Leather — Chemical tests for the determination of certain azo colourants in dyed leathers — Part 1: Determination of certain aromatic amines derived from azo colourants

This document specifies a method for determining the use of certain azo colourants which can release certain aromatic amines.

Cuir — Essais chimiques pour le dosage de certains colorants azoïques dans les cuirs teints — Partie 1: Dosage de certaines amines aromatiques dérivées des colorants azoïques

Le présent document spécifie une méthode permettant de déterminer l'utilisation de certains colorants azoïques qui peuvent libérer certaines amines aromatiques.

General Information

Status
Published
Publication Date
20-Aug-2020
Current Stage
6060 - International Standard published
Start Date
21-Aug-2020
Completion Date
21-Aug-2020
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INTERNATIONAL ISO
STANDARD 17234-1
IULTCS
IUC 20-1
Third edition
2020-08
Leather — Chemical tests for
the determination of certain azo
colourants in dyed leathers —
Part 1:
Determination of certain aromatic
amines derived from azo colourants
Cuir — Essais chimiques pour le dosage de certains colorants
azoïques dans les cuirs teints —
Partie 1: Dosage de certaines amines aromatiques dérivées des
colorants azoïques
Reference numbers
ISO 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 General ............................................................................................................................................................................................................................ 1

5 Principle ........................................................................................................................................................................................................................ 3

6 Safety precautions .............................................................................................................................................................................................. 3

7 Apparatus ..................................................................................................................................................................................................................... 3

8 Reagents ........................................................................................................................................................................................................................ 4

9 Sampling and preparation of samples ........................................................................................................................................... 5

10 Procedure..................................................................................................................................................................................................................... 5

10.1 Degreasing .................................................................................................................................................................................................. 5

10.2 Reductive cleavage ............................................................................................................................................................................... 5

10.3 Liquid-liquid extraction .................................................................................................................................................................. 5

10.4 Check of the analytical system ................................................................................................................................................... 6

11 Chromatographic analyses ......................................................................................................................................................................... 6

12 Calibration .................................................................................................................................................................................................................. 6

13 Evaluation .................................................................................................................................................................................................................... 6

13.1 Calculation of amine in the sample ....................................................................................................................................... 6

13.2 Reliability of the method ................................................................................................................................................................ 7

14 Test report ................................................................................................................................................................................................................... 7

Annex A (informative) Chromatographic analyses ................................................................................................................................ 9

Annex B (informative) Reliability of the method ..................................................................................................................................13

Annex C (informative) Assessment guide — Interpretation of analytical results ..............................................14

Annex D (informative) Procedure for liquid/liquid extraction without diatomaceous earth ..............20

Bibliography .............................................................................................................................................................................................................................23

© ISO 2020 – All rights reserved iii
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ISO 17234-1:2020(E)
IULTCS/IUC 20-1:2020(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

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

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

constitute an endorsement.

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 the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/

iso/ foreword .html.

This document was prepared by the Chemical Test Commission of the International Union of Leather

Technologists and Chemists Societies (IUC Commission, IULTCS) in collaboration with the European

Committee for Standardization (CEN) Technical Committee CEN/TC 289, Leather, the secretariat of

which is held by UNI, in accordance with the Agreement on technical cooperation between ISO and CEN

(Vienna Agreement). This method is technically similar to the method in IUC 20, which was declared an

official method at the IULTCS Delegates meeting on 31st May 2003 in Cancun, Mexico.

IULTCS, originally formed in 1897, is a world-wide organization of professional leather societies to

further the advancement of leather science and technology. IULTCS has three Commissions, which

are responsible for establishing international methods for the sampling and testing of leather. ISO

recognizes IULTCS as an international standardizing body for the preparation of test methods for

leather.

This third edition cancels and replaces the second edition (ISO 17234-1:2015), which has been

technically revised.
The main changes to the previous edition are as follows:
— a new Clause 3 added;
— technical changes to Clause 7 and Clause 8;
— changes to Clause 9 and Clause 10 to improve the method;

— Annex C expanded to give examples of false-positive results, suggested procedures and suggested

comments in the test report;
— a new informative Annex D added.
A list of all parts in the ISO 17234 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2020 – All rights reserved
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ISO 17234-1:2020(E)
INTERNATIONAL STANDARD
IULTCS/IUC 20-1:2020(E)
Leather — Chemical tests for the determination of certain
azo colourants in dyed leathers —
Part 1:
Determination of certain aromatic amines derived from
azo colourants
1 Scope

This document specifies a method for determining the use of certain azo colourants which can release

certain aromatic amines.
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 2418, Leather — Chemical, physical and mechanical and fastness tests — Sampling location

ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 4044, Leather — Chemical tests — Preparation of chemical test samples

ISO 17234-2, Leather — Chemical tests for the determination of certain azo colorants in dyed leathers —

Part 2: Determination of 4-aminoazobenzene
3 Terms and definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
4 General

Certain azo colourants can release, by reductive cleavage of azo group(s), one or more of the aromatic

[2] [3]

amines listed in EU Regulation 1907/2006, Annex XVII, Appendix 8 and GB 20400-2006 (see Table 1).

© ISO 2020 – All rights reserved 1
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ISO 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
[2]

Table 1 — Aromatic amines listed in EU Regulation 1907/2006, Annex XVII, Appendix 8 and

[3]
GB 20400-2006
No. CAS number Index number EC number Substances
1 92–67–1 612–072–00–6 202–177–1 biphenyl-4-ylamine
4-aminobiphenyl
xenylamine
2 92–87–5 612–042–00–2 202–199–1 benzidine
3 95–69–2 612–196–00–0 202–441–6 4-chloro-o-toluidine
4 91–59–8 612–022–00–3 202–080–4 2-naphthylamine
5 97–56–3 611–006–00–3 202–591–2 o-aminoazotoluene
4-amino-2',3-dimethylazobenzene
4-o-tolylazo-o-toluidine
6 99–55–8 612–210–00–5 202–765–8 5-nitro-o-toluidine
2-amino-4-nitrotoluene
7 106–47–8 612–137–00–9 203–401–0 4-chloroaniline
8 615–05–4 612–200–00–0 210–406–1 4-methoxy-m-phenylenediamine
2,4-diaminoanisole
9 101–77–9 612–051–00–1 202–974–4 4,4'-methylenedianiline
4,4'-diaminodiphenylmethane
10 91–94–1 612–068–00–4 202–109–0 3,3'-dichlorobenzidine
3,3'-dichlorobiphenyl-4,4'-ylenediamine
11 119–90–4 612–036–00-X 204–355–4 3,3'-dimethoxybenzidine
o-dianisidine
12 119–93–7 612–041–00–7 204–358–0 3,3'-dimethylbenzidine
4,4'-bi-o-toluidine
13 838–88–0 612–085–00–7 212–658–8 4,4'-methylenedi-o-toluidine
14 120–71–8 612–209–00–X 204–419–1 6-methoxy-m-toluidine
p-cresidine
15 101–14–4 612–078–00–9 202–918–9 4,4'-methylene-bis-(2-chloro-aniline)
2,2'-dichloro-4.4’-methylene-dianiline
16 101–80–4 612–199–00–7 202–977–0 4,4'-oxydianiline
17 139–65–1 612–198–00–1 205–370–9 4,4'-thiodianiline
18 95–53–4 612–091–00-X 202–429–0 o-toluidine
2-aminotoluene
19 95–80–7 612–099–00–3 202–453–1 4-methyl-m-phenylenediamine
2,4-toluylendiamine
2,4-diaminotoluene
20 137–17–7 612–197–00–6 205–282–0 2,4,5-trimethylaniline
21 90–04–0 612–035–00–4 201–963–1 o-anisidine
2-methoxyaniline
22 60–09–3 611–008–00–4 200–453–6 4-aminoazobenzene
23 95–68–1 612–027–00–0 202–440–0 2,4-xylidine
2,4-dimethylbenzene-1-amine
24 87–62–7 612–161–00-X 201–758–7 2,6-xylidine
2,6-dimethylbenzene-1-amine

The CAS-numbers 97–56–3 (no. 5) and 99–55–8 (no. 6) are further reduced to CAS-numbers 95–53–4 (no. 18) and

95–80–7 (no. 19).

Azo colourants that are able to form 4-aminoazobenzene generate under the condition of this method aniline CAS-

number 62–53–3) and 1,4-phenylenediamine (CAS number 106–50–3). The presence of these colourants shall be tested

using ISO 17234-2.
Additional aromatic amines in GB 20400–2006.
2 © ISO 2020 – All rights reserved
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ISO 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
5 Principle

After degreasing, the leather sample is treated with sodium dithionite in an aqueous buffer solution

(pH 6) at 70 °C in a closed vessel. The amines released in the process of reductive cleavage are transferred

to a t-butyl methyl ether (8.5) phase by means of liquid-liquid extraction using diatomaceous earth

columns. The t-butyl methyl ether (8.5) extract is then concentrated under mild conditions in a rotary

vacuum evaporator and the residue is dissolved in a suitable solvent, depending on the method used to

determine the amines (see Annex A).

Determination of the amines is performed by means of high-performance liquid chromatography

(HPLC) using a diode array detector (DAD) or mass selective detector (HPLC-MS), capillary gas

chromatography with a mass selective detector (GC-MS) or by capillary electrophoresis with a diode

array detector (CE-DAD), or qualitatively with thin layer chromatography (TLC, HPTLC).

The amines shall be identified by means of at least two different chromatographic separation methods

in order to avoid any possible misinterpretations caused by interfering substances (such as position

isomers of the amines to be identified) and hence any incorrect statements. Amine quantification shall

be performed by HPLC-DAD or GC-MS.

A screening method using liquid-liquid extraction without diatomaceous earth columns is described in

Annex D.
6 Safety precautions

WARNING — The aromatic amines listed in Clause 4 are classified as substances known to be or

suspected to be human carcinogens.

6.1 It is the user's responsibility to use safe and proper techniques when handling materials in this

test method. Consult manufacturers for specific details, such as material safety data sheets and other

recommendations.

6.2 Good laboratory practice should be followed. Wear safety glasses in all laboratory areas and a dust

respirator and single-use gloves while handling powder colourants and aromatic amines.

6.3 National and local safety regulations can apply.
7 Apparatus
The usual laboratory equipment and, in particular, the following:

7.1 Suitable reaction vessel, of temperature-resistant glass with a gas-tight closure.

7.2 Suitable heating system, at (70 ± 2) °C.

7.3 Polypropylene or glass column, inside diameter 25 mm to 30 mm, length 130 mm to 150 mm,

packed with 20 g of diatomaceous earth, fitted with glass fibre filter at the outlet.

The diatomaceous earth columns are either bought pre-packed and used as is, or 20 g of diatomaceous

earth can be packed into a glass or polypropylene column of the dimensions given.

7.4 Vacuum rotary evaporator with vacuum control and water bath.
7.5 Pipettes, in required sizes or variables pipettes.
7.6 Ultrasonic bath with thermostat.
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ISO 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
7.7 Chromatographic equipment, selected from the following:
7.7.1 High-performance liquid chromatography (HPLC) and DAD or MS.
7.7.2 Capillary gas chromatography (GC), with MS.
7.7.3 Capillary electrophoresis (CE), with DAD.

7.7.4 Thin layer chromatography (TLC) or high-performance thin layer chromatography

(HPTLC).
NOTE A description of the chromatographic equipment (7.7) is given in Annex A.
8 Reagents
Unless otherwise specified, analytical grade chemicals shall be used.
8.1 n-hexane.
8.2 Citrate buffer solution, 0,06 mol/l, pH = 6, preheated to (70 ± 2) °C.

8.3 Aqueous sodium dithionite solution, ρ = 200 mg/ml , freshly prepared, to be used immediately

after resting for 1 h in a closed vessel.
8.4 Sodium hydroxide aqueous solution, a mass fraction of 40 %.
8.5 t-butyl methyl ether.
8.6 Methanol.
8.7 Acetonitrile.
8.8 Amines, listed in Table 1 (highest available purity standard).
8.9 Standard solutions.
8.9.1 Stock solution of the amines (8.8), 400 µg/ml in ethyl acetate for TLC.

8.9.2 Stock solution of the amines (8.8), 200 µg/ml of each amine in an appropriate solvent.

NOTE Acetonitrile is an appropriate solvent for this stock solution, resulting in good stability of amines.

8.9.3 Standard solution for amine process control, 30 µg amine per millilitre solvent, freshly

prepared from stock solutions (8.9.1 or 8.9.2) depending on the analytical method

8.9.4 Internal standard in solution (IS), ρ = 10 μg of IS/ml of t-butyl methyl ether (8.5).

In the case of GC-MS analysis, one of the following internal standards can be used:

— IS1: naphthalene-d8, CAS no.: 1146-65-2;
— IS2: 2,4,5-trichloroaniline (TCA), CAS no.: 636-30-6;
1) ρ = mass concentration.
4 © ISO 2020 – All rights reserved
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ISO 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
— IS3: anthracene-d10, CAS no.: 1719-06-8.
8.10 Water, Grade 3 according to ISO 3696.
9 Sampling and preparation of samples

The leather shall be sampled in accordance with ISO 2418 and prepared in accordance with ISO 4044. If

sampling in accordance with ISO 2418 is not possible (e.g. in the case of leathers from finished products

such as shoes or garments), details about sampling shall be given in the test report. Any traces of

adhesives shall be removed mechanically.

In the case of leather patchwork fabrics with varicoloured patterns, the various colours shall be taken

into account separately as far as possible. For commodities consisting of various leather qualities,

specimens of the various qualities shall be analysed separately.

For the analytical procedure, accurately weigh a representative sample of 1,0 g of this leather sample in

the reaction vessel (7.1).
10 Procedure
10.1 Degreasing

Treat 1,0 g of the leather in a closed 50 ml vessel (7.1) with 40 ml n-hexane (8.1) in an ultrasonic bath

(7.6) at (40 ± 2) °C for 40 min.

Decant the n-hexane layer from the leather sample. Any loss of leather particles during decanting shall

be avoided. Evaporate the residual n-hexane at least overnight in the open vessel.

10.2 Reductive cleavage

Add a quantity of 15 ml buffer solution (8.9) preheated to (70 ± 2) °C to the sample.

Close the reaction vessel tightly and treat for (30 ± 1) min at (70 ± 2) °C.

Subsequently, add 3 ml aqueous sodium dithionite solution (8.3) for the reductive cleavage of the azo

groups to the reaction vessel, then shake vigorously and immediately keep at (70 ± 2) °C for another

(30 ± 1) min. Then cool to room temperature (20 °C to 25 °C) within 2 min with a cooling mixture of ice,

water and salt.
10.3 Liquid-liquid extraction

Add 1,5 ml of the NaOH solution (8.4) to the reaction solution and shake vigorously. Transfer the reaction

solution to the diatomaceous earth column (7.3) and allow it to be absorbed by the column for 15 min.

Meanwhile, add 10 ml t-butyl methyl ether (8.5) to the reaction vessel and shake vigorously. After the

15 min period decant the t-butyl methyl ether (8.5) onto the top of the column and collect the eluate in

a 250 ml round-bottom flask.

Rinse the reaction vessel with 10 ml t-butyl methyl ether (8.5) and transfer the solvent to the column.

Subsequently, pour 60 ml t-butyl methyl ether (8.5) directly onto the column.

For amine detection and quantification, the t-butyl methyl ether extract is concentrated to a volume

less than 5 ml (not to dryness) with a vacuum rotary at a temperature less than 50 °C and a pressure

of approximately 450 mbar. If it is necessary to change to another solvent, remove the remainder of the

solvent very carefully by means of a weak flow of inert gas.

NOTE 1 Removal of the solvent (concentration in the rotary vacuum evaporator, evaporation to dryness) can

lead to substantial amine losses if performed under uncontrolled conditions.
© ISO 2020 – All rights reserved 5
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ISO 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)

Make up the extract or residue to 2,0 ml with an appropriate solvent for detection and determination

of the amines using chromatography [acetonitrile (8.7), t-butyl methyl ether (8.5) or methanol (8.6)]

without delay. If the complete analysis cannot be performed within (24 ± 1) h, keep the extract at

(−18 ± 3) °C and warm carefully to room temperature before analysis.

NOTE 2 Owing to the matrix, individual amines such as 2,4-diaminotoluene and 2,4-diaminoanisole are likely

to exhibit a very poor stability, especially in methanol. Where delays occur in the work routine, amines can be no

longer detectable by the time of instrumental measurement.
10.4 Check of the analytical system

To check the analysis procedure, add a certain quantity of amines to obtain x mg/l as final concentration

in a reaction vessel containing 15 ml of buffer solution (8.9).
Then carry out the procedure set out in 10.3.
Amine recovery rates shall conform with the following minimum requirements:
— amines nos 1 to 4, 7, 9 to 17 and 20 to 21: recovery rate 70 %;
— amine no. 8: recovery rate 20 %;
— amines nos 18, 19, 23 and 24: recovery rate 50 %;
— amines nos 5, 6 and 22, see footnotes to Table 1.
11 Chromatographic analyses

The detection of the aromatic amines can be performed using the chromatographic techniques listed

in 7.7. Other validated methods can be used. The quantification of the aromatic amines is performed

by means of HPLC-DAD, HPLC-MS or GC-MS. Where gas chromatography is used, appropriate internal

standards as described in 8.9.4 shall be employed.

If any amine is detected by one chromatographic method, then confirmation shall be made using one or

more alternative methods. The result is positive only if both methods give a positive result.

12 Calibration

Use the standard solution (8.9.2) to prepare at least three calibration solutions in a range of 2 µg/ml to

30 µg/ml.
13 Evaluat ion
13.1 Calculation of amine in the sample

Calculate the amine concentration based on the peak areas of the individual amine components.

Calculate the content of the amine as a mass fraction, w, in milligrams of the individual component per

kilogram (mg/kg) of leather material according to Formula (1):
AV×
w=×ρ (1)
Am×
where
6 © ISO 2020 – All rights reserved
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ISO 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)

ρ is the concentration of the amine in the calibration solution, in micrograms per millilitre

(μg/ml);
A is the peak area of the amine in the sample solution, in area units;
A is the peak area of the amine in the calibration solution, in area units;

V is the volume of the specimen according to 10.3 (final sample volume), in millilitres (ml)

(here 2 ml);
m is the mass of the leather sample, in grams (g).
13.2 Reliability of the method
For the reliability of the method, see Annex B.
14 Test report

The test report shall refer to this official method and give information on at least the following aspects:

a) a reference to this document, i.e. ISO 17234-1:2020;
b) identification of the sample;
c) sampling procedure;

d) any deviations from the analytical procedure, particularly any additional steps performed;

e) declaration of analytical techniques used for detection and confirmation;
f) the date of the test;

g) the analytical results for the amines in milligrams per kilogram (see Clause 13), individually listed

and reported according to the identification threshold values as follows:
In the case of levels per amine component ≤ 30 mg/kg:

According to the analysis as carried out, azo colourants which release the listed aromatic amines were

not detected.
In the case of levels per amine component > 30 mg/kg:

The analysis result suggests that the leather submitted has been manufactured or treated using azo

colourants which release one or more of the listed amines.
© ISO 2020 – All rights reserved 7
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ISO 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
In the case of levels of 4-aminodiphenyl and/or 2-naphthylamine > 30 mg/kg:

Use of this analytical method has detected 4-aminodiphenyl and/or 2-naphthylamine. According to the

current state of knowledge it cannot be unequivocally confirmed without additional information that

azo colourants which release amines were used.

Care should be taken in the interpretation of less than 30 mg/kg of amines as these can be due to false-

positive results. For the interpretation of results, see Annex C.
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ISO 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
Annex A
(informative)
Chromatographic analyses
A.1 Preliminary remark

As the chromatographic equipment (7.7) of the laboratories can vary, no generally applicable

instructions can be provided for chromatographic analyses. The following parameters have been

successfully tested and used.
A.2 High-performance liquid chromatography (HPLC)
A.2.1 High-performance liquid chromatography/diode array detector (HPLC-DAD)
Eluent 1: methanol;
Eluent 2: 0,575 g of ammonium dihydrogen phosphate + 0,7 g of disodium hydrogen
phosphate in 1 000 ml of water, pH 6,9;

Stationary phase: LiChrospher 60 RP-select B (5 µm), length: 250 mm × inside diameter: 4,6 mm;

Flow rate: (0,7 to 1,0) ml/min;
Gradient: start: 15 % eluent 1, linear increase to 80 % eluent 1 within 45 min;
Column temperature: 40 °C;
Injection volume: 10,0 μl;
Detection: DAD, spectrograph;
Quantification: at 240 nm, 280 nm, and 305 nm.

NOTE LiChrospher 60 RP-select B is an example of a suitable product available commercially. This

information is given for the convenience of users of this document and does not constitute an endorsement by ISO

of this product. Equivalent products can be used if they can be shown to lead to the same results.

See example in Figure A.1.
© ISO 2020 – All rights reserved 9
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ISO 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
Key
X time in min
Y absorbance in mAU at 240 nm
1 1,4-phenylenediamine
2 aniline

NOTE For aromatic amines 1 to 21, see Table 1 (aromatic amines 23 and 24 are not shown).

Figure A.1 — HLPC-DAD-chromatogram
A.2.2 High-performance liquid chromatography/mass selective detector (HPLC-MS)
Eluent 1: acetonitrile;
Eluent 2: ammonium acetate in 1 000 ml of water, 5 mmol, pH 3,0;
Stationary phase: C18 (3,5 μm); length: 50 mm; inside diameter: 2,1 mm;
Flow rate: 300 μl/min;
Gradient: see Table A.1;
Column temperature 40 °C;
Injection volume: 2,0 μl;
Detection: quadrupole- and/or ion-trap mass detector, scanning mode and/or MS
daughter ion MS detection; DAD: for wavelengths, see A.2.1;
Spray gas: nitrogen (bottled/generator);
Ionization: API electrospray positive, fragmentor 120 V.
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ISO 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
Table A.1 — Gradient programme
Time Eluent 1 Eluent 2
min % %
0 10 90
1,5 20 80
7,5 90 10
A.3 Capillary gas chromatography (GC-MS)

Capillary column: medium polarity, e.g. SE 54 or DB 5, length: 50 m, inside diameter:

0,32 mm, film thickness: 0,5 μm;
Injector system: split/splitless;
Injector temperature: 250 °C;
Carrier gas: helium;

Temperature programme: 70 °C (2 min), 70 °C to 280 °C (at 10 °C/min), 280 °C (5 min);

Injection volume:
...

NORME ISO
INTERNATIONALE 17234-1
IULTCS
IUC 20-1
Troisième édition
2020-08
Cuir — Essais chimiques pour le
dosage de certains colorants azoïques
dans les cuirs teints —
Partie 1:
Dosage de certaines amines
aromatiques dérivées des colorants
azoïques
Leather — Chemical tests for the determination of certain azo
colourants in dyed leathers —
Part 1: Determination of certain aromatic amines derived from azo
colourants
Numéros de référence
ISO 17234-1:2020(F)
IULTCS/IUC 20-1:2020(F)
ISO 2020
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ISO 17234-1:2020(F)
IULTCS/IUC 20-1:2020(F)
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ISO 17234-1:2020(F)
IULTCS/IUC 20-1:2020(F)
Sommaire Page

Avant-propos ..............................................................................................................................................................................................................................iv

1 Domaine d’application ................................................................................................................................................................................... 1

2 Références normatives ................................................................................................................................................................................... 1

3 Termes et définitions ....................................................................................................................................................................................... 1

4 Généralités .................................................................................................................................................................................................................. 1

5 Principe .......................................................................................................................................................................................................................... 3

6 Précautions de sécurité ................................................................................................................................................................................. 3

7 Appareillage .............................................................................................................................................................................................................. 3

8 Réactifs ........................................................................................................................................................................................................................... 4

9 Échantillonnage et préparation des échantillons .............................................................................................................. 5

10 Mode opératoire.................................................................................................................................................................................................... 5

10.1 Dégraissage ................................................................................................................................................................................................ 5

10.2 Clivage réducteur .................................................................................................................................................................................. 5

10.3 Extraction liquide-liquide .............................................................................................................................................................. 5

10.4 Vérification du système d’analyse .......................................................................................................................................... 6

11 Analyses chromatographiques .............................................................................................................................................................. 6

12 Étalonnage .................................................................................................................................................................................................................. 6

13 Évaluation .................................................................................................................................................................................................................... 7

13.1 Calcul de la concentration en amine dans l’échantillon ...................................................................................... 7

13.2 Fiabilité de la méthode ..................................................................................................................................................................... 7

14 Rapport d’essai ....................................................................................................................................................................................................... 7

Annexe A (informative) Analyses chromatographiques ................................................................................................................... 9

Annexe B (informative) Fiabilité de la méthode ....................................................................................................................................13

Annexe C (informative) Guide d’appréciation — Interprétation des résultats d’analyse ..........................14

Annexe D (informative) Mode opératoire d’extraction liquide/liquide sans terre de diatomées ....20

Bibliographie ...........................................................................................................................................................................................................................23

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ISO 17234-1:2020(F)
IULTCS/IUC 20-1:2020(F)
Avant-propos

L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes

nationaux de normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est

en général confiée aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude

a le droit de faire partie du comité technique créé à cet effet. Les organisations internationales,

gouvernementales et non gouvernementales, en liaison avec l'ISO participent également aux travaux.

L'ISO collabore étroitement avec la Commission électrotechnique internationale (IEC) en ce qui

concerne la normalisation électrotechnique.

Les procédures utilisées pour élaborer le présent document et celles destinées à sa mise à jour sont

décrites dans les Directives ISO/IEC, Partie 1. Il convient, en particulier, de prendre note des différents

critères d'approbation requis pour les différents types de documents ISO. Le présent document a été

rédigé conformément aux règles de rédaction données dans les Directives ISO/IEC, Partie 2 (voir www

.iso .org/ directives).

L'attention est attirée sur le fait que certains des éléments du présent document peuvent faire l'objet de

droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour responsable

de ne pas avoir identifié de tels droits de propriété et averti de leur existence. Les détails concernant

les références aux droits de propriété intellectuelle ou autres droits analogues identifiés lors de

l'élaboration du document sont indiqués dans l'Introduction et/ou dans la liste des déclarations de

brevets reçues par l'ISO (voir www .iso .org/ brevets).

Les appellations commerciales éventuellement mentionnées dans le présent document sont données

pour information, par souci de commodité, à l’intention des utilisateurs et ne sauraient constituer un

engagement.

Pour une explication de la nature volontaire des normes, la signification des termes et expressions

spécifiques de l'ISO liés à l'évaluation de la conformité, ou pour toute information au sujet de l'adhésion

de l'ISO aux principes de l’Organisation mondiale du commerce (OMC) concernant les obstacles

techniques au commerce (OTC), voir www .iso .org/ avant -propos.

Le présent document a été élaboré par la Commission des essais chimiques de l’Union internationale des

sociétés de techniciens et chimistes du cuir (commission IUC, IULTCS), en collaboration avec le comité

technique du Comité européen de normalisation (CEN) CEN/TC 289, Cuir, dont le secrétariat est tenu

par l’UNI, conformément à l’Accord de coopération technique entre l’ISO et le CEN (Accord de Vienne).

Cette méthode est techniquement similaire à la méthode IUC 20, qui a été déclarée méthode officielle à

la réunion des délégués IULTCS de Cancun, Mexique, le 31 mai 2003.

L'IULTCS est une organisation mondiale de sociétés professionnelles des industries du cuir fondée en

1897 ayant pour mission de favoriser l'avancement des sciences et technologies du cuir. L'IULTCS a trois

commissions, qui sont responsables de l'établissement des méthodes internationales d'échantillonnage

et d'essai des cuirs. L'ISO reconnaît l'IULTCS en tant qu'organisme international à activités normatives

pour l'élaboration de méthodes d'essai relatives au cuir.

Cette troisième édition annule et remplace la deuxième édition (ISO 17234-1:2015), qui a fait l’objet

d’une révision technique.

Les principales modifications par rapport à l’édition précédente sont les suivantes:

— ajout d’un nouvel Article 3;
— apport de modifications techniques à l’Article 7 et à l’Article 8;

— apport de modifications à l’Article 9 et à l’Article 10 visant à améliorer la méthode;

— développement de l’Annexe C pour inclure des exemples de résultats faussement positifs, des

suggestions de modes opératoires et des suggestions d’observations à intégrer dans le rapport

d’essai;
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— ajout d’une nouvelle Annexe D, informative.

Une liste de toutes les parties de la série ISO 17234 se trouve sur le site web de l’ISO.

Il convient que l’utilisateur adresse tout retour d’information ou toute question concernant le présent

document à l’organisme national de normalisation de son pays. Une liste exhaustive desdits organismes

se trouve à l’adresse www .iso .org/ fr/ members .html.
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ISO 17234-1:2020(F)
NORME INTERNATIONALE
IULTCS/IUC 20-1:2020(F)
Cuir — Essais chimiques pour le dosage de certains
colorants azoïques dans les cuirs teints —
Partie 1:
Dosage de certaines amines aromatiques dérivées des
colorants azoïques
1 Domaine d’application

Le présent document spécifie une méthode permettant de déterminer l’utilisation de certains colorants

azoïques qui peuvent libérer certaines amines aromatiques.
2 Références normatives

Les documents suivants sont cités dans le texte de sorte qu’ils constituent, pour tout ou partie de leur

contenu, des exigences du présent document. Pour les références datées, seule l’édition citée s’applique.

Pour les références non datées, la dernière édition du document de référence s'applique (y compris les

éventuels amendements).

ISO 2418, Cuir — Essais chimiques, physiques, mécaniques et de solidité — Emplacement de l'échantillonnage

ISO 3696, Eau pour laboratoire à usage analytique — Spécification et méthodes d'essai

ISO 4044, Cuir — Essais chimiques - Préparation des échantillons pour essais chimiques

ISO 17234-2, Cuir — Essais chimiques pour le dosage de certains colorants azoïques dans les cuirs teints —

Partie 2: Dosage du 4-aminoazobenzène
3 Termes et définitions
Aucun terme n’est défini dans le présent document.

L’ISO et l’IEC tiennent à jour des bases de données terminologiques destinées à être utilisées en

normalisation, consultables aux adresses suivantes:

— ISO Online browsing platform: disponible à l’adresse https:// www .iso .org/ obp

— IEC Electropedia: disponible à l’adresse http:// www .electropedia .org/
4 Généralités

Certains colorants azoïques peuvent libérer, par clivage réducteur d’un ou de plusieurs groupes

azoïques, une ou plusieurs des amines aromatiques répertoriées dans l’Appendice 8 du Règlement

[2] [3]

européen 1907/2006, Annexe XVII , et dans la norme GB 20400-2006 (voir Tableau 1).

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Tableau 1 — Amines aromatiques répertoriées dans l’Appendice 8 du Règlement
[2] [3]
européen 1907/2006, Annexe XVII , et dans la norme GB 20400-2006
No Numéro CAS Numéro d’index Numéro CE Substances
1 92–67–1 612–072–00–6 202–177–1 biphényl-4-ylamine
4-aminobiphényle
xénylamine
2 92–87–5 612–042–00–2 202–199–1 benzidine
3 95–69–2 612–196–00–0 202–441–6 4-chloro-o-toluidine
4 91–59–8 612–022–00–3 202–080–4 2-naphtylamine
5 97–56–3 611–006–00–3 202–591–2 o-aminoazotoluène
4-amino-2’,3-diméthylazobenzène
4-o-tolylazo-o-toluidine
6 99–55–8 612–210–00–5 202–765–8 5-nitro-o-toluidine
2-amino-4-nitrotoluène
7 106–47–8 612–137–00–9 203–401–0 4-chloroaniline
8 615–05–4 612–200–00–0 210–406–1 4-méthoxy-m-phénylènediamine
2,4-diaminoanisole
9 101–77–9 612–051–00–1 202–974–4 4,4’-méthylènedianiline
4,4’-diaminodiphénylméthane
10 91–94–1 612–068–00–4 202–109–0 3,3’-dichlorobenzidine
3,3’-dichlorobiphényl-4,4’-ylènediamine
11 119–90–4 612–036–00-X 204–355–4 3,3’-diméthoxybenzidine
o-dianisidine
12 119–93–7 612–041–00–7 204–358–0 3,3’-diméthylbenzidine
4,4’-bi-o-toluidine
13 838–88–0 612–085–00–7 212–658–8 4,4’-méthylènedi-o-toluidine
14 120–71–8 612–209–00–X 204–419–1 6-méthoxy-m-toluidine
p-crésidine
15 101–14–4 612–078–00–9 202–918–9 4,4’-méthylène-bis-(2-chloro-aniline)
2,2’-dichloro-4.4’-méthylène-dianiline
16 101–80–4 612–199–00–7 202–977–0 4,4’-oxydianiline
17 139–65–1 612–198–00–1 205–370–9 4,4’-thiodianiline
18 95–53–4 612–091–00-X 202–429–0 o-toluidine
2-aminotoluène
19 95–80–7 612–099–00–3 202–453–1 4-méthyl-m-phénylènediamine
2,4-toluènediamine
2,4-diaminotoluène
20 137–17–7 612–197–00–6 205–282–0 2,4,5-triméthylaniline
21 90–04–0 612–035–00–4 201–963–1 o-anisidine
(2-méthoxyaniline)
22 60–09–3 611–008–00–4 200–453–6 4-aminoazobenzène
23 95–68–1 612–027–00–0 202–440–0 2,4-xylidine
2,4-diméthylbenzène-1-amine
24 87–62–7 612–161–00-X 201–758–7 2,6-xylidine
2,6-diméthylbenzène-1-amine
a o o o

Les numéros CAS 97–56–3 (n 5) et 99–55–8 (n 6) sont en outre réduits en numéros CAS 95–53–4 (n 18) et

95-80-7 (n 19).

Les colorants azoïques pouvant former du 4-aminoazobenzène génèrent, dans les conditions de la présente méthode,

de l’aniline (numéro CAS 62–53–3) et de la 1,4-phénylènediamine (numéro CAS 106–50–3). La présence de ces colorants

doit faire l’objet d’un essai selon l’ISO 17234-2.
Amines aromatiques supplémentaires figurant dans la norme GB 20400–2006.
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5 Principe

Après dégraissage, l’échantillon de cuir est traité par une solution de dithionite après extraction avec un

tampon aqueux (pH 6) à 70 °C dans un flacon étanche. Les amines libérées par le processus de clivage

réducteur sont transférées dans une phase éther t-butylméthylique (8.5) par extraction liquide/liquide

en utilisant des colonnes de terre de diatomées. L’extrait d’éther t-butylméthylique (8.5) est ensuite

concentré dans des conditions ménagées à l’aide d’un évaporateur rotatif sous vide et le résidu est repris

dans un solvant approprié, en fonction de la méthode utilisée pour doser les amines (voir Annexe A).

Le dosage des amines est réalisé par chromatographie en phase liquide haute performance (CLHP) avec

un détecteur à barrette de diodes (DAD) ou un spectromètre de masse (CLHP-SM), chromatographie

en phase gazeuse sur colonne capillaire avec un spectromètre de masse (CG-SM) ou électrophorèse

capillaire avec un détecteur à barrette de diodes (EC-DAD), ou qualitativement par chromatographie

sur couche mince (CCM, CCMHP).

Les amines doivent être identifiées à l’aide d’au moins deux méthodes de séparation chromatographique

différentes pour éviter les erreurs d’interprétation causées par les interférences possibles (telles que

les isomères de position des amines à identifier) et donc des déclarations incorrectes. La quantification

des amines doit être effectuée par chromatographie en phase liquide haute performance avec détecteur

à barrette de diodes ou par CG-SM.

Une méthode de criblage au moyen d’une extraction liquide/liquide sans colonne de terre de diatomées

est décrite à l’Annexe D.
6 Précautions de sécurité

AVERTISSEMENT — Les amines aromatiques répertoriées à l’Article 4 sont classées comme des

substances présentant un risque cancérigène connu ou suspecté pour l’homme.

6.1 Il incombe à l’utilisateur d’appliquer des techniques sûres et appropriées lors de la manipulation

des produits indiqués dans la présente méthode d’essai. S’adresser aux fabricants afin d’obtenir des

informations spécifiques, telles que les fiches de données de sécurité et autres recommandations.

6.2 Il convient de respecter les bonnes pratiques de laboratoire. Porter des lunettes de sécurité

dans toutes les zones du laboratoire ainsi qu’un masque anti-poussière et des gants jetables lors de la

manipulation des colorants en poudre et des amines aromatiques.

6.3 Des réglementations nationales et locales en matière de sécurité peuvent s’appliquer.

7 Appareillage
Matériel courant de laboratoire et, en particulier:

7.1 Flacon de réaction approprié, en verre résistant à la chaleur, avec bouchon étanche.

7.2 Système de chauffage approprié, opérant à (70 ± 2) °C.

7.3 Colonne en polypropylène ou en verre, de 25 mm à 30 mm de diamètre intérieur, de 130 mm à

150 mm de longueur, contenant 20 g de terre de diatomées, équipée d’un filtre en fibre de verre en sortie.

Il est possible soit d’acheter des colonnes de terre de diatomées préremplies et prêtes à l’emploi, soit

d’introduire 20 g de terre de diatomées dans une colonne en verre ou en polypropylène présentant les

dimensions susmentionnées.
7.4 Évaporateur rotatif sous vide avec contrôle du vide et bain-marie.
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7.5 Pipettes de tailles requises ou variables.
7.6 Bain à ultrasons avec thermostat.
7.7 Matériel de chromatographie choisi dans la liste suivante:

7.7.1 Système de chromatographie liquide haute performance (CLHP) avec détecteur à barrette

de diodes (DAD) ou spectromètre de masse (SM).

7.7.2 Appareil de chromatographie gazeuse à colonne capillaire (CG) avec spectromètre de

masse (SM).

7.7.3 Appareil d’électrophorèse capillaire (EC) avec détecteur à barrette de diodes (DAD).

7.7.4 Appareil de chromatographie en couche mince (CCM) ou appareil de chromatographie en

couche mince à haute performance (CCMHP).
NOTE L’Annexe A fournit une description des appareils de chromatographie (7.7).
8 Réactifs

Sauf spécification contraire, des produits chimiques de qualité analytique doivent être utilisés.

8.1 n-hexane.
8.2 Solution tampon de citrate, 0,06 mol/l, pH = 6, préchauffée à (70 ± 5) °C.

8.3 Dithionite de sodium en solution aqueuse, ρ = 200 mg/ml , préparée extemporanément, à

utiliser immédiatement après un temps de repos de 1 h dans un récipient fermé.
8.4 Solution aqueuse d’hydroxyde de sodium, 40 % en fraction massique.
8.5 Éther t-butylméthylique.
8.6 Méthanol.
8.7 Acétonitrile.
8.8 Amines, figurant dans le Tableau 1 (niveau de pureté maximal disponible).
8.9 Solutions étalons.

8.9.1 Solution mère des amines (8.8), 400 µg/ml dans de l’acétate d’éthyle pour CCM.

8.9.2 Solution mère des amines (8.8), 200 µg/ml de chaque amine dans un solvant approprié.

NOTE L’acétonitrile est un solvant approprié pour cette solution mère, assurant une bonne stabilité des amines.

8.9.3 Solution étalon pour le contrôle du traitement des amines, 30 µg d’amine par millilitre de

solvant, préparée extemporanément à partir de la solution mère (8.9.1 ou 8.9.2) correspondant à la

méthode d’analyse.
1) ρ = concentration massique.
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8.9.4 Étalon interne en solution, ρ = 10 μg d’étalon interne/ml d’éther t-butylméthylique (8.5).

En cas d’analyse par chromatographie en phase gazeuse et spectrométrie de masse (CG-SM), un des

étalons internes suivants peut être utilisé:
— étalon interne 1: naphtalène-d8, n CAS: 1146-65-2;
— étalon interne 2: 2,4,5-trichloroaniline (TCA), n CAS: 636-30-6;
— étalon interne 3: anthracène-d10, n CAS: 1719-06-8.
8.10 Eau, de qualité 3 conformément à l’ISO 3696.
9 Échantillonnage et préparation des échantillons

Le cuir doit être échantillonné conformément à l’ISO 2418 et préparé conformément à l’ISO 4044. Si

l’échantillonnage conformément à l’ISO 2418 n’est pas possible (par exemple, dans le cas de cuirs de

produits finis tels que des chaussures ou des vêtements), les informations relatives au prélèvement des

échantillons doivent être fournies dans le rapport d’essai. Toute trace d’adhésifs doit être éliminée par

voie mécanique.

Dans le cas de patchworks de cuirs de différentes couleurs, chaque couleur doit, dans la mesure du

possible, être prise en compte séparément. Pour les articles constitués de différentes qualités de cuir,

des éprouvettes des différentes qualités doivent être analysées séparément.

Pour l’analyse, peser avec précision un échantillon représentatif de 1,0 g de cet échantillon de cuir dans

le flacon de réaction (7.1).
10 Mode opératoire
10.1 Dégraissage

Traiter 1,0 g du cuir dans un flacon fermé de 50 ml (7.1) contenant 40 ml de n-hexane (8.1), dans un bain

à ultrasons (7.6) à (40 ± 2) °C, pendant 40 min.

Laisser décanter la couche de n-hexane imprégnant l’échantillon de cuir. Durant la décantation, toute

perte de particules de cuir doit être évitée. Laisser évaporer le n-hexane résiduel au moins toute la nuit

dans le flacon ouvert.
10.2 Clivage réducteur

Ajouter à l’échantillon 15 ml de solution tampon (8.9) préchauffée à (70 ± 2) °C.

Refermer hermétiquement le flacon de réaction et traiter pendant (30 ± 1) min à (70 ± 2) °C.

Ajouter ensuite 3 ml de solution aqueuse de dithionite de sodium (8.3), pour le clivage réducteur des

groupes azoïques, dans le flacon de réaction; agiter vigoureusement ce dernier avant de le replacer

immédiatement à (70 ± 2) °C pendant (30 ± 1) min supplémentaires. Refroidir ensuite jusqu’à

température ambiante (20 °C à 25 °C) dans un délai de 2 min, au moyen d’un mélange réfrigérant de

glace, d’eau et de sel.
10.3 Extraction liquide-liquide

Ajouter 1,5 ml de solution de NaOH (8.4) à la solution de réaction et agiter vigoureusement. Transférer

la solution de réaction dans la colonne de terre de diatomées (7.3) et laisser la colonne l’absorber

pendant 15 min.
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Pendant ce temps, ajouter 10 ml d’éther t-butylméthylique (8.5) dans le flacon de réaction et agiter

vigoureusement. À l’issue de la période de 15 min, transvaser l’éther t-butylméthylique (8.5) sur le

sommet de la colonne et recueillir l’éluat dans un ballon à fond rond de 250 ml.

Rincer le flacon de réaction avec 10 ml d’éther t-butylméthylique (8.5) et transférer le solvant dans la

colonne. Ensuite, verser 60 ml d’éther t-butylméthylique (8.5) directement dans la colonne.

Pour la détection et la quantification des amines, l’extrait d’éther t-butylméthylique est concentré

à un volume inférieur à 5 ml (pas jusqu’à siccité) au moyen d’un évaporateur rotatif sous vide, à une

température inférieure à 50 °C et à une pression d’environ 450 mbar. S’il est nécessaire d’utiliser un autre

solvant, éliminer avec beaucoup de précaution le reste du solvant au moyen d’un faible flux de gaz inerte.

NOTE 1 L’élimination du solvant (concentration dans l’évaporateur rotatif sous vide, évaporation à siccité)

peut entraîner des pertes importantes d’amines si elle est réalisée dans des conditions non contrôlées.

Compléter immédiatement l’extrait ou le résidu à 2,0 ml avec un solvant approprié pour la détection

et le dosage des amines par chromatographie [acétonitrile (8.7), éther t-butylméthylique (8.5) ou

méthanol (8.6)]. Si l’analyse complète ne peut être effectuée dans les (24 ± 1) h, conserver l’extrait

à (−18 ± 3) °C; le réchauffer avec précaution jusqu’à température ambiante avant de procéder à l’analyse.

NOTE 2 Du fait de la matrice, certaines amines, telles que le 2,4-diaminotoluène et le 2,4-diaminoanisole, sont

susceptibles d’être très peu stables, en particulier dans le méthanol. En cas de retards dans le programme de

travail, les amines peuvent ne plus être détectables au moment de procéder au mesurage avec les instruments.

10.4 Vérification du système d’analyse

Pour vérifier le mode opératoire d’analyse, ajouter, dans un flacon de réaction contenant 15 ml de

solution tampon (8.9), une certaine quantité d’amines de sorte à obtenir une concentration finale

de x mg/l.
Suivre ensuite le mode opératoire décrit en 10.3.

Les taux de récupération des amines doivent être conformes aux exigences minimales suivantes:

— amines n 1 à 4, 7, 9 à 17 et 20 à 21: taux de récupération de 70 %;
— amine n 8: taux de récupération de 20 %;
— amines n 18, 19, 23 et 24: taux de récupération de 50 %;
— amines n 5, 6 et 22: voir les notes du Tableau 1.
11 Analyses chromatographiques

La détection des amines aromatiques peut être réalisée au moyen des techniques chromatographiques

mentionnées en 7.7. D’autres méthodes validées peuvent être employées. La quantification des

amines aromatiques est réalisée par CLHP-DAD, par CLHP-SM ou par CG-SM. En cas d’utilisation de la

chromatographie en phase gazeuse, des étalons internes appropriés (voir 8.9.4) doivent être employés.

En cas de détection d’une amine par une méthode de chromatographie, une confirmation doit être

effectuée en utilisant une ou plusieurs autres méthodes. Le résultat n’est positif que si les deux

méthodes donnent un résultat positif.
12 Étalonnage

Utiliser la solution étalon (8.9.2) pour préparer au moins trois solutions d’étalonnage de concentrations

comprises entre 2 µg/ml et 30 µg/ml.
6 © ISO 2020 – Tous droits réservés
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ISO 17234-1:2020(F)
IULTCS/IUC 20-1:2020(F)
13 Évaluation
13.1 Calcul de la concentration en amine dans l’échantillon

La concentration en amine est calculée sur la base de l’aire des pics de chaque composant amine. Calculer

la teneur en amine sous forme de fraction massique, w, en milligrammes de composant individuel par

kilogramme (mg/kg) de cuir à l’aide de la Formule (1):
AV×
w=×ρ (1)
Am×

ρ est la concentration de l’amine dans la solution d’étalonnage, en microgrammes par millilitre

(μg/ml);

A est l’aire du pic de l’amine, en unités de surface, dans la solution échantillon;

A est l’aire du pic de l’amine, en unités de surface, dans la solution d’étalonnage;

V est le volume de l’échantillon selon 10.3 (volume final d’échantillon), en millilitres (ml) (en

l’occurrence, 2 ml);
m est la masse de l’échantillon de cuir, en grammes (g).
13.2 Fiabilité de la méthode
Pour ce qui concerne la fiabilité de la méthode, voir l’Annexe B.
14 Rapport d’essai

Le rapport d’essai doit faire référence à la présente méthode officielle et fournir des informations au

minimum sur les points suivants:
a) une référence au présent document, c’est-à-dire l’ISO 17234-1:2020;
b) l’identification de l’échantillon;
c) le mode opératoire d’échantillonnage;

d) tout écart par rapport au mode opératoire d’analyse, notamment toutes les opérations

complémentaires effectuées;

e) la mention des techniques d’analyse utilisées pour la détection et la confirmation;

f) la date de l’essai;

g) les résultats d’analyse pour les amines, en milligrammes par kilogramme (voir l’Article 13),

consignés individuellement suivant les valeurs des seuils d’identification comme suit:

Dans le cas de teneurs par composant amine ≤ 30 mg/kg:

Conformément au type d’analyse effectuée, l’analyse n’a pas détecté de colorants azoïques ayant libéré

les amines aromatiques répertoriées.
Dans le cas de teneurs par composant amine > 30 mg/kg:

Le résultat d’analyse suggère que le cuir soumis à essai a été fabriqué ou traité en utilisant des colorants

azoïques qui libèrent une ou plusieurs des amines répertoriées.
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ISO 17234-1:2020(F)
IULTCS/IUC 20-1:2020(F)
Dans le cas de teneurs en 4-aminodiphényl et/ou 2-naphtylamine > 30 mg/kg:

L’utilisation de cette méthode d’analyse a permis de détecter du 4-aminodiphényl et/ou 2-naphtylamine.

Dans l’état actuel des connaissances techniques, il ne peut pas être indiqué de façon affirmative sans

...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 17234-1
IULTCS/IUC 20-1
IULTCS
Leather — Chemical tests for the
Voting begins on:
2020-05-01
determination of certain azo colorants
Voting terminates on: in dyed leathers —
2020-06-26
Part 1:
Determination of certain aromatic
amines derived from azo colorants
Cuir — Essais chimiques pour le dosage de certains colorants
azoïques dans les cuirs teints —
Partie 1: Dosage de certaines amines aromatiques dérivées des
colorants azoïques
ISO/CEN PARALLEL PROCESSING
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference numbers
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/FDIS 17234-1:2020(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
IULTCS/IUC 20-1:2020(E)
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. ISO 2020
---------------------- Page: 1 ----------------------
ISO/FDIS 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
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ISO/FDIS 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 General ............................................................................................................................................................................................................................ 1

5 Principle ........................................................................................................................................................................................................................ 3

6 Safety precautions .............................................................................................................................................................................................. 3

7 Apparatus ..................................................................................................................................................................................................................... 3

8 Reagents ........................................................................................................................................................................................................................ 4

9 Sampling and preparation of samples ........................................................................................................................................... 5

10 Procedure..................................................................................................................................................................................................................... 5

10.1 Degreasing .................................................................................................................................................................................................. 5

10.2 Reductive cleavage ............................................................................................................................................................................... 5

10.3 Liquid-liquid extraction .................................................................................................................................................................. 5

10.4 Check of the analytical system ................................................................................................................................................... 6

11 Chromatographic analyses ......................................................................................................................................................................... 6

12 Calibration .................................................................................................................................................................................................................. 6

13 Evaluation .................................................................................................................................................................................................................... 6

13.1 Calculation of amine in the sample ....................................................................................................................................... 6

13.2 Reliability of the method ................................................................................................................................................................ 7

14 Test report ................................................................................................................................................................................................................... 7

Annex A (informative) Chromatographic analyses ................................................................................................................................ 9

Annex B (informative) Reliability of the method ..................................................................................................................................13

Annex C (informative) Assessment guide — Interpretation of analytical results ..............................................14

Annex D (informative) Procedure for liquid/liquid extraction without diatomaceous earth ..............20

Bibliography .............................................................................................................................................................................................................................23

© ISO 2020 – All rights reserved iii
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ISO/FDIS 17234-1:2020(E)
IULTCS/IUC 20-1:2020(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

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

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

constitute an endorsement.

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 the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/

iso/ foreword .html.

This document was prepared by the Chemical Test Commission of the International Union of Leather

Technologists and Chemists Societies (IUC Commission, IULTCS) in collaboration with the European

Committee for Standardization (CEN) Technical Committee CEN/TC 289, Leather, the secretariat of

which is held by UNI, in accordance with the Agreement on technical cooperation between ISO and CEN

(Vienna Agreement). This method is technically similar to the method in IUC 20, which was declared an

official method at the IULTCS Delegates meeting on 31st May 2003 in Cancun, Mexico.

IULTCS, originally formed in 1897, is a world-wide organization of professional leather societies to

further the advancement of leather science and technology. IULTCS has three Commissions, which

are responsible for establishing international methods for the sampling and testing of leather. ISO

recognizes IULTCS as an international standardizing body for the preparation of test methods for

leather.

This third edition cancels and replaces the second edition (ISO 17234-1:2015), which has been

technically revised.
The main changes to the previous edition are as follows:
— a new Clause 3 added;
— technical changes to Clause 7 and Clause 8;
— changes to Clause 9 and Clause 10 to improve the method;

— Annex C expanded to give examples of false-positive results, suggested procedures and suggested

comments in the test report;
— a new informative Annex D added.
A list of all parts in the ISO 17234 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2020 – All rights reserved
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ISO/FDIS 17234-1:2020(E)
FINAL DRAFT INTERNATIONAL STANDARD
IULTCS/IUC 20-1:2020(E)
Leather — Chemical tests for the determination of certain
azo colorants in dyed leathers —
Part 1:
Determination of certain aromatic amines derived from
azo colorants
1 Scope

This document specifies a method for determining the use of certain azo colorants which can release

certain aromatic amines.
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 2418, Leather — Chemical, physical and mechanical and fastness tests — Sampling location

ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 4044, Leather — Chemical tests — Preparation of chemical test samples

ISO 17234-2, Leather — Chemical tests for the determination of certain azo colorants in dyed leathers —

Part 2: Determination of 4-aminoazobenzene
3 Terms and definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
4 General

Certain azo colorants can release, by reductive cleavage of azo group(s), one or more of the aromatic

[2] [3]

amines listed in EU Regulation 1907/2006, Annex XVII, Appendix 8 and GB 20400-2006 (see Table 1).

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ISO/FDIS 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
[2]

Table 1 — Aromatic amines listed in EU Regulation 1907/2006, Annex XVII, Appendix 8 and

[3]
GB 20400-2006
No. CAS number Index number EC number Substances
1 92–67–1 612–072–00–6 202–177–1 biphenyl-4-ylamine
4-aminobiphenyl
xenylamine
2 92–87–5 612–042–00–2 202–199–1 benzidine
3 95–69–2 612–196–00–0 202–441–6 4-chloro-o-toluidine
4 91–59–8 612–022–00–3 202–080–4 2-naphthylamine
5 97–56–3 611–006–00–3 202–591–2 o-aminoazotoluene
4-amino-2',3-dimethylazobenzene
4-o-tolylazo-o-toluidine
6 99–55–8 612–210–00–5 202–765–8 5-nitro-o-toluidine
2-amino-4-nitrotoluene
7 106–47–8 612–137–00–9 203–401–0 4-chloroaniline
8 615–05–4 612–200–00–0 210–406–1 4-methoxy-m-phenylenediamine
2,4-diaminoanisole
9 101–77–9 612–051–00–1 202–974–4 4,4'-methylenedianiline
4,4'-diaminodiphenylmethane
10 91–94–1 612–068–00–4 202–109–0 3,3'-dichlorobenzidine
3,3'-dichlorobiphenyl-4,4'-ylenediamine
11 119–90–4 612–036–00-X 204–355–4 3,3'-dimethoxybenzidine
o-dianisidine
12 119–93–7 612–041–00–7 204–358–0 3,3'-dimethylbenzidine
4,4'-bi-o-toluidine
13 838–88–0 612–085–00–7 212–658–8 4,4'-methylenedi-o-toluidine
14 120–71–8 612–209–00–X 204–419–1 6-methoxy-m-toluidine
p-cresidine
15 101–14–4 612–078–00–9 202–918–9 4,4'-methylene-bis-(2-chloro-aniline)
2,2'-dichloro-4.4’-methylene-dianiline
16 101–80–4 612–199–00–7 202–977–0 4,4'-oxydianiline
17 139–65–1 612–198–00–1 205–370–9 4,4'-thiodianiline
18 95–53–4 612–091–00-X 202–429–0 o-toluidine
2-aminotoluene
19 95–80–7 612–099–00–3 202–453–1 4-methyl-m-phenylenediamine
2,4-toluylendiamine
2,4-diaminotoluene
20 137–17–7 612–197–00–6 205–282–0 2,4,5-trimethylaniline
21 90–04–0 612–035–00–4 201–963–1 o-anisidine
2-methoxyaniline
22 60–09–3 611–008–00–4 200–453–6 4-aminoazobenzene
23 95–68–1 612–027–00–0 202–440–0 2,4-xylidine
2,4-dimethylbenzene-1-amine
24 87–62–7 612–161–00-X 201–758–7 2,6-xylidine
2,6-dimethylbenzene-1-amine

The CAS-numbers 97–56–3 (No. 5) and 99–55–8 (No. 6) are further reduced to CAS-numbers 95–53–4 (No. 18) and

95–80–7 (No. 19).

Azo colorants that are able to form 4-aminoazobenzene generate under the condition of this method aniline CAS-

number 62–53–3) and 1,4-phenylenediamine (CAS number 106–50–3). The presence of these colorants shall be tested

using ISO 17234-2.
Additional aromatic amines in GB 20400–2006.
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ISO/FDIS 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
5 Principle

After degreasing, the leather sample is treated with sodium dithionite in an aqueous buffer solution

(pH 6) at 70 °C in a closed vessel. The amines released in the process of reductive cleavage are transferred

to a t-butyl methyl ether (8.5) phase by means of liquid-liquid extraction using diatomaceous earth

columns. The t-butyl methyl ether (8.5) extract is then concentrated under mild conditions in a rotary

vacuum evaporator and the residue is dissolved in a suitable solvent, depending on the method used to

determine the amines (see Annex A).

Determination of the amines is performed by means of high-performance liquid chromatography

(HPLC) using a diode array detector (DAD) or mass selective detector (HPLC-MS), capillary gas

chromatography with a mass selective detector (GC-MS) or by capillary electrophoresis with a diode

array detector (CE-DAD), or qualitatively with thin layer chromatography (TLC, HPTLC).

The amines shall be identified by means of at least two different chromatographic separation methods

in order to avoid any possible misinterpretations caused by interfering substances (such as position

isomers of the amines to be identified) and hence any incorrect statements. Amine quantification shall

be performed by HPLC-DAD or GC-MS.

A screening method using liquid-liquid extraction without diatomaceous earth columns is described in

Annex D.
6 Safety precautions

WARNING — The aromatic amines listed in Clause 4 are classified as substances known to be or

suspected to be human carcinogens.

6.1 It is the user's responsibility to use safe and proper techniques when handling materials in this

test method. Consult manufacturers for specific details, such as material safety data sheets and other

recommendations.

6.2 Good laboratory practice should be followed. Wear safety glasses in all laboratory areas and a dust

respirator and single-use gloves while handling powder colorants and aromatic amines.

6.3 National and local safety regulations can apply.
7 Apparatus
The usual laboratory equipment and, in particular, the following:

7.1 Suitable reaction vessel, of temperature-resistant glass with a gas-tight closure.

7.2 Suitable heating system, at (70 ± 2) °C.

7.3 Polypropylene or glass column, inside diameter 25 mm to 30 mm, length 130 mm to 150 mm,

packed with 20 g of diatomaceous earth, fitted with glass fibre filter at the outlet.

The diatomaceous earth columns are either bought pre-packed and used as is, or 20 g of diatomaceous

earth can be packed into a glass or polypropylene column of the dimensions given.

7.4 Vacuum rotary evaporator with vacuum control and water bath.
7.5 Pipettes, in required sizes or variables pipettes.
7.6 Ultrasonic bath with thermostat.
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ISO/FDIS 17234-1:2020(E)
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7.7 Chromatographic equipment, selected from the following:
7.7.1 High-performance liquid chromatography (HPLC) and DAD or MS.
7.7.2 Capillary gas chromatography (GC), with MS.
7.7.3 Capillary electrophoresis (CE), with DAD.

7.7.4 Thin layer chromatography (TLC) or high-performance thin layer chromatography

(HPTLC).
NOTE A description of the chromatographic equipment (7.7) is given in Annex A.
8 Reagents
Unless otherwise specified, analytical grade chemicals shall be used.
8.1 n-hexane.
8.2 Citrate buffer solution, 0,06 mol/l, pH = 6, preheated to (70 ± 5) °C.

8.3 Aqueous sodium dithionite solution, ρ = 200 mg/ml , freshly prepared, to be used immediately

after resting for 1 h in a closed vessel.
8.4 Sodium hydroxide aqueous solution, a mass fraction of 40 %.
8.5 t-butyl methyl ether.
8.6 Methanol.
8.7 Acetonitrile.
8.8 Amines, listed in Table 1 (highest available purity standard).
8.9 Standard solutions.
8.9.1 Stock solution of the amines (8.8), 400 µg/ml in ethyl acetate for TLC.

8.9.2 Stock solution of the amines (8.8), 200 µg/ml of each amine in an appropriate solvent.

NOTE Acetonitrile is an appropriate solvent for this stock solution, resulting in good stability of amines.

8.9.3 Standard solution for amine process control, 30 µg amine per millilitre solvent, freshly

prepared from stock solutions (8.9.1 or 8.9.2) depending on the analytical method

8.9.4 Internal standard in solution (IS), ρ = 10 μg of IS/ml of t-butyl methyl ether (8.5).

In the case of GC-MS analysis, one of the following internal standards can be used:

— IS1: naphthalene-d8, CAS No.: 1146-65-2;
— IS2: 2,4,5-trichloroaniline (TCA), CAS No.: 636-30-6;
1) ρ = mass concentration.
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ISO/FDIS 17234-1:2020(E)
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— IS3: anthracene-d10, CAS No.: 1719-06-8.
8.10 Water, Grade 3 according to ISO 3696.
9 Sampling and preparation of samples

The leather shall be sampled in accordance with ISO 2418 and prepared in accordance with ISO 4044. If

sampling in accordance with ISO 2418 is not possible (e.g. in the case of leathers from finished products

such as shoes or garments), details about sampling shall be given in the test report. Any traces of

adhesives shall be removed mechanically.

In the case of leather patchwork fabrics with varicoloured patterns, the various colours shall be taken

into account separately as far as possible. For commodities consisting of various leather qualities,

specimens of the various qualities shall be analysed separately.

For the analytical procedure, accurately weigh a representative sample of 1,0 g of this leather sample in

the reaction vessel (7.1).
10 Procedure
10.1 Degreasing

Treat 1,0 g of the leather in a closed 50 ml vessel (7.1) with 40 ml n-hexane (8.1) in an ultrasonic bath

(7.6) at (40 ± 2) °C for 40 min.

Decant the n-hexane layer from the leather sample. Any loss of leather particles during decanting shall

be avoided. Evaporate the residual n-hexane at least overnight in the open vessel.

10.2 Reductive cleavage

Add a quantity of 15 ml buffer solution (8.9) preheated to (70 ± 2) °C to the sample.

Close the reaction vessel tightly and treat for (30 ± 1) min at (70 ± 2) °C.

Subsequently, add 3 ml aqueous sodium dithionite solution (8.3) for the reductive cleavage of the azo

groups to the reaction vessel, then shake vigorously and immediately keep at (70 ± 2) °C for another

(30 ± 1) min. Then cool to room temperature (20 °C to 25 °C) within 2 min with a cooling mixture of ice,

water and salt.
10.3 Liquid-liquid extraction

Add 1,5 ml of the NaOH solution (8.4) to the reaction solution and shake vigorously. Transfer the reaction

solution to the diatomaceous earth column (7.3) and allow it to be absorbed by the column for 15 min.

Meanwhile, add 10 ml t-butyl methyl ether (8.5) to the reaction vessel and shake vigorously. After the

15 min period decant the t-butyl methyl ether (8.5) onto the top of the column and collect the eluate in

a 250 ml round-bottom flask.

Rinse the reaction vessel with 10 ml t-butyl methyl ether (8.5) and transfer the solvent to the column.

Subsequently, pour 60 ml t-butyl methyl ether (8.5) directly onto the column.

For amine detection and quantification, the t-butyl methyl ether extract is concentrated to a volume

less than 5 ml (not to dryness) with a vacuum rotary at a temperature less than 50 °C and a pressure

of approximately 450 mbar. If it is necessary to change to another solvent, remove the remainder of the

solvent very carefully by means of a weak flow of inert gas.

NOTE 1 Removal of the solvent (concentration in the rotary vacuum evaporator, evaporation to dryness) can

lead to substantial amine losses if performed under uncontrolled conditions.
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ISO/FDIS 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)

Make up the extract or residue to 2,0 ml with an appropriate solvent for detection and determination

of the amines using chromatography [acetonitrile (8.7), t-butyl methyl ether (8.5) or methanol (8.6)]

without delay. If the complete analysis cannot be performed within (24 ± 1) h, keep the extract at

(−18 ± 3) °C and warm carefully to room temperature before analysis.

NOTE 2 Owing to the matrix, individual amines such as 2,4-diaminotoluene and 2,4-diaminoanisole are likely

to exhibit a very poor stability, especially in methanol. Where delays occur in the work routine, amines can be no

longer detectable by the time of instrumental measurement.
10.4 Check of the analytical system

To check the analysis procedure, add a certain quantity of amines to obtain x mg/l as final concentration

in a reaction vessel containing 15 ml of buffer solution (8.9).
Then carry out the procedure set out in 10.3.
Amine recovery rates shall conform with the following minimum requirements:
— amines Nos. 1 to 4, 7, 9 to 17 and 20 to 21: recovery rate 70 %;
— amine No. 8: recovery rate 20 %;
— amines Nos. 18, 19, 23 and 24: recovery rate 50 %;
— amines Nos. 5, 6 and 22, see footnotes to Table 1.
11 Chromatographic analyses

The detection of the aromatic amines can be performed using the chromatographic techniques listed

in 7.7. Other validated methods can be used. The quantification of the aromatic amines is performed

by means of HPLC-DAD, HPLC-MS or GC-MS. Where gas chromatography is used, appropriate internal

standards as described in ISO 17234-2 shall be employed.

If any amine is detected by one chromatographic method, then confirmation shall be made using one or

more alternative methods. The result is positive only if both methods give a positive result.

12 Calibration

Use the standard solution (8.9.2) to prepare at least three calibration solutions in a range of 2 µg/ml to

30 µg/ml.
13 Evaluat ion
13.1 Calculation of amine in the sample

Calculate the amine concentration based on the peak areas of the individual amine components.

Calculate the content of the amine as a mass fraction, w, in milligrams of the individual component per

kilogram (mg/kg) of leather material according to Formula (1):
AV×
w=×ρ (1)
Am×
where
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ISO/FDIS 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)

ρ is the concentration of the amine in the calibration solution, in micrograms per millilitre

(μg/ml);
A is the peak area of the amine in the sample solution, in area units;
A is the peak area of the amine in the calibration solution, in area units;

V is the volume of the specimen according to 10.3 (final sample volume), in millilitres (ml)

(here 2 ml);
m is the mass of the leather sample, in grams (g).
13.2 Reliability of the method
For the reliability of the method, see Annex B.
14 Test report

The test report shall refer to this official method and give information on at least the following aspects:

a) a reference to this document, i.e. ISO 17234-1:—;
b) identification of the sample;
c) sampling procedure;

d) any deviations from the analytical procedure, particularly any additional steps performed;

e) declaration of analytical techniques used for detection and confirmation;
f) the date of the test;

g) the analytical results for the amines in milligrams per kilogram (see Clause 13), individually listed

and reported according to the identification threshold values as follows:
In the case of levels per amine component ≤ 30 mg/kg:

According to the analysis as carried out, azo colorants which release the listed aromatic amines were

not detected.
In the case of levels per amine component > 30 mg/kg:

The analysis result suggests that the leather submitted has been manufactured or treated using azo

colorants which release one or more of the listed amines.
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ISO/FDIS 17234-1:2020(E)
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In the case of levels of 4-aminodiphenyl and/or 2-naphthylamine > 30 mg/kg:

Use of this analytical method has detected 4-aminodiphenyl and/or 2-naphthylamine. According to the

current state of knowledge it cannot be unequivocally confirmed without additional information that

azo colorants which release amines were used.

Care should be taken in the interpretation of less than 30 mg/kg of amines as these can be due to false-

positive results. For the interpretation of results, see Annex C.
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Annex A
(informative)
Chromatographic analyses
A.1 Preliminary remark

As the chromatographic equipment (7.7) of the laboratories can vary, no generally applicable

instructions can be provided for chromatographic analyses. The following parameters have been

successfully tested and used.
A.2 High-performance liquid chromatography (HPLC)
A.2.1 High-performance liquid chromatography/diode array detector (HPLC-DAD)
Eluent 1: methanol;
Eluent 2: 0,575 g of ammonium dihydrogen phosphate + 0,7 g of disodium hydrogen
phosphate in 1 000 ml of water, pH 6,9;

Stationary phase: LiChrospher 60 RP-select B (5 µm), length: 250 mm × inside diameter: 4,6 mm;

Flow rate: (0,7 to 1,0) ml/min;
Gradient: start: 15 % eluent 1, linear increase to 80 % eluent 1 within 45 min;
Column temperature: 40 °C;
Injection volume: 10,0 μl;
Detection: DAD, spectrograph;
Quantification: at 240 nm, 280 nm, and 305 nm.

NOTE LiChrospher 60 RP-select B is an example of a suitable product available commercially. This

information is given for the convenience of users of this document and does not constitute an endorsement by ISO

of this product. Equivalent products can be used if they can be shown to lead to the same results.

See example in Figure A.1.
© ISO 2020 – All rights reserved 9
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ISO/FDIS 17234-1:2020(E)
IULTCS/IUC 20-1:2020(E)
Key
X time in min
Y absorbance in mAU at 240 nm
1 1,4-phenylenediamine
2 aniline

NOTE For aromatic amines 1 to 21, see Table 1 (aromatic amines 23 and 24 are not shown).

Figure A.1 — HLPC-DAD-chromatogram
A.2.2 High-performance liquid chromatography/mass selective detector (HPLC-MS)
Eluent 1: acetonitrile;
Eluent 2: ammonium acetate in 1 000 ml of water, 5 mmol, pH 3,0;
Stationary phase: C18 (3,5 μm); length: 50 mm; inside diameter: 2,1 mm;
Flow rate: 300 μl/min;
Gradient: see Table A.1;
Column temperature 40 °C;
Injection volume: 2,0 μl;
Detection: quadrupole- and/or ion-trap mass detector, scanning mode and/or MS
daughter ion MS detection; DAD: for wavelengths, see A.2.1;
Spray ga
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