Rubber and rubber products — Identification of antidegradants — Thin layer chromatographic methods

This document describes two methods for identification of antidegradants (antioxidants, antiozonants and stabilizers) which can be present in raw rubber, unvulcanized compounded rubber, or rubber products, by thin layer chromatography. Method A is a simplified method that provides for the identification of known materials and can be used to check the presence or absence of a particular antidegradant which is expected to be present. Method B is a more detailed method that enables a greater degree of separation of the spots to be obtained and therefore can be used to detect and identify an unknown antidegradant. Antidegradants to which these methods are applicable include phosphited polyalkyl phenols, substituted bisphenols, secondary amines, substituted cresols and substituted p-phenylenediamines. Examination for other types of antidegradants is possible under the same condition when there is a standard chromatogram.

Caoutchouc et produits à base de caoutchouc — Identification des agents de protection — Méthodes par chromatographie en couche mince

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INTERNATIONAL ISO
STANDARD 4645
Second edition
2022-05
Rubber and rubber products —
Identification of antidegradants —
Thin layer chromatographic methods
Caoutchouc et produits à base de caoutchouc — Identification des
agents de protection — Méthodes par chromatographie en couche
mince
Reference number
ISO 4645:2022(E)
© ISO 2022
---------------------- Page: 1 ----------------------
ISO 4645:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022

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

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© ISO 2022 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 4645:2022(E)
Contents Page

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

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

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

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

4 Principle ........................................................................................................................................................................................................................ 1

5 Reagents ........................................................................................................................................................................................................................ 2

6 Apparatus .................................................................................................................................................................................................................... 4

7 Preparation of developing tank and plates ............................................................................................................................. 5

7.1 Preparation of developing tank ............................................................................................................................................... 5

7.2 Preparation of plates ......................................................................................................................................................................... 5

7.3 Preparation of pre-coated plates ........................................................................................................................................... 6

8 Preparation of test portion ....................................................................................................................................................................... 6

9 Plate spotting ...................................................................... ..................................................................................................................................... 6

9.1 General ........................................................................................................................................................................................................... 6

9.2 Quantity of solution to apply ........................................................................................................................................... ........... 6

9.3 Spotting technique .............................................................................................................................................................................. 6

10 Plate development .............................................................................................................................................................................................. 7

10.1 Method A ...................................................................................................................................................................................................... 7

10.2 Method B ...................................................................................................................................................................................................... 7

11 Colour development on the plate ........................................................................................................................................................ 7

12 Expression of results .......................................................................................................................................................................................7

12.1 Method A ...................................................................................................................................................................................................... 7

12.1.1 For amine type antidegradants ................................... .......................................................................................... 7

12.1.2 For phenolic type antidegradants ....................................................................................................................... 8

12.2 Method B ...................................................................................................................................................................................................... 8

12.3 Confirmation tests .............................................................................................................................................................................. 8

13 Standard chromatograms ..........................................................................................................................................................................8

14 Test report .................................................................................................................................................................................................................. 9

Annex A (informative) Preliminary spot tests .......................................................................................................................................10

Annex B (informative) Example procedure when oil is present .........................................................................................12

iii
© ISO 2022 – All rights reserved
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ISO 4645:2022(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 Technical Committee ISO/TC 45, Rubber and rubber products,

Subcommittee SC 2, Testing and analysis.

This second edition cancels and replaces the first edition (ISO 4645:1984), which has been technically

revised. It also incorporates the Technical Corrigendum ISO 4645:1984/Cor.1:1991.

The main changes are as follows:
— the description of the principle has been improved;
— method A has been modified.

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.
© ISO 2022 – All rights reserved
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INTERNATIONAL STANDARD ISO 4645:2022(E)
Rubber and rubber products — Identification of
antidegradants — Thin layer chromatographic methods
1 Scope

This document describes two methods for identification of antidegradants (antioxidants, antiozonants

and stabilizers) which can be present in raw rubber, unvulcanized compounded rubber, or rubber

products, by thin layer chromatography.

Method A is a simplified method that provides for the identification of known materials and can be used

to check the presence or absence of a particular antidegradant which is expected to be present.

Method B is a more detailed method that enables a greater degree of separation of the spots to be

obtained and therefore can be used to detect and identify an unknown antidegradant.

Antidegradants to which these methods are applicable include phosphited polyalkyl phenols,

substituted bisphenols, secondary amines, substituted cresols and substituted p-phenylenediamines.

Examination for other types of antidegradants is possible under the same condition when there is a

standard chromatogram.
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 1407, Rubber — Determination of solvent extract

ISO 4661-2, Rubber, vulcanized — Preparation of samples and test pieces — Part 2: Chemical tests

3 Terms and definitions
No terms and definitions are listed in this document.

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

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

Due to the different chemical structures of antioxidants and their transformations, the partition

coefficients in the liquid-solid phase are different. Antidegradants are extracted from the rubber

with a solvent. The extraction solution is deposited in the form of spots on a thin layer silica gel

chromatographic plate or a glass plate coated with silica gel.

If extender oil is present, the oil is removed either by column chromatography of the extract prior to

the completion of the evaporation of the original solvent or by the development of the plate in light

petroleum prior to the normal development in an appropriate solvent.

The colour and the shape are reported. The ratio shift value R of the spots of the corresponding

antioxidant in the colour map is calculated
© ISO 2022 – All rights reserved
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ISO 4645:2022(E)

Identification of the unknown antidegradant is carried out by comparing its chromatogram with its

standard chromatograms.
5 Reagents

During the analysis, use only reagents of recognized analytical grade, and only distilled water or water

of equivalent purity.

Whenever there appears turbidity, precipitation, or colour change in the solution (in 5.5 and 5.6), it

shall be prepared again.

WARNING — Use of fume hoods when handling volatile and toxic solvents is mandatory.

Approved health and safety precautions shall be observed when using any solvent or chemical

mentioned in this document.

5.1 Plate adsorbent: silica gel, particle size 2 μm to 50 μm, with or without calcium sulfate binder.

Silica gel containing a fluorescent indicator is useful in many cases to observe spots under ultraviolet

radiation before spraying.
®1) ®1)

It is also possible to use commercial silica gel, Silica-G or Silica-HF for example.

5.2 Column adsorbent: silica gel, to pass a sieve of aperture 200 μm to 600 μm activated by drying,

either:
— for at least 2 h at 110 °C, if the product is dry after that period; or
— overnight (approximately 16 h at 110 °C) for convenience.

5.3 Silica gel plate adhesive. A solution of sodium carboy-methyl cellulose with a mass fraction

of 0,1 % to 0,2 %, prepared the day before the test. Use the clear solution (with self-made thin layer

chromatography silica gel plates).
5.4 Solvents
5.4.1 Propanol.
5.4.2 Light petroleum, boiling range 35 °C to 60 °C.
5.4.3 Dichloromethane or trichloromathane.
5.4.4 Toluene.
5.4.5 Ethyl acetate.
5.4.6 n-Hexane.
5.4.7 n-Heptane.
5.4.8 Cyclohexane.
5.4.9 Diethylamine.

1) Silica-G and Silica-HF are examples of suitable products available commercially. This information is given for the

convenience of users of this document and does not constitute an endorsement by ISO of these products.

© ISO 2022 – All rights reserved
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ISO 4645:2022(E)

5.4.10 Ammonium hydroxide, 25 % to 30 % (mass fraction) of NH , solution (ρ = 0,9 Mg/m ).

5.4.11 Ethanol.
5.4.12 Acetone.
5.5 Developing solvents:
5.5.1 For method A:

5.5.1.1 n-Heptane-ethyl acetate solution: add 90 parts by volume of heptane (5.4.7) to 10 parts by

volume of ethyl acetate (5.4.5).

5.5.1.2 Ethanol-ammonium hydroxide-toluene solution: add 0,5 parts by volume of ethanol (5.4.11)

and 0,05 parts by volume of ammonium hydroxide (5.4.10) to 100 parts by volume of toluene (5.4.4).

5.5.1.3 Acetone-ammonium hydroxide-toluene solution: add 10 parts by volume of acetone (5.4.12)

and 0,2 parts by volume of ammonium hydroxide (5.4.10) to 100 parts by volume of toluene (5.4.4).

5.5.2 For method B:

5.5.2.1 Ethyl acetate-toluene solution: add 5 parts by volume of ethyl acetate (5.4.5) to 95 parts by

volume of toluene (5.4.4).

5.5.2.2 Diethylamine-cyclohexane solution: add 25 parts by volume of diethylamine to 75 parts by

volume of cyclohexane.

5.5.2.3 Toluene-n-heptane solution: add 50 parts by volume of toluene to 50 parts by volume of

heptane.
5.6 Colour development and spray reagents

Most of the spray reagents are suitable for colour development of both amines and phenols. The

following suggestion gives an example from which analytical expertise may be developed.

5.6.1 For colour development of most antidegradants
5.6.1.1 2,6-Dichloroquinone chlorimine ethanol solution (0,2 %)

Dissolve 0,2 g of 2,6-dichloroquinone chlorimide in 100 g of anhydrous ethanol (it is recommended to

be used fresh and stored in the refrigerator after preparation).
5.6.1.2 Buffer spray reagent for use with 5.6.1.1

Dissolve 3,5 g of sodium hydroxide and 23,4 g of sodium tetraborate decahydrate in water, dilute to 1 l.

5.6.2 Special for colour development of amines (optional)
5.6.2.1 Diazotised sulphanilic acid.

Dissolve 1 g of sulphanilic acid and 1 g of potassium nitrite in 200 cm of hydrochloric acid solution,

c(HCl) = 1 mol/l. Make fresh daily.
5.6.2.2 Benzoyl peroxide, 40 g in 1 l of toluene.
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ISO 4645:2022(E)

WARNING — Benzoyl peroxide is a powerful oxidizer which can explode spontaneously.

5.6.2.3 Bismuth nitrate, solution.

Dissolve 7,5 g of anhydrous bismuth nitrate in a mixture of 1 ml of concentrated nitric acid and 150 ml

of water.

5.6.2.4 Tetracyanoethylene (ethenetetracarbonitrile), saturated solution in dichloromethane.

5.6.3 Special for colour development of phenols(optional)

5.6.3.1 Overspray, after the use of the reagent specified in 5.6.2.1: with sodium hydroxide,

c(NaOH) = 1 mol/l.

5.6.3.2 p-Nitrophenyldiazonium fiuoborate, 1 % (mass fraction) solution in methanol containing

0,5 % (mass fraction) of hydrochloric acid.

5.6.3.3 Dichloroquinonechlorimide (Gibb's Reagent) or 2,6-dibromoquinonechlorimide, 0,1 %

solution in methanol.

5.6.3.4 Buffer spray, for use with reagent 5.6.3.3, dissolve 23,4 g of sodium tetraborate decahydrate,

and 3,3 g of sodium hydroxide in 1 l of water.
5.6.3.5 Tollen's reagent.

Mix 0,5 cm of 5 % silver nitrate solution and 2 drops of sodium hydroxide, c(NaOH) = 2 mol/l. Dissolve

the precipitate in as a mass fraction of 2 % ammonium hydroxide solution as possible and add an equal

volume of a volume fraction of 96 % ethanol solution.

WARNING — Prepare this reagent immediately before use and dispose of within 12 h.

6 Apparatus
Ordinary laboratory apparatus and the following.

6.1 Glass plates, of any convenient and adequate size, for example 200 mm x 200 mm,

200 mm × 50 mm, 200 mm × 70 mm, 3 mm to 5 mm thin.

6.2 Device for spreading a coating, 0,2 mm to 0,3 mm thick on the glass plates (6.1).

6.3 Pre-coated plates, covered with a layer of silica gel, 0,25 mm to 0, 30 mm thick.

These may be used as an altern
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