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Holzforschung, Vol. 65, pp. 601604, 2011 Copyright
by Walter de Gruyter Berlin Boston. DOI 10.1515/HF.2011.102

Simplified determination of total lignin content in kraft lignin

samples and black liquors
11th EWLP, Hamburg, Germany, August 1619, 2010

Fredrik Aldaeus*, Hannah Schweinebarth,

Per Torngren and Anna Jacobs
Innventia AB, Stockholm, Sweden
*Corresponding author.
Innventia AB, Box 5604, SE-114 86 Stockholm, Sweden
Phone: q46-8-6767188
E-mail: fredrik.aldaeus@innventia.com

Abstract
Determination of total lignin content in lignin samples and
black liquors using methods with reduced or omitted acid
hydrolysis has been compared to the standard method, and
results show that the simplified methods yield equivalent
results.
Keywords: acid hydrolysis; acid-soluble lignin; biorefinery;
black liquor; Klason lignin; kraft lignin.

Introduction
To close a lignin mass balance in an industrial process, for
instance concerning lignin precipitation in a biorefinery, it is
of central importance to accurately measure the total lignin
content in both black liquors and precipitated lignin samples.
The most commonly used analytical methods for the determination of lignin content are based on an acid hydrolysis
protocol published by Klason (1893). In the standard methods (TAPPI T 222 om-02, TAPPI T 249 cm-00, TAPPI UM
250, SCAN-CM 71:09) the sample is pre-hydrolyzed by concentrated sulfuric acid, further hydrolyzed in diluted sulfuric
acid, and subsequently filtered. Several aspects of lignin
determination are well illuminated by Dence (1992). The
gravimetrically measured retained insoluble constituent is
commonly referred to as acid-insoluble-lignin or Klason
lignin, and the lignin concentration in the filtrate is measured spectrophotometrically and denoted acid-soluble lignin. The sum of these two lignin entities is assumed to
represent the total lignin content. However, these standard
methods are developed for samples of extractive-free wood
and wood pulp. Nevertheless, these methods are commonly
used without adjustment for other materials, such as lignin
samples and black liquors, in everyday routine analysis in
the industry and in academic research as well as during international standardization activities (Gosselink et al. 2004).
The acid hydrolysis method is labor intensive. Previous
attempts to improve it include, for instance, finding an optimal wavelength for different wood types (Maekawa et al.
1989) or improving the precision by employing secondary

analytical methods (Schwanninger and Hinterstoisser 2002).

Willfor et al. (2009) compared different methods for hydrolytic pre-treatments of plant materials. However, few studies
have been performed aiming at reducing the complexity of
the standard methods for lignin determination in kraft pulping streams while maintaining acceptable analytical precision.
When subjected to acid hydrolysis, acid-soluble lignin
may be released from the acid-insoluble lignin during
extended acidic treatment (Kaar and Brink 1991), and
degraded acid-soluble lignin and carbohydrate degradation
products may be condensed under acidic conditions (Lundquist 1973; Yasuda et al. 2001), thus forming insoluble polymeric matter. It may hence be assumed that even though the
ratio between acid-insoluble and acid-soluble lignin varies
depending on acid treatment conditions, the total lignin content should remain the same. The pre-treatment with concentrated acid is commonly regarded to enable hydrolysis of
polysaccharides in cell walls (Willfor et al. 2009). It should
be possible to omit this step for raw materials without such
structures.
In this paper, a comparison is presented between the standard method and some time-saving methods for the determination of lignin and carbohydrate content in black liquors
and kraft lignin samples.

Material and methods

Samples
Three black liquors and four lignin samples (Innventia AB, Stockholm, Sweden) were investigated. The black liquors are from kraft
pulping of mixed hardwood (Sweden), pure eucalyptus (Portugal),
and mixed softwood (Sweden). One lignin sample was prepared
from each of the black liquors by means of the LignoBoost process
hman et al. 2007a,b). The lignin samples from mixed hardwood
(O
and eucalyptus were both precipitated and washed, whereas the
mixed softwood lignin sample was precipitated but not washed, thus
having a high content of ash. In addition, the commercially available
pine lignin (Indulin AT, MeadWestvaco Corporation, Richmond,
VA, USA) was included in this study. All lignin samples were airdried for at least 1 week prior to analysis.

Determination of dry matter, ash, and extractives

The dry matter contents of the black liquors and lignin samples were
determined in duplicates by oven-drying at 1058C (SCAN-N 22:96,
ISO 638:2008), and the ash was determined in duplicates by annealing at 5258C (ISO 1762:2001). The black liquors were extracted
with petroleum ether after dilution with an aqueous-methanol-acetone mixture (Saltsman and Kuiken 1959) (triplicates), whereas the
lignin samples were extracted with petroleum ether in Soxtec
(FOSS, Hillerd, Denmark) equipment (SCAN-CM 67:03) (duplicates). The yields were gravimetrically measured.

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602 F. Aldaeus et al.

Table 1 Comparison between the standard method and the proposed simplified methods.
Methods tested for lignin determination
Steps in protocol

Standard
method

Reduced
hydrolysis

Without
hydrolysis

1h
1 hb
Yes

No
1 min
Yes

No
No
Yes

Pre-hydrolysis with 72% H2SO4

Autoclave hydrolysis time at a final temperature of 1218Ca
Hot filtering
a

Time for heating and cooling (in total approximately 30 min) is not included.
If the objective is the determination of acid-insoluble lignin, boiling at normal pressure for 4 h may be used
instead of autoclave treatment for 1 h (TAPPI T 222 om-02).
b

ing and cooling is approximately 1 h longer (depending on details

of heating, cooling, and autoclave model).

Determination of lignin and carbohydrates in one

step
Prior to determination of lignin and carbohydrates, the samples
were, unless otherwise stated, subjected to acid hydrolysis consisting of a pre-hydrolysis with 72% H2SO4 followed by dilute acid
hydrolysis in an autoclave (TAPPI T 249 cm-00, SCAN-CM 71:09)
for 1 h at final temperature. For the lignin samples, the analyses
were scaled down (i.e., keeping the same solid/liquid ratio) for ca.
100 mg of solid material. After hydrolysis, the suspensions were
filtered while hot, and the retentates were measured gravimetrically
(TAPPI T 222 om-02) and reported as acid-insoluble lignin. The
acid-soluble lignin contents in the permeates were determined at
205 nm (spectrometer: Agilent Technologies Sweden AB, Kista,
Sweden; absorptivity coefficient: 110 l g-1 cm-1 according to TAPPI
UM 250; quadruple determination).
The carbohydrate contents were determined by high performance
anion exchange chromatography equipped with a pulsed amperometric detector (Dionex Sweden, Vastra Frolunda, Sweden) (SCANCM 71:09). All determinations were performed in quadruples,
except the carbohydrate contents determined without hydrolysis that
were determined only once.

Simplified methods
Two simplified methods for carbohydrate and lignin determination
were addressed: 1. The pre-hydrolysis step was omitted, and the
autoclave time at final temperature was reduced to 1 min. 2. No
hydrolysis was performed, i.e., the sample was directly suspended
in sulfuric acid, diluted and filtered hot. A comparison between the
standard and these simplified methods is given in Table 1. Note that
hydrolysis time in the autoclave presented in Table 1 is the time at
final temperature, and that the total hydrolysis time including heat-

Results and discussion

Initial chemical characterization of the samples

The chemical compositions of the samples were first determined (Table 2) by the standard methods described in the
Material and methods section. As normal, approximately half
of the dry content of the black liquors consists of ash. Only
the hardwood samples contained a conspicuous amount of
extractives. The lignin samples contained, with the exception
of the unwashed mixed softwood lignin, only minor amounts
of ash. Expectedly, the (unwashed) softwood lignin contains
a lower amount of acid-insoluble residue than the other hardwood lignins as the content of acid-soluble lignin is increasing with increasing syringyl content (Musha and Goring
1975).
A common feature of the lignin samples here is that the
sum is more than 1000 mg g-1, which is due to the inherent
inaccuracy of summative analysis of complex biomaterials
such as wood, and the inaccuracy of the absorptivity coefficient for determination of acid-soluble lignin.
Simplified determination of lignin and carbohydrates

Total lignin was calculated as a sum of acid-insoluble and

acid-soluble lignins. It was found that the ash remaining after

Table 2 Chemical composition of the samples determined according to SCAN-CM 71:09,

TAPPI T 222 om-02, TAPPI UM 250, ISO 1762:2001 and Saltsman and Kuiken (1959).

Sample name
BL, mixed HW
BL, eucalyptus
BL, mixed HW
Lignin, mixed HW
Lignin, eucalyptus
Lignin, mixed SW
Indulin AT (SW)

Lignin content as

Total CH
content
(mg g-1)

Linsol
(mg g-1)

Lsol
(mg g-1)

Ash
content
(mg g-1)

Extractives
content
(mg g-1)

32
35
16
15
17
13
9

223
299
312
885
831
689
870

104
111
54
112
150
42
47

498
497
497
7
12
238
29

25
3
5
41
4
-1
-1

BL, black liquor; HW, hardwood; SW, softwood; CH, carbohydrate; Linsol., lignin insoluble;
Lsol., lignin soluble in diluted acid.
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Lignin determination in context of kraft pulping 603

Table 3 Comparison of results for various lignin determination methods.

Standard method
Sample name
BL, mixed HW
BL, eucalyptus
BL, mixed HW
Lignin, mixed HW
Lignin, eucalyptus
Lignin, mixed SW
Indulin AT (SW)

Reduced hydrolysis method

a

Method without hydrolysis

Ltot
(mg g-1)

Ratio
Linsol/lsol

Ltot
(mg g-1)

Rel. diff.
(%)

Ratio
Linsol/lsol

Ltot
(mg g-1)

Rel. diff.a
(%)

Ratio
Linsol/lsol

327
410
366
997
980
731
918

2.1
2.7
5.7
7.9
5.5
16.5
18.3

325
415
371
991
984
728
939

-1"3
1"9
1"2
-1"2
0"2
0"3
2"3

1.8
2.4
5.3
5.9
5.2
9.6
9.7

335
410
368
960
999
737
927

2"5
0"12
0"1
-4"3
2"2
1"3
1"3

1.6
1.6
3.5
7.6
5.2
11.3
28.2

BL, black liquor; HW, hardwood; SW, softwood; Ltot, total lignin contents, i.e., the sum of acid-insoluble (Linsol) and acid-soluble
lignin (Lsol). Linsol and Lsol as determined by the methods outlined in Table 1.
a
Compared with standard method, i.e., 100%=(Ltot for simplified method Ltot for standard method)/Ltot for standard method
"95% confidence limits for the difference; degrees of freedoms6.

annealing is soluble in acid, and hence no correction for ash

in the acid-insoluble residue was necessary. As the black
liquors and lignin samples only contain small amounts of
carbohydrates, it was decided that no correction for carbohydrate degradation products in the absorbance measurements was needed. Calculated total lignin contents for the
standard and simplified methods are reported in Table 3. Furthermore, no correction for acid-insoluble extractives was
made due to the analytical difficulty in this regard.
It can be concluded that there is no significant difference
in the total lignin contents determined by the simplified and
standard methods, respectively. However, there is a difference in lignin composition obtained by the two methods.
Even though the UV spectra of all acid-soluble fractions are
similar, the simplified methods yielded typically more acidsoluble and less acid-insoluble lignin content. This statement
is not valid for the Indulin AT sample. The interpretation is
that in concentrated sulfuric acid condensation occurs and
this reduces the solubility in diluted acid. This finding is in
agreement with that of Lundquist (1973).

The total carbohydrate content generally decreases when

employing milder conditions (Table 4). However, the difference was clearly significant only for the low carbohydrate
content in the Indulin AT lignin sample (wherein both the
pentose and the hexose content decreased). Generally, under
mild hydrolysis conditions in the simplified method, the pentose content increased slightly (compared to the standard
method), whereas the hexose content decreased slightly.
Without hydrolysis, the carbohydrate contents were close to
the limit of detection, and hence no significance tests were
made.
For the determinations of total lignin content, the standard
method yielded a median relative standard deviation (2.1%)
similar to the simplified methods (0.9% and 1.6%, respectively). The median relative standard deviations in the carbohydrate measurements were 6.5% for the standard method
and 8.6% for the simplified hydrolysis method. Accordingly,
the simplified methods did not remarkably affect the precisions of the determinations of total lignin content.

Table 4 Comparison of various methods for lignin determination.

Standard method

Sample name
BL, mixed HW
BL, eucalyptus
BL, mixed HW
Lignin, mixed HW
Lignin, eucalyptus
Lignin, mixed SW
Indulin AT (SW)

Reduced hydrolysis method

Method without hydrolysis

Total CH
content
(mg g-1)

Ratio
P/H

Total CH
content
(mg g-1)

Rel. diff.a in
CH content

Ratio
P/H

Total CH
content
(mg g-1)

Rel. diff.a in
CH content

32
35
16
15
17
13
9

3.8
3.1
1.0
4.0
3.7
0.6
1.2

32
34
15
13
16
14
7

0%"14%
-2%"11%
-3%"11%
-17%"17%
-5%"26%
3%"11%
-23%"17%

3.4
2.8
0.9
1.2
2.0
0.5
0.7

2
1
2
1
4
2
1

-93%
-99%
-88%
-95%
-76%
-87%
-87%

Ratio
P/H
b

2.5
2.2
b

8.5
3.8
3.2

BL, black liquor; HW, hardwood; SW, softwood; total carbohydrate (CH) content, the sum of the anhydro monosaccharides in
hydrolysate as determined by HPAEC-PAD; the pentose/hexose ratio (P/H) determined by methods outlined in Table 1.
a
Compared with standard method, i.e., 100%=(Total CH content for simplified method Total CH content for standard method)/
Total CH content for standard method "95% confidence limits for the difference; degrees of freedoms6.
b
No hexoses detected.
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604 F. Aldaeus et al.

Considering the time consumption, it should be noted that

without hydrolysis a slightly prolonged time for the filtering
step was needed. As this method did not require autoclave
treatment, the overall analysis time is still shorter.

Conclusion
The lignin contents in kraft lignin and black liquors can be
determined by simplified methods without primary hydrolysis with 72% sulfuric acid. These methods are faster and of
equal precision. For lignin determination, a protocol with
only hot filtering is recommended. For the simultaneous
determination of carbohydrate and lignin content, a protocol
is recommended without pre-hydrolysis but with a shortened
autoclave treatment (1 min at final temperature).

Acknowledgements
We wish to acknowledge Hoshyar Rada for skillful technical assis kerlund and Dr Fredrik O
hman for supplying the
tance, Lars-Erik A
black liquors and lignin samples, and Dr Elisabeth Sjoholm for
interesting discussions regarding this topic. Research Institutes of
Sweden (RISE) is gratefully acknowledged for financial support.

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Received November 5, 2010. Accepted May 9, 2011.

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