Milk and milk products - Determination of the sugar contents - High performance anion exchange chromatography with pulsed amperometric detection method (HPAEC-PAD) (ISO 22184:2021)

This International Standard describes the quantitative liquid chromatographic determination of specific sugars (galactose, glucose, fructose, sucrose, lactose, and maltose) in various milk and milk products, applying arabinose or fucose as internal standards. The method is applicable for the following different dairy matrices: milk, milk powder, chees, whey powder, infant formula, dessert and yogurt. Soy containing dairy products are excluded. The determination of the lactose content in low lactose milk products is excluded.
A sophisticated high performance anion exchange chromatographic method in combination with
pulsed amperometric detection (HPAEC-AD) is applied. With this method the following 13 different
mono- and disaccharides can be separated: fucose, arabinose, galactose, glucose, fructose, sucrose, lactose, lactulose, maltose, melobiose, trehalose, platinose (maltulose) and maltotriose.

Milch und Milcherzeugnisse - Bestimmung des Zuckergehalts - Verfahren der Hochleistungs-Anionenaustausch-Chromatographie mit gepulster amperometrischer Detektion (HPAEC PAD) (ISO 22184:2021)

Dieses Dokument legt die quantitative Bestimmung spezifischer Zucker (Galactose, Glucose, Fructose, Saccharose, Lactose und Maltose) in Milch und verschiedenen Milcherzeugnissen mittels Flüssigkeits-Chromatographie unter Anwendung von Arabinose als einen internen Standard fest.
Das Verfahren ist für die folgenden Molkerei-Matrizes anwendbar: Milch, gezuckerte Kondensmilch, Milchpulver, Käse, Molkenpulver, Säuglingsnahrung, Milchdessert und Joghurt.
Das Verfahren ist nicht für Molkereierzeugnisse, die Soja enthalten oder für die Bestimmung des Lactosegehalts in lactosearmen Milcherzeugnissen mit einem Gehalt unter 1 mg/g anwendbar.
Es wird ein Verfahren der Hochleistungs-Anionenaustausch-Chromatographie mit gepulster amperometrischer Detektion (HPAEC-PAD) angewendet [5][3][4]. Mit diesem Verfahren können dreizehn verschiedene Monosaccharide, Disaccharide und Trisaccharide getrennt werden: Fucose, Arabinose, Galactose, Glucose, Fructose, Saccharose, Lactose, Lactulose, Maltose, Melibiose, Trehalose, Isomaltulose und Maltotriose.
Das Verfahren ist für die Kennzeichnung der sechs wichtigsten Zucker anwendbar, die in Milch und Milcherzeugnissen natürlich oder durch Zusatz vorkommen können. Das Verfahren ist bei Zuckergehalten kleiner 0,1 % nicht anwendbar.

Lait et produits laitiers - Détermination de la teneur en sucre - Chromatographie d’échange d’anions haute performance couplée à la détection par ampérométrie pulsée (HPAEC-PAD) (ISO 22184:2021)

Le présent document spécifie la détermination quantitative par chromatographie en phase liquide de certains sucres (galactose, glucose, fructose, saccharose, lactose et maltose) dans divers laits et produits laitiers, en utilisant l'arabinose comme étalon interne.
Cette méthode s'applique aux matrices laitières suivantes: lait, lait concentré sucré, lait en poudre, fromage, lactosérum en poudre, formule infantile, dessert lacté et yaourt.
Cette méthode ne s'applique pas aux produits laitiers contenant du soja ni à la détermination de la teneur en lactose des produits laitiers à faible teneur en lactose inférieure à 0,1 mg/g.
Cette méthode s'appuie sur la chromatographie d'échange d'anions à haute performance couplée à la détection par ampérométrie pulsée (HPAEC-PAD)[5][3][4]. Treize monosaccharides, disaccharides et trisaccharides différents peuvent être séparés par cette méthode: fucose, arabinose, galactose, glucose, fructose, saccharose, lactose, lactulose, maltose, mélibiose, tréhalose, isomaltulose et maltotriose.
Cette méthode s'applique à l'étiquetage des six principaux sucres qui peuvent être présents naturellement ou ajoutés dans le lait et les produits laitiers. Cette méthode ne s'applique pas aux teneurs en sucre inférieures à 0,1 %.

Mleko in mlečni proizvodi - Določanje vsebnosti sladkorjev - Anionska izmenjevalna kromatografija z visoko ločljivostjo s pulzno amperometrično detekcijo (HPAEC-PAD) (ISO 22184:2021)

General Information

Status
Published
Public Enquiry End Date
19-Dec-2019
Publication Date
16-Mar-2021
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
02-Mar-2021
Due Date
07-May-2021
Completion Date
17-Mar-2021

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SLOVENSKI STANDARD
SIST EN ISO 22184:2021
01-april-2021
Mleko in mlečni proizvodi - Določanje vsebnosti sladkorjev - Anionska
izmenjevalna kromatografija z visoko ločljivostjo s pulzno amperometrično
detekcijo (HPAEC-PAD) (ISO 22184:2021)

Milk and milk products - Determination of the sugar contents - High performance anion

exchange chromatography with pulsed amperometric detection method (HPAEC-PAD)
(ISO 22184:2021)
Milch und Milcherzeugnisse - Bestimmung des Zuckergehalts - Verfahren der
Hochleistungs-Anionenaustausch-Chromatographie mit gepulster amperometrischer
Detektion (HPAEC PAD) (ISO 22184:2021)

Lait et produits laitiers - Détermination de la teneur en sucre - Chromatographie

d’échange d’anions haute performance couplée à la détection par ampérométrie pulsée

(HPAEC-PAD) (ISO 22184:2021)
Ta slovenski standard je istoveten z: EN ISO 22184:2021
ICS:
67.100.01 Mleko in mlečni proizvodi na Milk and milk products in
splošno general
SIST EN ISO 22184:2021 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 22184:2021
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SIST EN ISO 22184:2021
EN ISO 22184
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2021
EUROPÄISCHE NORM
ICS 67.100.01
English Version
Milk and milk products - Determination of the sugar
contents - High performance anion exchange
chromatography with pulsed amperometric detection
method (HPAEC-PAD) (ISO 22184:2021)

Lait et produits laitiers - Détermination de la teneur en Milch und Milcherzeugnisse - Bestimmung des

sucre - Chromatographie d'échange d'anions haute Zuckergehalts - Verfahren der Hochleistungs-

performance couplée à la détection par ampérométrie Anionenaustausch-Chromatographie mit gepulster

pulsée (HPAEC-PAD) (ISO 22184:2021) amperometrischer Detektion (HPAEC PAD) (ISO
22184:2021)
This European Standard was approved by CEN on 30 October 2020.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 22184:2021 E

worldwide for CEN national Members.
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SIST EN ISO 22184:2021
EN ISO 22184:2021 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 22184:2021
EN ISO 22184:2021 (E)
European foreword

This document (EN ISO 22184:2021) has been prepared by Technical Committee ISO/TC 34 "Food

products" in collaboration with Technical Committee CEN/TC 302 “Milk and milk products - Methods of

sampling and analysis” the secretariat of which is held by NEN.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by August 2021, and conflicting national standards shall

be withdrawn at the latest by August 2021.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
Endorsement notice

The text of ISO 22184:2021 has been approved by CEN as EN ISO 22184:2021 without any modification.

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SIST EN ISO 22184:2021
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SIST EN ISO 22184:2021
INTERNATIONAL ISO
STANDARD 22184
IDF 244
First edition
2021-02
Milk and milk products —
Determination of the sugar contents
— High performance anion exchange
chromatography with pulsed
amperometric detection method
(HPAEC-PAD)
Lait et produits laitiers — Détermination de la teneur en sucre —
Chromatographie d’échange d’anions haute performance couplée à la
détection par ampérométrie pulsée (HPAEC-PAD)
Reference numbers
ISO 22184:2021(E)
IDF 244:2021(E)
ISO and IDF 2021
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SIST EN ISO 22184:2021
ISO 22184:2021(E)
IDF 244:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO and IDF 2021

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 International Dairy Federation
CP 401 • Ch. de Blandonnet 8 Silver Building • Bd Auguste Reyers 70/B
CH-1214 Vernier, Geneva B-1030 Brussels
Phone: +41 22 749 01 11 Phone: +32 2 325 67 40
Fax: +32 2 325 67 41
Email: copyright@iso.org Email: info@fil-idf.org
Website: www.iso.org Website: www.fil-idf.org
Published in Switzerland
ii © ISO and IDF 2021 – All rights reserved
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SIST EN ISO 22184:2021
ISO 22184:2021(E)
IDF 244:2021(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 Sampling ........................................................................................................................................................................................................................ 6

8 Preparation of the test sample ............................................................................................................................................................... 6

8.1 General ........................................................................................................................................................................................................... 6

8.2 Sample preparation of sweetened condensed milk ................................................................................................ 6

8.2.1 Samples of recently manufactured products in which no appreciable

separation of components can be expected ............................................................................................. 6

8.2.2 Samples of older products and samples in which separation of

components can be expected ................................................................................................................................ 6

9 Procedure..................................................................................................................................................................................................................... 7

9.1 Sample extraction and clean up ............................................................................................................................................... 7

9.1.1 General...................................................................................................................................................................................... 7

9.1.2 Sample extraction and clean-up ......................................................................................................................... 7

9.2 Chromatographic analysis ............................................................................................................................................................. 9

10 Calculation and expression of the results ...............................................................................................................................10

11 Precision ....................................................................................................................................................................................................................11

11.1 General ........................................................................................................................................................................................................11

11.2 Repeatability ..........................................................................................................................................................................................11

11.3 Reproducibility ....................................................................................................................................................................................13

12 Test report ................................................................................................................................................................................................................16

Annex A (informative) Precision data ..............................................................................................................................................................17

Annex B (informative) Accuracy data ...............................................................................................................................................................23

Bibliography .............................................................................................................................................................................................................................25

© ISO and IDF 2021 – All rights reserved iii
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SIST EN ISO 22184:2021
ISO 22184:2021(E)
IDF 244:2021(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 34, Food products, Subcommittee SC

5, Milk and milk products, in collaboration with the European Committee for Standardization (CEN)

Technical Committee CEN/TC 302, Milk and milk products — Methods of sampling and analysis, in

accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement),

and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF.

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 and IDF 2021 – All rights reserved
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SIST EN ISO 22184:2021
ISO 22184:2021(E)
IDF 244:2021(E)

IDF (the International Dairy Federation) is a non-profit private sector organization representing the

interests of various stakeholders in dairying at the global level. IDF members are organized in National

Committees, which are national associations composed of representatives of dairy-related national

interest groups including dairy farmers, dairy processing industry, dairy suppliers, academics and

governments/food control authorities.

ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis

and sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International

Standards using the logos and reference numbers of both organizations.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. IDF 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.

This document was prepared by the IDF Standing Committee on Analytical Methods for Composition and

ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is

being published jointly by ISO and IDF.

The work was carried out by the IDF/ISO Action Team C22 of the Standing Committee on Analytical

Methods for Composition under the aegis of its project leader Mr H. Cruijsen (NL).

© ISO and IDF 2021 – All rights reserved v
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SIST EN ISO 22184:2021
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SIST EN ISO 22184:2021
ISO 22184:2021(E)
INTERNATIONAL STANDARD
IDF 244:2021(E)
Milk and milk products — Determination of the
sugar contents — High performance anion exchange
chromatography with pulsed amperometric detection
method (HPAEC-PAD)
1 Scope

This document specifies the quantitative liquid chromatographic determination of specific sugars

(galactose, glucose, fructose, sucrose, lactose and maltose) in various milk and milk products, applying

arabinose as an internal standard.

The method is applicable to the following dairy matrices: milk, sweetened condensed milk, milk powder,

cheese, whey powder, infant formula, milk dessert and yoghurt.

The method does not apply to dairy products containing soy or to the determination of the lactose

content in low-lactose milk products at levels below 1 mg/g.

A high performance anion exchange chromatography method in combination with pulsed amperometric

[5][3][4]

detection (HPAEC-PAD) method is applied . With this method, thirteen different monosaccharides,

disaccharides and trisaccharides can be separated: fucose, arabinose, galactose, glucose, fructose,

sucrose, lactose, lactulose, maltose, melibiose, trehalose, isomaltulose and maltotriose.

The method is applicable to labelling for the six most important sugars that can be present by nature

or by addition in milk and milk products. The method does not apply to sugar contents less than 0,1 %.

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 3696, Water for analytical laboratory use — Specification and test methods
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 Principle

The sugars present in the sample are extracted with an aqueous ethanol buffer solution in order to

inhibit potential probiotic activities. The obtained extract is deproteinized with a Carrez clarification.

After clarification, the solution is diluted and the sugars present are separated and quantified by

HPAEC. HPAE allows carbohydrates separation at high pH. In order to improve sensitivity and stability,

post-column sodium hydroxide solution is added to the HPAEC-PAD. GOS (galacto-oligosaccharides) and

[5]

fructans do not interfere with the analysis of the sugars . Arabinose is applied as an internal standard

for the quantification of the sugars.
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SIST EN ISO 22184:2021
ISO 22184:2021(E)
IDF 244:2021(E)
5 Reagents

Use only reagents of recognized analytical grade and water in accordance with ISO 3696, unless

otherwise specified.
5.1 Water, conforming to ISO 3696, grade 3 and grade 1.
5.2 Sodium hydroxide (NaOH) pellets.
5.3 Aqueous sodium hydroxide solution, substance concentration c = 1 mol/l.

Add to a 1 000 ml volumetric flask 40 g ± 1 g NaOH pellets (5.2), dissolve in about 500 ml of water and,

after cooling down, dilute with water to the mark and homogenize.
5.4 Sodium hydroxide solution, mass fraction w(NaOH) = 33 % in water.
5.5 Sodium hydroxide solution, mass fraction w(NaOH) = 50 % in water.

The amount of carbonate and mercury in the reagent should be minimized. Do not shake or stir the

solution before use. A suitable commercially available carbonate sodium hydroxide solution may also

be used.

5.6 Concentrated hydrochloric acid (HCl), mass fraction of 36 % to 38 % in water.

5.7 Aqueous hydrochloric acid solution, c = 1 mol/l.

Add to a 1 000 ml volumetric flask (6.2) 500 ml of water followed by 83 ml of concentrated HCl (5.6)

and, after cooling down, dilute with water to the mark and homogenize.
5.8 Acetonitrile (HPLC quality).
5.9 Acetonitrile in water, a volume fraction of 5 % in water.

Add to a 1 000 ml volumetric flask (6.2) 50 ml of acetonitrile (5.8), dilute with water grade 3 to the

mark and homogenize.
5.10 Anhydrous sodium acetate (CH COONa) (HPLC quality).

5.11 Eluent 1 (E1), aqueous solution of sodium acetate (CH COONa), c = 1,0 mol/l.

Add to a 1 000 ml volumetric flask (6.2) about 800 ml of degassed water grade 1 (eluent 3, 5.13) followed

by 82,0 g sodium acetate (5.10). Then dilute the aqueous solution with degassed water (eluent 3, 5.13)

to the mark and homogenize. Store the eluent under an inert atmosphere.

5.12 Eluent 2 (E2), aqueous solution of carbonate free sodium hydroxide (NaOH), c = 0,2 mol/l.

Add to a 1 000 ml volumetric flask (6.2) about 800 ml of degassed water grade 1 (eluent 3, 5.13) and

purge for 15 min with helium. Add 16,0 g of sodium hydroxide solution (5.5). Then quickly dilute the

aqueous solution with degassed water grade 1 (eluent 3, 5.13) to the mark, immediately close the bottle

and homogenize. Store the eluent under an inert atmosphere.
5.13 Eluent 3 (E3), degassed water grade 1, stored under an inert atmosphere.
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SIST EN ISO 22184:2021
ISO 22184:2021(E)
IDF 244:2021(E)

5.14 Eluent 4 (E4), aqueous solution of sodium acetate (CH COONa), c = 0,025 mol/l.

Add to a 1 000 ml volumetric flask (6.2) about 800 ml of degassed water grade 1 (eluent 3, 5.13) followed

by 2,05 g of sodium acetate (5.10). Then dilute the aqueous solution with degassed water (eluent 3,

5.13) to the mark and homogenize. Store the eluent under an inert atmosphere.
5.15 Post column reagent, aqueous solution of sodium hydroxide, c = 0,3 mol/l.

Add to a 1 000 ml volumetric flask (6.2) about 800 ml of degassed water grade 1 (eluent 3, 5.13). Purge

for 15 min with helium. Add 24,0 g of sodium hydroxide solution (5.5) and quickly fill up to the mark

with the degassed water grade 1 (eluent 3, 5.13). Immediately close the flask and homogenize. Store the

post column reagent under an inert atmosphere.

IMPORTANT — It is extremely important to remove dissolved carbon dioxide from the eluents

and post column reagent prior to use and during use to avoid fast reduction in detector

sensitivity. The eluents and post column reagent are maintained under an inert gas during use.

5.16 Mixture of a volume fraction of 95 % of ethanol (with a volume fraction of 96 % ethanol and

4 % of water) and a volume fraction of 5 % methanol.
5.17 Potassium hexacyanoferrate (II) trihydrate, K Fe(CN) ·3H O.
4 6 2
5.18 Zinc acetate dihydrate, Zn(CH COO) ·2H O.
3 2 2
5.19 Glacial acetic acid.
5.20 Carrez reagent I.

Weigh 106 g of K Fe(CN) ·3H O (5.17) in a 1 000 ml volumetric flask (6.2), dissolve in 800 ml of water

4 6 2

(5.1) and dilute with water grade 3 to the mark. Store the Carrez reagent I in the refrigerator.

5.21 Carrez reagent II.

Weigh 220 g of Zn(CH COO) ·2H O (5.18) in a 1 000 ml volumetric flask (6.2), dissolve in 800 ml water,

3 2 2

add 30 ml of glacial acetic acid (5.19) and dilute with water grade 3 to the mark. Store the Carrez reagent

II in the refrigerator.
IMPORTANT — Do not use Carrez reagent II with zinc sulfate.

5.22 Buffer solution of piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) (c = 1,5 mol/l and

pH = 6,9).

Add 22,5 g of the PIPES buffer solution to a 100 ml conical flask and add 20 ml of sodium hydroxide

solution (5.3). Adjust the pH to pH = 6,9 with NaOH 33 % (5.4) in water. Transfer the PIPES buffer

solution quantitatively into a 50 ml calibrated tube and fill up with water grade 3 till 50 ml. The pH of

the obtained buffer solution shall be within the range of 6,8 to 7,0.
5.23 Arabinose.
5.24 Galactose.
5.25 Glucose.
5.26 Fructose.
5.27 Sucrose.
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SIST EN ISO 22184:2021
ISO 22184:2021(E)
IDF 244:2021(E)
5.28 Lactose.
5.29 Maltose.
5.30 Internal standard stock solution arabinose.

Weigh, to the nearest mg, approximately 7 g of arabinose (5.23) into a 50 ml volumetric flask (6.2). Add

about 30 ml of water grade 3 and dissolve the arabinose. Add 2,5 ml of acetonitrile (5.8), fill up to the

mark with water and homogenize the solution.
5.31 Sugar standard stock solution.

Weigh, to the nearest 0,1 mg, approximately 260 mg of the monosaccharides galactose (5.24), glucose

(5.25) and fructose (5.26), and approximately 400 mg of the disaccharide sucrose (5.27), lactose (5.28)

and maltose (5.29) into a 500 ml volumetric flask (6.2). Add about 200 ml of water grade 3 and dissolve

the sugars. Add 25 ml of acetonitrile (5.8), fill up to the mark with water grade 3 and homogenize the

solution.
5.32 Sugar standard solutions for calibration.

Prepare the different dilutions of the sugar calibration standards as specified in Table 1. Mix the

specified volumes of the internal standard stock solution arabinose (5.30) and sugar standard stock

solution (5.31) in a 200 ml volumetric flask, add about 50 ml of water grade 3 and homogenize. Add

10 ml of acetonitrile (5.8), fill up to the mark with water and homogenize.
Table 1 — Preparation of the sugar standard solutions for calibration
Volume of sugar standard Volume of arabinose internal
Sugar standard solution stock solution (5.31) standard stock solution (5.30)
ml ml
1 0,2 0,050
2 1,0 0,050
3 6,0 0,050
4 10,0 0,050
5 20,0 0,050
6 40,0 0,050
7 80,0 0,050
8 100,0 0,050
6 Apparatus
6.1 Analytical balance, capable of weighing to an accuracy of ±0,1 mg.
6.2 Volumetric flask, volume of 50 ml, 500 ml and 1 000 ml.
6.3 pH meter.
6.4 Black band filter paper.
6.5 Centrifugation tubes.
4 © ISO and IDF 2021 – All rights reserved
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SIST EN ISO 22184:2021
ISO 22184:2021(E)
IDF 244:2021(E)
6.6 Homogenizer .
6.7 Vortex mixer.
6.8 Screw cap closed graduated tubes, 50 ml volume.
6.9 Positive displacement multipipette.
6.10 Dispensers, resistant to organic solvent and adjusted to 2,5 ml and 25 ml.
6.11 HPLC vials.

6.12 Metal-free liquid chromatographic system, e.g. Thermo Dionex ICS 3000 , applicable for a

(quaternary) gradient elution.

6.13 Column oven, with a temperature stability of ±1 °C with an operating temperature of 20 °C to 35 °C.

6.14 High performance anion exchange analytical column , filled with pellicular polystyrene-

divinylbenzene resin or filled with an anion exchanger resin enabling the required separation.

6.15 High performance anion exchange guard column , filled with pellicular polystyrene-

divinylbenzene resin or filled with an anion exchanger resin enabling the required separation.

6.16 Pulsed amperometric detector (PAD) with a stability of ±1 °C and an operating temperature

range of 20 °C to 35 °C.

Use pulsed amperometric detection and potential settings and waveforms recommended by the

instrument supplier. Example detector potential settings (versus Ag/AgCl reference) are given in

Table 2.
6.17 Metal-free post column reagent pump .

1) An Ultra turrax with an appropriate probe is an example of a suitable homogenizer available commercially. This

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

or IDF of this product.

2) Thermo Dionex ICS 3000 is an example of a suitable homogenizer available commercially. This information is

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

product.

3) A Thermo-Dionex Carbopac PA1 analytical column (2 mm × 250 mm) is an example of a suitable high

performance anion exchange column available commercially. This information is given for the convenience of users

of this document and does not constitute an endorsement by ISO or IDF of this product.

4) A Dionex Carbopac PA1 guard column (2 mm × 50 mm) is an example of a suitable high performance anion

exchange column available commercially. This information is given for the convenience of users of this document

and does not constitute an endorsement by ISO or IDF of this product.

5) A Thermo-Dionex model PAD is an example of a suitable PAD available commercially. This information is given

for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of this product.

6) A single piston Thermo Dionex Axp pump is an example of a suitable metal-free post column re

...

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