (2003) 69–74

New Method for Quantitative Preparation of

Lignin-Carbohydrate Complex from Unbleached
Softwood Kraft Pulp: Lignin-Polysaccharide Networks I
By Martin Lawoko, Gunnar Henriksson and Göran Gellerstedt
Department of Fiber and Polymer Technology, Royal Institute of Technology, Stockholm, Sweden

Key words Summary

Lignin-carbohydrate A new method for the quantitative preparation of pulp representative lignin-carbohydrate com-
complex (LCC) plexes (LCC) has been developed, in which LCC has been systematically prepared at quantitative
Endoglucanase yield, fractionated and qualitatively determined. At least 90% of residual lignin in softwood kraft
Residual lignin pulp is proposed to be chemically bonded to carbohydrates. A major part of LCC (92%) in soft-
wood kraft pulp was observed between lignin, xylan and glucomannan, whereas a minor part (8%)
was linked to cellulose. Half of the hemicelullosic LCC is a lignin-glucomannan complex. The oth-
er half is lignin-xylan complex and xylan-lignin-glucomannan complex. Thus, part of the residual
lignin in softwood kraft pulp crosslinks xylan and glucomannan. The proposed linkages are of co-
valent type. At most 10% of the residual lignin is not bonded covalently to carbohydrates.

Introduction therefore hydrolysed during the kraft pulping process.

Covalent linkages between lignin and polysaccharides,
traditionally known as lignin-carbohydrate complexes
(LCC), have been suggested to exist in native wood or
to be formed during the pulping process. From an indus-
trial perspective, there is reason to believe that the
rather sluggish delignification, loss in yield during the
residual delignification phase and the difficulties en-
countered in the bleaching process (at least in TCF-
bleaching) are partly attributable to the stability of LCC
in pulp. Detailed information about the quantity and
quality of these complexes can be used to modify the
pulping process and to improve the bleachability of
pulp. Preparation of LCC for characterisation is a prob-
lem since some of the chemical bonds might be sensitive
to certain treatments and the fibre morphology is com-
plex, at least for native wood. Both chemical and enzy-
matic methods have been used to isolate LCC (Pew
1957; Chang et al. 1975; Obst 1982) and both have draw-
backs and advantages. However, it is generally believed
that enzymatically isolated lignin is more representative
of native lignin than the chemically or mechanically iso-
lated lignin. Enzymatically isolated native lignin has
been suggested to contain 5–10% chemically-linked
carbohydrates (Lai and Sarkanen 1971; Lundquist et al.
1983). The most frequently suggested linkages in native
LCC are benzyl esters, benzyl ethers and phenyl glyco-
sidic linkages. The benzyl ester is alkaline labile and is
The latter two are alkaline stable and would therefore
survive the pulping process (Minor 1986). Methylation
analysis has shown bonds between lignin and sugars in
enzymatically isolated lignin (Iversen and Wännström
1986), model compound experiments support the possi-
bility of LCC being formed during the pulping process
(Gierer and Wännström 1986). Studies have revealed
associations between lignin and cellulose for pine kraft
pulp (Karlsson and Westermark 1996) and lignin-hemi-
cellulose complexes for birch kraft pulp (Karlsson and
Westermark 1994). Lignin-xylan linkages for birch kraft
pulp and cellulose, mannan and xylan complexes in pine
kraft pulp (Tenkanen et al. 1999) have been suggested.
However, there is much controversy concerning the
methods used in isolation and the results obtained. To
obtain reliable and representative information, the
method used to prepare LCC is a vital factor. There is
very little concrete quantitative information. It is conse-
quently important that the results obtained are quanti-
tatively and qualitatively representative of wood/pulp.
Possible network formation by LCC has not been stud-
ied because degradation techniques have been used in
most of the previous studies.
When the lignin is isolated chemically, an almost pure
fraction is obtained (Gellerstedt et al. 1994), however,
the yield is low (50–60%). The hemicelluloses are de-
graded in the process and there is a risk that chemical
reactions could modify the substrate. Therefore, acid hy-

Holzforschung / Vol. 57 / 2003 / No. 1

© Copyright 2003 Walter de Gruyter · Berlin · New York

Brought to you by | Bibliotheque de l'Universite Laval

Authenticated | 132.203.227.62
Download Date | 7/1/14 1:25 AM
70 M. Lawoko et al.: Quantitative Preparation of Lignin-Carbohydrate Complex from Kraft Pulp

drolysis is not suitable for the isolation of lignin-carbo-

hydrate complexes (LCC). Commercially available cel-
lulase mixtures from softrot and mould fungi are com-
monly used. These cellulase mixtures not only contain
several types of synergistically acting cellulases but also
other enzymes such as hemicellulases and pectinases.
The product of enzyme hydrolysis is an insoluble lignin
residue. This product is, however, contaminated with
carbohydrate residues and protein (which has a high
affinity for lignin) and there is a risk of losing the hemi-
cellulose-lignin complexes, since low molecular weight
hemicellulose products (oligosaccharides), including
those to which lignin is bound, could solubilize. The lat-
ter problem could be resolved by using specific cellulose
hydrolysing enzymes followed by chemical extraction
methods. In this work, we present a method in which
LCC representative of softwood kraft pulp is prepared,
and quantitatively and qualitatively characterized.

Materials and Methods

Laboratory prepared unbleached pine kraft pulp (kappa num-
ber 15) was used. Mono-component endoglucanase (Novozym
476) was a kind gift from Novozyme (Bogsvaerd, Denmark).
Carboxymethyl cellulose was purchased from Fluka Biochem-
ica. Locust bean gum mannan and birchwood xylan were pur-
chased from Sigma (St. Louis, USA). All other chemicals
where of analytical grade.

Activity measurements
The endoglucanase was tested for hemicellulase activity on
1% polysaccharide suspensions (carboxymethyl cellulose, xy-
lan and mannan) in a pH 6 buffer system. The substrates were
incubated with enzyme for 30 min. As control, the above pro-
cedure was also performed on substrates without inclusion of
enzyme. Released sugars were detected by DNS reagent
(Miller 1959) (dinitrosalicylic acid, 1% NaOH, 10% NaK tar-
tate, 0.05% Na2SO3 and 0.2% phenol). Glucose standards
(concentration range 0.5–4 mM) were used for calibration.
Addition of DNS reagent to samples and standards and subse-
quent boiling (5 min), initiates the DNS- released sugar reduc-
tion reaction. The UV-absorbance of the cooled solutions is
then measured at 575 nm. The activity is calculated as catalysis
per minute. (Mol of liberated sugars/moles of enzyme) × (1/in-
cubation time).
Enzyme hydrolysis and fractionation of pulp components
Unbleached softwood kraft pulp (kappa number 15) was sub-
jected to enzyme hydrolysis using a mono component endoglu-
canase (Novozym 476). The partial pulp hydrolysis was per-
formed at a 5% pulp consistency and at pH 6 using a 10 mM
bistris buffer system at 50 °C, for 48 h. 3.2 ml enzyme /g pulp
was used.
The resulting hydrolysate was centrifuged and the residue
properly washed with water. The obtained residue (residue 1 in
Fig. 1) was washed with 8 M urea solution at pH 8.8 (80 ml/g
pulp) overnight with stirring at room temperature, centrifuged
and was properly washed with water (>100 ml/g pulp). The
residue (residue 2 in Fig. 1) was treated with an alkaline borate
solution (18% NaOH with 4% H3BO3) with stirring and at
room temperature for 4 h. The solution obtained after centrifu-
gation (solution 3 in Fig. 1) was subjected to a pH fractionation
whereby two fractions were obtained. The first fraction was
formed as a precipitate at pH~12, and the other remained in
Fig. 1. Extraction and fractionation scheme used for the pre-
paration of LCC.
solution over the whole pH range (14–1). For future analyses,
however, the pH of the final solution was only reduced to ~7,
since low pH could lead to modification and bond cleavage.
The residue after the alkaline borate treatment (residue 3 in
Fig. 1) was washed with water until the pH of the wash water
was neutral, and a repeat of the whole procedure described
above was performed on the residue (residue 3 in Fig. 1). A to-
tal of four loops (each loop represented by loop B in Fig. 1)
where performed. To the neutral fraction, twice its volume of
5% aqueous barium hydroxide was added over a 3-h period.
The precipitate (precipitate 2 in Fig. 1) formed was recovered
on the centrifuge, dialysed overnight and freeze-dried. The su-
pernant after centrifugation was shaken with 50% aqueous
acetic acid and poured into 4 times its volume of ethanol. The
obtained precipitate (precipitate 3 in Fig. 1) was recovered on
the centrifuge, dialysed and freeze-dried. The supernant (solu-
tion 5 in Fig. 1) was dialysed and freeze-dried.
Analyses
Nitrogen analysis was performed by Mikro Kemi AB on 1) the
endoglucanase treated and water washed material (residue 1
in Fig. 1) and 2) urea treated and water washed material
(residue 2 in Fig. 1). Part of the alkaline borate dissolved mate-
rial (solution 3 in Fig. 1) was dialysed and freeze-dried. The in-
trinsic viscosity of this material was determined according to
SCAN-CM 15:99. Carbohydrate analyses were performed
(Theander and Westerlund 1986) on the starting material
(pulp in Fig. 1), Residue 3, pH 12 fraction and neutral fraction,
barium hydroxide precipitated fraction, ethanol precipitated

Holzforschung / Vol. 57 / 2003 / No. 1

Brought to you by | Bibliotheque de l'Universite Laval

Authenticated | 132.203.227.62
Download Date | 7/1/14 1:25 AM
M. Lawoko et al.: Quantitative Preparation of Lignin-Carbohydrate Complex from Kraft Pulp 71

fraction and the ethanol unprecipitated part (all shown in
Fig. 1). Dissolved lignin was monitored by UV-Vis spectrome-
try and absorbance value at 280 nm used for quantitative cal-
culation. The mass balances were monitored by gravimetry.
Results and Discussion
Activity measurements
It is evident from the results shown in Table 1 that hemi-
cellulase activity is minimal as was expected. The activi-
ty towards carboxymethyl cellulose is expected from an
endoglucanase.
Enzyme hydrolysis and urea treatment
The enzyme hydrolysis of pulp was performed at a tem-
perature of 50 °C and pH 6. These conditions are mild
and as such, the modification of pulp components dur-
ing the hydrolysis is unlikely.
After the enzymatic hydrolysis, the residue was
washed with water. The resulting residue (residue 1 in
Fig. 1) was analysed for nitrogen. The obtained N-value
was converted by calculation to protein content (N-val-
ue × 6.25). Pulp contamination by enzyme was 7.5%.
This value suggests that 30% of the enzyme adsorbs
onto the residual material after the enzyme hydrolysis.
The 8 M-urea solution has a threefold effect on the pulp.
Firstly, it effectively removes enzyme contaminant from
pulp after the enzyme hydrolysis. The contamination at-
tributable to enzyme in the residual pulp material after
the urea and subsequent water wash (residue 2 in Fig. 1)
was as little as 1.2%. This is calculated from the results
obtained from the nitrogen analysis (N-value × 6.25).
However, it must be pointed out that this value could
Table 1. Substrate specificity of endoglucanase measured as
catalysis per minutes. DNS assay used for detection
Substrate Activity (min– 1)
Carboxymethyl Cellulose 112.25
Birchwood Xylan 1.28
Locust Bean Mannan 4.22
represent an overestimation of the protein content be-
cause urea contains nitrogen as well and could con-
tribute to the obtained N-value. The second function of
the urea solution is that it swells the pulp into a gel (in
contrast to untreated fibres). When observed under a
microscope, the pulp material had a different form as
compared to the rather characteristic fibril structure
seen in untreated kraft pulp. Thirdly, it dissolves any
lignin that is not covalently linked to polysaccharides. It
was observed that when pulp was maximally hydrolysed
using a cellulase mixture (culture filtrate containing
both cellulases and hemicellulases), some lignin was dis-
solved when the urea solution was applied. It is there-
fore not recommendable to use the urea solution as an
enzyme contaminant remover from maximally hydrol-
ysed pulps since a loss in lignin yield is observed. It is
most unlikely that the urea chemically derivatises the
pulp material, since the effect of derivatisation would
have been expressed as a high nitrogen content (high N-
value) on urea-treated pulp. The urea solution, there-
fore, serves a physical purpose and is not chemically in-
corporated into the pulp. However, when pulp which
was not incubated with endoglucanase was treated with
urea solution, no changes on the material were ob-
served. It is therefore plausible that the swelling effect
of urea on the fibril structure is facilitated by a low de-
gree of polymerisation of cellulose.
Alkaline borate extraction and fractionation of pulp
components
After the urea treated material was thoroughly washed
with water to remove any traces of urea, the residue
(residue 2 in Fig. 1) was treated with a solution contain-
ing 18% NaOH and 4% boric acid. This solvent system
was originally designed for the extraction of glucoman-
nans from holocellulose (Jones et al. 1956). Softwood
pulps are known to have a higher content of glucoman-
nans than hardwood pulps. The poor solubility of soft-
wood pulps in most known cellulose solvent systems is
partly attributed to the high content of glucomannans in
the latter. The alkaline borate solvent has, however,
been observed to dissolve the glucomannans. The mech-
anism of this dissolution has been suggested to involve

Table 2. The relative amounts of reduced sugars from different fractions in the extraction in Figure 1

Sample L Xyl Man Gal Glu Ara

P 0 9.18 7.58 0.53 81.6 1.12

pH12 1 3.23 4.64 – 91.5 0.63
Neut 1 42.5 40.0 1.72 11.9 3.90
pH12 2 1.15 1.21 – 97.2 0.39
Neut 2 29.1 43.7 4.19 19.4 3.55
Res 4 0.27 0.48 0.22 99.0 –
BaOH precipitate 1+2 4.92 69.71 4.41 19.41 1.59
Ethanol precipitate 1+2 49.52 34.2 0.89 12.21 3.19
Ethanol soluble 1+2 58.72 20.32 – 16.48 6.49

P = pulp in scheme, L = loop number, pH12 = fraction precipitated at pH12, Neut = the unprecipitated neutral fraction. Res = the
residual material left after 4 loops.

Holzforschung / Vol. 57 / 2003 / No. 1

Brought to you by | Bibliotheque de l'Universite Laval

Authenticated | 132.203.227.62
Download Date | 7/1/14 1:25 AM
72 M. Lawoko et al.: Quantitative Preparation of Lignin-Carbohydrate Complex from Kraft Pulp

Table 3. Mass balance of the fractions in Figure 1 solved components are thus fractionated by pH-gradi-
ent as shown in Figure 1. Two fractions are obtained,
L no 1 2 3 4 5 one that is precipitated at pH 12 and another that is not
start 20 13 6 3 1
precipitated over the whole pH-range (14–1). However,
Tot lig 0.45 – – – – to avoid possible modification at low pH values, the fi-
res2 20 10 4.2 1.5 – nal pH of the second fraction was only reduced to 7 pre-
sol2 0 3 1.9 1.5 – analysis. This fraction contained most of the dissolved
Uv-lig sol3 0.3 0.1 .01 – – lignin.
sol3 6 3.9 1.2 0.3 –
pH 12 0.3 3 1 0.3
neut 5.6 0.9 0.1 – – Carbohydrate analysis and mass balance
k-lig pH 12 .03
k-lig neut .37 In general, from the carbohydrate analyses (Table 2), it
is observed that the two fractions from the pH-fraction-
L no = loop number, start = amount of pulp material at begin- ation step can be seen as a hemicellulose fraction with
ning of every loop, res2 = pulp material in residue 2, sol2 = pulp xylan and glucomannan being the main components
material in solution 2, Uv-lig sol3 = lignin content in solution 3 (neutral fraction in Fig. 1) and a cellulose fraction (frac-
calculated using absorbance at 280nm, sol3 = pulp material in
tion precipitated at pH 12, Fig. 1). More specifically, as
solution 3, pH12 = pulp material in the pH 12 precipitated frac-
tion, neut = pulp material in the neutral fraction, k-lig pH 12 = the loop number increases, the purity of cellulose in the
total klason lignin of all pH 12 fractions, k-lig neut = total kla- pH 12 fraction increases. It is also observed that nearly
son lignin of all neutral fractions. All amounts are in grams. all the xylan and mannan are extracted in the earlier
loops. In fact, combining the carbohydrate analysis re-
complexion between the borate anion and the cis-hy- sults with the mass balance, it is quite evident that nearly
droxyl groups of the mannose units. Such complexes are all the hemicelluloses together with a small amount of
more acidic than the non-complexed glucomannans and cellulose are extracted by the alkaline borate solvent
would thus be more soluble in alkali. system in the first loop. This result supports suggestions
In our work, this solvent system is applied to enzyme that dissolved hemicelluloses, especially xylan in the
hydrolysed, urea swelled pulp, which, unlike holocellu- black liquor, are re-precipitated on the fibre surfaces
lose, contains residual pulp lignin. The extractability of during the kraft cook (Yllner and Enström 1956). As
this solvent together with the mass balances of the dif- such, the limited extraction of cellulose in the first loop
ferent fractions in Figure 1, are represented in Table 3. of the extraction scheme (Fig. 1) could be due to its inac-
From the viscosity measurement, the average degree of cessibility. Only when the hemicelluloses are extracted
polymerisation (DP) was determined (Evans and Wallis will the quantitative extraction of cellulose begin. The
1989) to be ~200. A UV-Vis spectrum of the alkaline bo- alkaline borate solvent system dissolves cellulose, xylan,
rate solution (solution 3 in Fig. 1) was taken and the ab- glucomannan and lignin alike. Recalling that the cellu-
sorbance value at 280 nm was used to calculate the lose is severely degraded (DP~200), this observed disso-
lignin content. Results showed that a significant amount lution pattern implies that that the reactivity of cellulose
of lignin was dissolved. The alkaline conditions together begins to resemble that of hemicelluloses when its mo-
with the high temperatures involved in the kraft pulping lecular weight approaches that of the latter. More than
process are harsh. The lignin-carbohydrate complexes 90% of the pulp lignin was dissolved in alkaline borate
(LCC) have survived such conditions or have in some solution in a total of 3 loops (Table 3). Interestingly,
cases possibly been formed under such conditions. It is >90% of the alkaline borate dissolved lignin was in the
therefore unlikely that the conditions under which the neutral fraction (i.e. the xylan and glucomannan frac-
alkaline borate extraction is performed in this work tion). Since the hydrophobic nature of isolated residual
could lead to cleavage of LC-bonds or modification of lignin is known, this result is a strong indicator of cova-
the pulp components. The somewhat higher alkalinity in lent bonds between lignin and the hemicelluloses. It is
this case is compensated for by the significantly lower unlikely that lignin would dissolve in aqueous solvent if
working temperatures (room temperature) as com- it were attached to the hemicelluloses by physical forces.
pared to 170 °C in the kraft process. Thus, with the ex- Moreover, it is known for black liquor that alkali dis-
ception of the partial hydrolysis of glycosidic bonds in solved lignins are precipitated at a pH <10 when the
cellulose during the enzyme hydrolysis, the urea wash phenolate anions are protonated to phenols. The reason
and alkaline borate treatment do not chemically modify for this rather untypical behaviour of pulp lignin in
the pulp components. As such, the alkaline borate solu- aqueous solvent is that it is chemically bonded to the
tion is qualitatively representative of pulp. comparatively more hydrophilic hemicelluloses. There
was no precipitation observed even when the ionic
strength of the solution was eliminated by dialysis. To
pH-fractionation
confirm the presence of a chemical bond, a simple ex-
The working hypothesis with pH-fractionation is based periment was carried out. Dioxane:water (9:1 v:v) is
on the idea that each pulp component is unique with re- known to be a solvent for pure pulp lignin (Gellerstedt
spect to structure and reactivity. The alkaline borate dis- et al. 1994). This solvent system was applied on the origi-

Holzforschung / Vol. 57 / 2003 / No. 1

Brought to you by | Bibliotheque de l'Universite Laval

Authenticated | 132.203.227.62
Download Date | 7/1/14 1:25 AM
M. Lawoko et al.: Quantitative Preparation of Lignin-Carbohydrate Complex from Kraft Pulp
Table 4. Shows the distribution of lignin in the fractionated
components
Fraction Main pulp % of total
components pulp lignin
in fraction in fraction
Urea soluble fraction Lignin 10
pH12 precipitate Cellulose 8 used for other pulp cellulose related studies. It is also
BaOH precipitate Glucomannan 42
Ethanol precipitate Xylan* and 28
Glucomannan
Ethanol soluble part Xylan* and 12
Glucomannan
* represents the dominant component in fraction for the cases
where two components are given.
nal pulp and on the urea-swelled material (residue 2 in
Fig. 1) at room temperature with stirring overnight. No
lignin was extracted in either case. Recalling that the
crystalline structure of cellulose has been destroyed and
replaced by a more swollen, solvent accessible structure
by the urea treatment, it would be expected that if there
was any pure lignin fraction left, such material would
quite easily be extracted by the dioxane/water system.
Moreover, our experiments suggest that urea is a good
solvent for free pulp lignin (Table 4). It is therefore rea-
sonable to state that at least 90% lignin in softwood
kraft pulp is chemically bonded to carbohydrates. The
total lignin in each fraction was calculated from the kla-
son lignin contents in the cellulose fraction and the xy-
lan + glucomannan fraction (Table 3) together with the
mass balances. It was found that 8% of the residual
lignin in softwood kraft pulp is chemically linked to cel-
lulose and 82% is bonded to xylan and glucomannan
(Table 4). Separation of glucomannan from a mixture of
hemicelluloses obtained from holocellulose in solution,
by aqueous barium hydroxide precipitation has been
published (Meier 1958). In that procedure, a pure gluco-
mannan fraction is obtained. In this work, when such a
fractionation was applied to the hemicellulose-lignin
complexes, the results were rather different. The lignin
analysis of the barium hydroxide precipitated fraction
(Table 4) shows that about half of the lignin in this frac-
tion is precipitated as well, suggesting that about half of
the hemicellulose-lignin complex is a glucomannan-
lignin complex. The unprecipitated part contains the
other half of the lignin together with most of the xylan
and a significant amount of glucomannan. This latter re-
sult implies that some part of residual pulp lignin
crosslinks glucomannan and xylan, since part of the glu-
comannan is not precipitated even when an excess of
aqueous barium hydroxide is added. Interestingly, al-
though the differences in lignin content between the
two fractions were small, the difference in colour be-
tween the two fractions was significant. The glucoman-
nan-lignin complex was much darker than the xylan
containing complex. Whether this is an indication of the
Brought to you by | Bibliotheque de l'Universite Laval
Authenticated | 132.203.227.62
Download Date | 7/1/14 1:25 AM
73
lignin structures involved in complexion being different
in the two fractions or that the kraft cook modified
structures in the glucomannan are responsible, is still
unclear. After four loops of the analytical scheme in Fig-
ure 1, it is observed that the residue is a cellulose materi-
al with high purity (99%). This residual material ac-
counts for 5% of the total pulp material and could be
observed that 30% of the total pulp material is lost dur-
ing the urea wash to solution 2 in Figure 1. This material
was determined by mass balance to be cellulose.
Conclusions
Lignin-carbohydrate complexes (LCC) were systemati-
cally prepared. Unique to this new method is the partial
enzyme hydrolysis of cellulose and subsequent swelling
which facilitates the dissolution of pulp components at
high yields. The reactivity of cellulose has been observed
to resemble that of hemicelluloses when its molecular
weight approaches that of the latter. Importantly, the
solutes are polymeric materials. The isolation procedure
involved is non-modifying to pulp components and the
fractionated components are nearly free from contami-
nation (at most 1.2% contamination from enzyme). A
high yield of residual lignin-carbohydrate complexes
(>90%) is obtained. At least 90% of the residual lignin
in kraft pulp is proposed to be covalently linked to car-
bohydrates. Of this, 92% is bound to xylan and gluco-
mannan and 8% is bound to cellulose. About half of the
hemicellulose-lignin complex is a glucomannan-lignin
complex. The other half comprises xylan-lignin and xy-
lan-lignin-glucomannan complexes. Thus, part of the
residual lignin is suggested to crosslink xylan and gluco-
mannan forming a network structure. A residue contain-
ing almost pure cellulose (99% purity) is obtained. This
residue accounts for 5% of the total material. Though
time consuming, this method provides a systematic
preparation and fractionation of pulp-representative
components. For lignin studies, a high yield (90%) is ob-
tained already after 2 loops of the scheme in Figure 1.
Acknowledgements
Financial support from Skogindustrins Forskningsstiftelse is
gratefully acknowledged. Thanks are due to Novozyme for
their gift of the enzyme Novozym 476.
References
Chang, H.-m., E.B. Cowling, W. Brown, E. Adler and G.
Miksche. 1975. Comparative studies on cellulolytic enzyme
lignin and milled wood lignin in sweetgum and spruce.
Holzforschung 29(5), 153–159.
Evans, R. and A.F.A. Wallis. 1989. Cellulose molecular weights
determined by Viscometry. J. appl. Polymer Sci. 37, 2331–
2340.
Gellerstedt, G., J. Pranda and E.-L. Lindfors. 1994. Structural
and molecular properties of residual birch kraft lignins. J.
Wood Chem. Technol. 14(4), 467–482.
Gierer, J and S. Wännström. 1986. Formation of ether bonds
Holzforschung / Vol. 57 / 2003 / No. 1
74 M. Lawoko et al.: Quantitative Preparation of Lignin-Carbohydrate Complex from Kraft Pulp

between lignin and carbohydrate fragments during alkaline
pulping processes. Holzforschung 40(6), 347–352.
Iversen, T. and S. Wännström. 1986. Lignin-carbohydrate
bonds in residual lignin isolated from pine kraft pulp. Holz-
forschung 40, 19–22.
Jones, J.K.N., L.E. Wise and J.P. Jappe. 1956. The action of al-
kali containing metaborates on wood cellulose. Tappi 39,
139.
Karlsson, O. and U. Westermark. 1994. Condensation reactions
between wood polymers during kraft pulping. Proc. TAPPI
Pulping Conf., San Diego, 1–4.
Karlsson, O. and U. Westermark. 1996. Evidence for chemical
bonds between lignin and cellulose in kraft pulps. J. Pulp
Paper Sci. 22, 397–401.
Lai, Y.Z. and K.V. Sarkanen. 1971. Isolation and structural
studies. In: Lignin – Occurence, Formation, Structure and
Reactions. Eds. K.V. Sarkanen, C. Ludwig. Wiley Inter-
science, New York. pp. 165–240.
Lundquist, K., R. Simonson and K. Tingsvik. 1983. Lignin car-
bohydrate linkages in milled wood lignin preparations from
spruce wood. Svensk papperstidn. 86(6), R44-R47.
Meier, H. 1958. Barium hydroxide as a selective precipitating
agent for hemicelluloses. Acta Chem. Scand. 12, 144–146.
Miller, G.L. 1959. Dinitrosalicylic acid reagens for the determi-
nation of reducing sugar. Anal. Chem. 31, 426–428.
Minor J.L. 1986. Chemical linkage of polysaccharides to resid-
ual lignin in loblolly pine kraft pulps., J. Wood Chem. Tech-
nol. 6(2), 185–201.
Obst, J.R. 1982. Frequency and alkali resistance of lignin-car-
bohydrate bonds in wood. Tappi 65(4), 109–112.
Pew, J.C. 1957. Properties of powdered wood and isolation of
lignin by cellulolytic enzymes. Tappi 40(7), 553–558.
Tenkanen, M., T. Tamminen and B. Hortling. 1999. Investiga-
tion of lignin-carbohydrate complexes in kraft pulps by se-
lective enzymatic treatments. Appl. Microbiol. Biotechnol.
51, 241–248.
Theander, O. and E.A. Westerlund. 1986. Improved procedure
for the analysis of dietry fiber. J. Agric. Food Chem. 34,
330–336.
Yllner, S. and B. Enström. 1956. Adsorption of xylan on cellu-
lose fibers during the sulphate cook. 1. Svensk Papperstidn.
59, 229–232.
Received September 3rd, 2001
Martin Lawoko
Gunnar Henriksson
Göran Gellerstedt
Royal Institute of Technology
Department of Fiber and Polymer Technology
Drottning Kristinas väg 53
100 44 Stockholm
Sweden

Holzforschung / Vol. 57 / 2003 / No. 1

Brought to you by | Bibliotheque de l'Universite Laval

Authenticated | 132.203.227.62
Download Date | 7/1/14 1:25 AM