SIST EN ISO 22184:2021

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

2

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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.

3

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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.
© ISO and IDF 2021 – All rights reserved 1

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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).
3
5.11 Eluent 1 (E1), aqueous solution of sodium acetate (CH COONa), c = 1,0 mol/l.
3
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.
2 © ISO and IDF 2021 – All rights reserved

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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.
3
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.
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SIST EN ISO 22184:2021
ISO 22184:2021(E)
IDF 244:2021(E)
1)
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.
2)
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.
3)
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.
4)
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.
5)
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)
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
...