ISO-21436-2020 - Lignin in Pulp

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INTERNATIONAL ISO

STANDARD 21436

First edition
2020-12

Pulps — Determination of lignin


content — Acid hydrolysis method
Pâtes — Détermination de la teneur en lignine — Méthode
d’hydrolyse acide

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ISO 21436:2020
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b869923c01dd/iso-21436-2020

Reference number
ISO 21436:2020(E)

© ISO 2020
ISO 21436:2020(E)


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ISO 21436:2020
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b869923c01dd/iso-21436-2020

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ISO 21436:2020(E)


Contents Page

Foreword......................................................................................................................................................................................................................................... iv
Introduction...................................................................................................................................................................................................................................v
1 Scope.................................................................................................................................................................................................................................. 1
2 Normative references....................................................................................................................................................................................... 1
3 Terms and definitions...................................................................................................................................................................................... 1
4 Principle......................................................................................................................................................................................................................... 2
5 Apparatus...................................................................................................................................................................................................................... 2
6 Reagents......................................................................................................................................................................................................................... 2
7 Sampling......................................................................................................................................................................................................................... 3
8 Test Specimens........................................................................................................................................................................................................ 3
9 Procedure..................................................................................................................................................................................................................... 3
9.1 Hydrolysis..................................................................................................................................................................................................... 3
9.1.1 General...................................................................................................................................................................................... 3
9.1.2 Hydrolysis procedure A.............................................................................................................................................. 4
9.1.3 Hydrolysis procedure B............................................................................................................................................... 4
9.2 Filtration........................................................................................................................................................................................................ 5
9.3 Acid-insoluble lignin determination..................................................................................................................................... 5
9.4 iTeh STANDARD PREVIEW
Acid-soluble lignin determination.......................................................................................................................................... 5
10 Calculation...................................................................................................................................................................................................................
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11 Precision........................................................................................................................................................................................................................ 6
ISO 21436:2020
12 Test Reporthttps://standards.iteh.ai/catalog/standards/sist/8cacea0c-711b-4dc1-9072-
................................................................................................................................................................................................................... 6
Annex A (informative) Precision................................................................................................................................................................................
b869923c01dd/iso-21436-2020 8
Bibliography.............................................................................................................................................................................................................................. 12

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ISO 21436:2020(E)


Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
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ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www​.iso​.org/​directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www​.iso​.org/​patents).
Any trade name used in this document is information given for the convenience of users and does not
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For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
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This document was prepared by Technical Committee ISO/TC 6, Paper, board and pulps.
ISO 21436:2020
Any feedback or questions on this document should be directed to the user’s national standards body. A
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ISO 21436:2020(E)


Introduction
The main objective of measuring lignin in pulp is to assess the effect of a particular pulping or bleaching
process on the degree of delignification. In chemical pulping, the goal is to remove lignin from wood
with minimum degradation of the carbohydrates. The higher the level of residual lignin in any type of
unbleached pulp, the greater the amount of bleaching chemicals that is applied in order to achieve a
target brightness.
Comprehensive textbooks and reviews have been written on methods of lignin determination[1]-[3].
This document specifies one such method, commonly used for the determination of the total lignin
content of pulp. In this method, a pulp sample is treated with sulfuric acid, in a two-step (primary and
secondary) hydrolysis process, to solubilize the carbohydrates. Most of the lignin remains insoluble at
the end of the treatment and is filtered off, dried and weighed. This acid-insoluble lignin is also referred
to as “Klason lignin”.
A small portion of lignin is dissolved during acid hydrolysis of the pulp. This so-called acid-soluble
lignin is determined spectrophotometrically, from the UV absorbance at 205 nm of the filtrate from the
acid-insoluble lignin determination[4]-[6]. The total lignin content is determined as the sum of the acid-
insoluble and acid-soluble lignin.
Two hydrolysis procedures are described in this document. In procedure A[7]-[9], the primary hydrolysis
is performed with 72 % sulfuric acid at 30 °C for one hour, followed by dilution with water to 4 %
sulfuric acid, and secondary hydrolysis in an autoclave at 120 °C for one hour. In procedure B[10][11], the
primary hydrolysis is done at 15-20 °C for two hours, followed by secondary hydrolysis at 3 % sulfuric
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acid in a water bath at 100 °C for four hours. In procedure A, the use of 4 % sulfuric acid, instead of 3 %,
for secondary hydrolysis has no impact on the lignin analysis and is accepted when both lignin and
(standards.iteh.ai)
carbohydrates need to be analysed in the same sample.
Both procedures have been shown to giveISO the21436:2020
same results; thus, either one can be used for determining
acid-insoluble lignin. However, procedure A is considerably more rapid, and the use of an autoclave
https://standards.iteh.ai/catalog/standards/sist/8cacea0c-711b-4dc1-9072-
allows multiple samples to be hydrolysed simultaneously
b869923c01dd/iso-21436-2020 with minimum supervision. As such, it is now
more commonly used in laboratories equipped with an autoclave. It is therefore the preferred method
and should be used when analysis of carbohydrates is required in addition to the determination of lignin.

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ISO 21436:2020
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b869923c01dd/iso-21436-2020
INTERNATIONAL STANDARD ISO 21436:2020(E)

Pulps — Determination of lignin content — Acid


hydrolysis method
WARNING — This method involves the use of hazardous chemicals. Care should be taken to
ensure that the relevant precautions are taken.

1 Scope
The method is applicable to unbleached, bleached and semi-bleached wood pulp with a lignin content
above 1 %. It is not generally intended for fully bleached chemical pulp, because the lignin content in
these pulps is too low to be determined accurately.

2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 638, Paper, board and pulps — Determination of dry matter content — Oven-drying method
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ISO 1762, Paper, board, pulps and cellulose nanomaterials — Determination of residue (ash content) on
ignition at 525 °C
(standards.iteh.ai)
ISO 7213, Pulps — Sampling for testing
ISO 21436:2020
ISO 14453, Pulps —https://standards.iteh.ai/catalog/standards/sist/8cacea0c-711b-4dc1-9072-
Determination of acetone-soluble matter
b869923c01dd/iso-21436-2020
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://​w ww​.iso​.org/​obp
— IEC Electropedia: available at http://​w ww​.electropedia​.org/​
3.1
lignins
class of complex, organic macromolecules, containing aromatic sub-units, that play a key role in the
formation of cell walls in wood and bark, conferring mechanical strength and rigidity to cell walls and
to plants as a whole
Note 1 to entry: Lignin is the main non-carbohydrate constitutent of wood.

3.2
acid-insoluble (Klason) lignin
residue after treating wood or pulp with sulfuric acid in a two-step hydrolysis procedure to solubilize
the carbohydrates into monosaccharides
3.3
acid-soluble lignin
portion of lignin (3.1) which is soluble during the acid-insoluble lignin determination

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ISO 21436:2020(E)


3.4
acetone-soluble matter
amount of material that can be extracted with acetone from a sample of pulp by the method specified
in ISO 14453

4 Principle
A pulp sample is treated with sulfuric acid in a two-step (primary and secondary) hydrolysis process to
dissolve the carbohydrates. The residue after hydrolysis is filtered off, dried, and weighed, and referred
to as acid-insoluble or Klason lignin. A small amount of lignin is dissolved during acid hydrolysis. This
so-called acid-soluble lignin is determined by measuring the absorbance at 205 nm of the filtrate from
the acid-insoluble lignin determination. The total lignin content is determined as the sum of the acid-
insoluble lignin and acid-soluble lignin.

5 Apparatus

5.1 Filtration equipment: filtering flask; filtering crucible, fritted glass, medium or fine porosity,
30 ml; adapter for the filtering crucible, siphon tube (optional).
NOTE The choice of fritted glass porosity depends on the rate of filtration of the particular type of sample.
For slow filtering samples, the use of medium (M) porosity is preferable. In low-yield sulfite pulps especially,
lignin forms a fine dispersion and clogs the pores of the filter. Filtration can be facilitated by using a medium
porosity crucible with a disc of fine porosity glass-fibre filter paper fitted over the sintered glass in the crucible.
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Other types of filtering crucibles, such as alundum or porous porcelain crucibles lined with a mat of
glass fibres can also be used. (standards.iteh.ai)
5.2 Constant temperature water bath, capable of maintaining a temperature of 30 ± 1 °C
ISO 21436:2020
(Procedure A); or 20 ± 1 °Chttps://standards.iteh.ai/catalog/standards/sist/8cacea0c-711b-4dc1-9072-
(Procedure B).
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5.3 Flask, Erlenmeyer, 1 000 ml, or smaller depending on the size of the pulp specimen.

5.4 Procedure A only: Autoclave, capable of maintaining a temperature of 120 ± 3 °C.

5.5 Drying oven, convection type, maintained at 105 ± 2 °C.

5.6 Analytical balance, accurate to 0,1 mg.

5.7 Spectrophotometer, UV-visible, diode array or simple wavelength, with high purity quartz
cuvettes of pathlength 1 cm.

6 Reagents

6.1 Water, distilled or deionized.

6.2 Sulfuric acid, 72 % w/w (specific gravity 1,633 8 at 20 °C). 72 % sulfuric acid is available
commercially. It can also be prepared from concentrated sulfuric acid as follows:

Slowly add 650 ml of concentrated sulfuric acid (H2SO4 sp gr 1,84) to 400 ml of water, while cooling
under a cold water trap. When the temperature has reached equilibrium with the ambient temperature,
adjust the specific gravity of the sulfuric acid solution to 1,633 8, with the use of a hydrometer, by
careful addition of concentrated sulfuric acid or water.

6.3 Acetone, only if extraction of the sample is required prior to hydrolysis with sulfuric acid.

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ISO 21436:2020(E)


7 Sampling
If the analysis is being made to evaluate a lot of a consignment of pulp, the sample shall be taken in
accordance with ISO 7213. If the analysis is made on another type of sample, report the origin of the
sample, and if possible the sampling procedure.
Obtain a representative sample of pulp equivalent to about 10 g moisture-free pulp. Air dry the pulp
and disintegrate in a household blender, or grind in a Wiley mill to pass a No. 20 (0,85 mm) screen.
Groundwood and high yield pulps containing a significant amount of resins shall be extracted with
acetone (6.3) according to ISO 14453 before testing.
NOTE Resins, if not extracted from the pulp prior to analysis, would remain insoluble in acid and be weighed
as lignin.

NOTE Acetone is considered an effective solvent for extracting resin from pulp. Dichloromethane and
ethanol/benzene (1:2), as specified in other methods, are not recommended due to health hazards. In particular,
benzene is a confirmed carcinogen.

Determine the moisture content of the pulp according to ISO 638 by drying a 2-3 g specimen in an
oven at 105 ± 3 °C to constant weight. If the pulp shall be pre-extracted, the moisture content shall be
determined on the extracted pulp.

8 Test Specimens
Weigh (5.6) a test specimen, equivalent to 300 mg to 1,0 g of oven-dried mass of pulp to the nearest
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0,1 mg (see NOTE) and transfer to a 200 ml beaker. Make sure that the test specimens taken are
representative of the sample received.
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The mass of the test specimen is such as to provide a minimum of 20 mg of lignin in the residue
remaining after acid hydrolysis, for accurate weighing. The following can be used as a guide to the
ISO 21436:2020
amount of pulp thathttps://standards.iteh.ai/catalog/standards/sist/8cacea0c-711b-4dc1-9072-
should be selected for analysis, based on the lignin content:
b869923c01dd/iso-21436-2020
— >10 % lignin: 300 mg pulp
— 5-10 % lignin: 500 mg pulp
— <5 % lignin: 1,0 g pulp
NOTE The amount of pulp selected for analysis also depends on whether hydrolysis procedure A or
hydrolysis procedure B is used.

9 Procedure
Run the entire procedure in duplicate.

9.1 Hydrolysis

9.1.1 General

Two hydrolysis procedures are described in this Standard. In procedure A[7]-[9], the primary hydrolysis
is performed at 30 °C for 1 h, followed by secondary hydrolysis in an autoclave at 120 °C for 1 h. In
procedure B[10][11], the primary hydrolysis is performed at 15 - 20 °C for 2 h, followed by secondary
hydrolysis in a water bath at 100 °C for 4 h .
NOTE Boiling under reflux is not allowed if acid-soluble lignin is to be determined in the solution (TAPPI
T222, Section 9.4, NOTE 6). This is due to the possible contribution of furfural compounds to the acid-soluble
lignin, if not removed in this step (Ref.2, p. 192).

Both procedures have been shown to give the same results[7]; thus either one can be used for
determining acid-insoluble lignin. However, procedure A is considerably more rapid, and the use of

© ISO 2020 – All rights reserved  3


ISO 21436:2020(E)


an autoclave allows multiple samples to be hydrolysed simultaneously with minimum supervision. As


such, it is now more commonly used in laboratories equipped with an autoclave.
NOTE Procedure A is therefore the preferred method and is used when analysis of carbohydrates is required,
in addition to the determination of lignin.

9.1.2 Hydrolysis procedure A

Hydrolysis procedure A is based on the use of 300 mg of pulp. This procedure is more commonly used
in laboratories equipped with an autoclave, and is similar to that described in other methods[7]-[9] in
situations in which analysis of carbohydrates is required, in addition to analysis of lignin.
For larger pulp specimens, the acid concentrations for both primary and secondary hydrolysis must
remain the same as those used for 300 mg of pulp; thus the volume of solutions must be adjusted
accordingly.
Add 3,0 ml of 72 % sulfuric acid (6.2) to the test specimen in the beaker. Add the acid gradually in small
increments while stirring and macerating the material with a glass rod. To avoid losses, ensure that no
material is sticking to the glass rod when it is removed.
NOTE Some pulps do not absorb the acid and therefore do not disperse readily. In such cases, after addition
of acid, place the beaker under vacuum, in a vacuum desiccator for at least 15 minutes to facilitate wetting and
absorption.

Place the beaker in a water bath adjusted to 30 ± 1,0 °C for 1 h. Stir occasionally.
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Add 84,0 ml of water. Mix, cover the beaker with aluminium foil and place it in an autoclave at (120 ± 3) °C
for 1 h. Allow the insoluble lignin to settle and for the beaker and contents to cool to approximately 80 °C.
(standards.iteh.ai)
NOTE In some cases, the lignin requires an overnight or a longer period to settle, especially if it is finely
dispersed. ISO 21436:2020
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9.1.3 Hydrolysis procedure B b869923c01dd/iso-21436-2020
Hydrolysis procedure B is based on the use of 1,0 g of pulp, and is similar to that described in other
methods[10][11]. This procedure is also used in some laboratories.
NOTE Hydrolysis procedure B is not used when carbohydrate analysis is also required.

For smaller pulp specimens, the acid concentrations for both primary and secondary hydrolysis must
remain the same as those used for 1,0 g of pulp; thus the solution volumes shall be adjusted accordingly.
Place the beaker with the test specimen in a water bath (5.2) at 20 ± 1 °C and add gradually 20,0 ml of
72 % sulfuric acid (6.2), maintaining a reaction temperature of 20 ± 1 °C Stir thoroughly with a glass rod
for about 1 min. To avoid losses, ensure that no material is sticking to the glass rod when it is removed.
NOTE Some pulps do not absorb the acid and therefore do not disperse readily. In such cases, after addition
of acid, place the beaker under vacuum, in a vacuum desiccator for at least 15 min to facilitate wetting and
absorption.

Keep the beaker in the bath for 120 ± 10 min with occasional stirring.
Transfer the material quantitatively from the beaker into a 1 000 ml Erlenmeyer flask and dilute with
water to 750 ml.
Boil the solution for 4 h, maintaining a constant volume of solution by frequent addition of hot (80-90 °C)
water. Allow the insoluble lignin to settle and for the flask and contents to cool to approximately 80 °C.
NOTE In some cases, the lignin requires an overnight or a longer period to settle, especially if it is finely
dispersed.

4  © ISO 2020 – All rights reserved


ISO 21436:2020(E)


9.2 Filtration
Without stirring the insoluble lignin residue, decant or siphon off the supernatant solution through a
pre-weighed filtering crucible (5.1) placed on a 250 ml filtering flask (5.1) (hydrolysis procedure A) or
a 1 000 ml filtering flask (5.1) (hydrolysis procedure B). Proceed to filtration procedure A if hydrolysis
procedure A is used, or to filtration procedure B if hydrolysis procedure B is used.
Filtration procedure A: Transfer the filtrate to a 250 ml volumetric flask. Wash the precipitate
with 2 × 30 ml warm water (6.1) and add the washings to the volumetric flask. Allow to cool to room
temperature and fill up to the mark with water (6.1). This filtrate will be used for the acid-soluble lignin
determination.
NOTE Dilution to precisely 250 ml also allows accurate quantification of carbohydrates in the filtrate, if
required.

Filtration procedure B: Proceed as in filtration procedure A, but transfer the filtrate to a 1 l volumetric
flask, wash the precipitate with 2 × 100 ml warm water (6.1), and add the washings to the volumetric
flask. Allow to cool to room temperature and fill up to the mark with water. This filtrate will be used for
the acid-soluble lignin determination.

9.3 Acid-insoluble lignin determination


Quantitatively transfer the insoluble lignin, from 9.1.2 or 9.1.3, to the pre-weighed filtering crucible
(5.1) using hot water (6.1) and a rod with rubber policeman. Wash the lignin with hot water (6.1) until
the pH of the filtrate is in the range of 6-7, as confirmed with the use of a pH indicator paper or pH
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meter. Discard this filtrate.

(standards.iteh.ai)
Dry the crucible with lignin in an oven (5.5) at 105 ± 2 °C to constant weight. Cool in a desiccator and
weigh with an analytical balance(5.6).
ISO 21436:2020
If a correction for ash is required, transfer the lignin from the previous step to a small pre-weighed
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platinum or porcelain crucible and determine the ash content according to ISO 1762.
b869923c01dd/iso-21436-2020
9.4 Acid-soluble lignin determination
Use the filtrate from the acid-insoluble lignin determination (9.2) as a test specimen.
Measure the absorbance of the filtrate at 205 nm in the 1,0 cm pathlength cuvette of the
spectrophotometer (5.7). Use 4 % sulfuric acid (hydrolysis procedure A) or 3 % sulfuric acid (hydrolysis
procedure B), as a blank. If necessary, dilute the filtrate with 4 % sulfuric acid (hydrolysis procedure
A) or 3 % sulfuric acid (hydrolysis procedure B), such that its absorbance is in the range of 0,2-0,7 AU.
NOTE Degradation products from carbohydrates can contribute to the measured acid-soluble lignin[6].
However, this contribution is generally small and can be neglected.

NOTE In the round robin study (Annex A), labs using the autoclave (hydrolysis procedure A) reported
levels of acid-soluble lignin that were on average 0,2 % higher than those obtained when using the water bath
(hydrolysis procedure B). This could indicate that furfural compounds are not completely removed when using
the autoclave, due to the fact that the beaker is covered with aluminium foil during the secondary hydrolysis.
However, since these differences are relatively small, they are unlikely to have a significant impact on the total
lignin results.

10 Calculation
Calculate the mean of duplicate determinations
m⋅100
Acid-insoluble lignin, % =
M
where

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