Animal feeding stuffs - Methods of sampling and analysis - Determination of organic acids by Ion Chromatography with Conductivity Detection (IC-CD)

This document specifies a method for the determination of organic acids in animal feeding stuffs by Ion Chromatography with conductivity detection (IC-CD).
The method is intended to be used for the determination of formic acid, lactic acid, propionic acid, citric acid, fumaric acid and malic acid as active substances in feed additives, premixtures, compound feed and water and for screening of acetic acid in the same matrices. This method determines the total extractable concentration of the above mentioned organic acids and their salts.
The working range of the method must be determined for each organic acid by the user of this standard. The lower limit of the working range depends on the matrix and the interferences encountered. A working range between 10 [mg/l] and 100 [mg/l] should be achievable.
The method was successfully tested in an inter-laboratory study in concentrations between 0,02 % up to 27 % of the above mentioned organic acids.
NOTE   Limitation occurs during simultaneous determination of high concentration of lactic acid and low concentration of acetic acid. If the ratio of concentration of lactic acid to acetic acid exceeds factor 20, the determination of acetic acid is not guaranteed.
On the basis of the referred working range, sample weight and extraction volume,  limits of quantification (LOQ), as calculated (Table 1) should be achievable.

Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung organischer Säuren mittels Ionenchromatographie mit Leitfähigkeitsdetektion (IC-CD)

Dieses Dokument legt ein Verfahren zur Bestimmung organischer Säuren in Futtermitteln mittels Ionen-chromatographie mit Leitfähigkeitsdetektion (en: Ion Chromatography with conductivity detection, IC CD) fest.
Das Verfahren ist für die Verwendung zur Bestimmung von Ameisensäure, Milchsäure, Propionsäure, Citronensäure, Fumarsäure und Apfelsäure als Wirkstoffe in Futtermittelzusatzstoffen, Vormischungen, Mischfuttermitteln und Wasser sowie für das Screening von Essigsäure in denselben Matrices vorgesehen. Dieses Verfahren ermittelt die extrahierbare Gesamtkonzentration der vorstehend aufgeführten organischen Säuren und ihrer Salze.
Dem Anwender dieser Norm wird empfohlen, den Arbeitsbereich des Verfahrens für jede organische Säure zu ermitteln. Der untere Grenzwert des Arbeitsbereiches hängt von der Matrix und den aufgetretenen Störungen ab. Es wird empfohlen, dass ein Arbeitsbereich zwischen 10 [mg/l] und 100 [mg/l] eingehalten wird.
Das Verfahren wurde im Rahmen einer Ringversuchsstudie bei Konzentrationen von 0,02 % bis 27 % der vorgenannten organischen Säuren erfolgreich geprüft.
ANMERKUNG   Bei der gleichzeitigen Bestimmung einer hohen Konzentration von Milchsäure und einer niedrigen Konzentration von Essigsäure tritt eine Einschränkung auf. Wenn das Verhältnis der Konzentration der Milchsäure zu der von Essigsäure den Faktor 20 überschreitet, ist die Bestimmung der Essigsäure nicht sichergestellt.
Auf der Grundlage des zugehörigen Arbeitsbereichs, der Probenmasse und des Extraktionsvolumens sollten Bestimmungsgrenzen (en: limits of quantification, LOQ) wie berechnet (Tabelle 1) erreichbar sein.
Tabelle 1 - Bestimmungsgrenzen (LOQ)
Organische Säure   LOQ
[mg/kg]
Ameisensäure   200
Milchsäure   200
Propionsäure   200
Citronensäure   200
Fumarsäure   200
Essigsäure   200
Apfelsäure   200

Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Dosage des acides organiques par chromatographie ionique avec détection conductimétrique (CI-DC)

Le présent document spécifie une méthode de dosage des acides organiques dans les aliments pour animaux par chromatographie ionique avec détection conductimétrique (CI-DC).
La méthode est destinée à permettre le dosage de l’acide formique, de l’acide lactique, de l’acide propionique, de l’acide citrique, de l’acide fumarique et de l’acide malique présents comme substances actives dans les additifs pour l’alimentation animale, les prémélanges, les aliments composés pour animaux et l’eau, ainsi que la détection de l’acide acétique dans les mêmes matrices. Cette méthode détermine la concentration totale extractible des acides organiques susmentionnés et de leurs sels.
Il est recommandé à l’utilisateur de la présente norme de déterminer le domaine de mesure de la méthode pour chaque acide organique. La limite inférieure du domaine de mesure dépend de la matrice et des interférences rencontrées. Il convient de pouvoir atteindre un domaine de mesure compris entre 10 [mg/l] et 100 [mg/l].
La méthode a été soumise à essai avec succès lors d’une étude interlaboratoires à des concentrations allant de 0,02 % à 27 % pour les acides organiques susmentionnés.
NOTE   La détermination simultanée d’une concentration élevée d’acide lactique et d’une faible concentration d’acide acétique présente des limites. Si le rapport de la concentration d’acide lactique à la concentration d’acide acétique dépasse un facteur de 20, le dosage de l’acide acétique n’est pas garanti.
En s’appuyant sur le domaine de mesure mentionné, le poids de l’échantillon et le volume d’extraction, il convient de pouvoir atteindre les limites de quantification (LQ), telles que calculées (Tableau 1).
Tableau 1 - Limites de quantification (LQ)
Acide organique   LQ [mg/kg]
Acide formique   200
Acide lactique   200
Acide propionique   200
Acide citrique   200
Acide fumarique   200
Acide acétique   200
Acide malique   200

Krma - Metode vzorčenja in analize - Določevanje organskih kislin z ionsko kromatografijo in detekcijo na osnovi prevodnosti (IC-CD)

Ta dokument določa metodo za določevanje organskih kislin v krmi z ionsko kromatografijo in detekcijo na osnovi prevodnosti (IC-CD).
Ta metoda je predvidena za določevanje mravljične kisline, mlečne kisline, propionske kisline, citronske kisline, fumarne kisline in jabolčne kisline kot aktivnih snovi v dodatkih za krmo, premiksih, krmnih mešanicah in vodi ter za varnostne preglede ocetne kisline pri istih matrikah. Ta metoda določa skupno koncentracijo omenjenih organskih kislin in njihovih soli, ki jo je mogoče ekstrahirati.
Uporabnik tega standarda mora določiti delovni razpon metode za vsako organsko kislino. Spodnja meja delovnega razpona je odvisna od matrice in motenj, ki se pojavijo. Delovni razpon med 10 [mg/l] in 100 [mg/l] naj bi bil dosegljiv.
Metoda je bila uspešno preskušena v medlaboratorijski študiji pri koncentracijah od 0,02 % do 27 % omenjenih organskih kislin.
OPOMBA:   Omejitev se pojavi pri hkratnem določevanju velike koncentracije mlečne kisline in majhne koncentracije ocetne kisline. Če razmerje med koncentracijo mlečne in ocetne kisline presega faktor 20, potem določevanje ocetne kisline ni zagotovljeno.
Na podlagi omenjenega delovnega razpona, teže vzorca in volumna ekstrakta naj bi bile mejne vrednosti kvantifikacije (LOQ) dosegljive, kot so izračunane (preglednica 1).

General Information

Status
Published
Public Enquiry End Date
24-Oct-2018
Publication Date
01-Sep-2019
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
21-Aug-2019
Due Date
26-Oct-2019
Completion Date
02-Sep-2019

Buy Standard

Standard
EN 17294:2019
English language
33 pages
sale 10% off
Preview
sale 10% off
Preview
e-Library read for
1 day
Draft
prEN 17294:2018
English language
34 pages
sale 10% off
Preview
sale 10% off
Preview
e-Library read for
1 day

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN 17294:2019
01-oktober-2019
Krma - Metode vzorčenja in analize - Določevanje organskih kislin z ionsko
kromatografijo in detekcijo na osnovi prevodnosti (IC-CD)
Animal feeding stuffs - Methods of sampling and analysis - Determination of organic
acids by Ion Chromatography with Conductivity Detection (IC-CD)
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung organischer
Säuren mittels Ionenchromatographie mit Leitfähigkeitsdetektion (IC-CD)
Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Dosage des acides
organiques par chromatographie ionique avec détection conductimétrique (CI-DC)
Ta slovenski standard je istoveten z: EN 17294:2019
ICS:
65.120 Krmila Animal feeding stuffs
71.040.40 Kemijska analiza Chemical analysis
SIST EN 17294:2019 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN 17294:2019

---------------------- Page: 2 ----------------------
SIST EN 17294:2019


EN 17294
EUROPEAN STANDARD

NORME EUROPÉENNE

August 2019
EUROPÄISCHE NORM
ICS 65.120; 71.040.40
English Version

Animal feeding stuffs - Methods of sampling and analysis -
Determination of organic acids by Ion Chromatography
with Conductivity Detection (IC-CD)
Aliments des animaux - Méthodes d'échantillonnage et Futtermittel - Probenahme- und
d'analyse - Dosage des acides organiques par Untersuchungsverfahren - Bestimmung organischer
chromatographie ionique avec détection Säuren mittels Ionenchromatographie mit
conductimétrique (CI-DC) Leitfähigkeitsdetektion (IC-CD)
This European Standard was approved by CEN on 24 June 2019.

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
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17294:2019 E
worldwide for CEN national Members.

---------------------- Page: 3 ----------------------
SIST EN 17294:2019
EN 17294:2019 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 6
4 Principle . 6
5 Reagents . 6
6 Apparatus . 8
7 Sampling . 11
8 Preparation of test sample . 11
9 Procedure. 11
9.1 General . 11
9.2 Calibration . 13
9.3 Measurement of the calibration standards and sample solutions . 13
9.4 Confirmation of identity . 13
10 Calculation and expression of results . 14
11 Precision . 14
11.1 Interlaboratory test . 14
11.2 Repeatability . 15
11.3 Reproducibility . 15
12 Test report . 19
Annex A (normative) Flow Chart . 20
Annex B (informative) Examples of measuring equipment and appropriate eluents . 21
Annex C (informative) Precision data . 25
Bibliography . 33

2

---------------------- Page: 4 ----------------------
SIST EN 17294:2019
EN 17294:2019 (E)
European foreword
This document (EN 17294:2019) has been prepared by Technical Committee CEN/TC 327 “Animal
feeding stuffs - 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 February 2020, and conflicting national standards shall
be withdrawn at the latest by Feburary 2020.
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.
This document has been prepared under a standardization request given to CEN by the European
Commission and the European Free Trade Association.
According to the CEN-CENELEC Internal Regulations, the national standards organisations 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.
3

---------------------- Page: 5 ----------------------
SIST EN 17294:2019
EN 17294:2019 (E)
Introduction
Organic acids and their salts such as citric acid, formic acid, lactic acid, acetic acid, propionic acid, fumaric
acid, benzoic acid and sorbic acid are animal feed additives which play an important role in animal feeding
by improving the animals’ performance and decreasing the development of (pathogenic) microorganisms
in the intestine especially in the pig production. Concerning the feed legislation the substances can be
used for different purposes depending on its functions and properties. According to their functional
principle or their function, the relevant organic acid could be allocated within one or more of the
functional groups mentioned in Annex I of Regulation (EC) no. 1831/2003 as preservative, acidity
regulators, flavouring compounds, silage additives or other zoo-technical additives.
4

---------------------- Page: 6 ----------------------
SIST EN 17294:2019
EN 17294:2019 (E)
1 Scope
This document specifies a method for the determination of organic acids in animal feeding stuffs by Ion
Chromatography with conductivity detection (IC-CD).
The method is intended to be used for the determination of formic acid, lactic acid, propionic acid, citric
acid, fumaric acid and malic acid as active substances in feed additives, premixtures, feed materials,
compound feed and water and for acetic acid in a limited manner in the same matrices. This method
determines the total extractable concentration of the above mentioned organic acids and their salts.
It is advisable that the user of this standard determines the working range of the method for each organic
acid. The lower limit of the working range depends on the matrix and the interferences encountered. It is
advisable that a working range between 10 mg/l and 100 mg/l is achievable.
The method was successfully tested in an inter-laboratory study in concentrations between 0,02 % up to
27 % of the above mentioned organic acids.
NOTE Limitation occurs during simultaneous determination of high concentration of lactic acid and low
concentration of acetic acid. If the ratio of concentration of lactic acid to acetic acid exceeds factor 20, the
determination of acetic acid is not guaranteed.
On the basis of the referred working range, sample weight and extraction volume, limits of
quantification (LOQ), as calculated (Table 1) can be achievable.
Table 1 — Limits of quantification (LOQ)
Organic acid LOQ
mg/kg
Formic acid 200
Lactic acid 200
Propionic acid 200
Citric acid 200
Fumaric acid 200
Acetic acid 200
Malic acid 200
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.
EN ISO 3696, Water for analytical laboratory use — Specification and test methods (ISO 3696)
EN ISO 6498, Animal feeding stuffs — Guidelines for sample preparation (ISO 6498)
EN ISO 10304-1, Water quality — Determination of dissolved anions by liquid chromatography of ions —
Part 1: Determination of bromide, chloride, fluoride, nitrate, nitrite, phosphate and sulfate (ISO 10304-1)
5

---------------------- Page: 7 ----------------------
SIST EN 17294:2019
EN 17294:2019 (E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
3.1
feed additives
substances, micro-organisms or preparations, other than feed material and premixtures, which are
intentionally added to feed or water
[SOURCE: Regulation (EC) No 1831/2003/Article 2/2 a [1]]
3.2
animal feeding stuffs
any substance or product,including additives, whether processed, partially processed or unprocessed,
intended to be used for oral feeding to animals
[SOURCE: Regulation (EC) No 178/2002/Article 2/4 [2]]
4 Principle
The sample is extracted with water. The extract is filtered or centrifuged and – if necessary – diluted. The
amount of organic acids extracted from the sample is determined with ion chromatography (IC) in
conjunction with conductivity detection (CD) using external calibration.
When using CD it is essential that the eluent shows a sufficiently low conductivity. For this reason, CD is
usually combined with a suppressor device (cation exchanger), which will reduce the conductivity of the
eluent and transform the sample acids into their respective salts.
The method description follows a proven approach where the chromatographic resolution R shall be
checked to ensure that it complies with the required separation conditions in accordance with
EN ISO 10304-1.
WARNING — The use of this European Standard can involve hazardous materials, operations and
equipment. This standard does not purport to address all the safety problems associated with its use. It
is the responsibility of the user of this European Standard to establish appropriate safety and health
practices and determine the applicability of regulatory limitations prior to use.
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
5.1 Water, grade 1 in accordance with EN ISO 3696
5.2 Formic acids, lactic acid, propionic acid, citric acid, acetic acid, malic acid standard solution,
c = 1 000 [mg/l]
Single acids standard solutions with adequate and required specification are commercially available
(ready-to-use solutions).
6

---------------------- Page: 8 ----------------------
SIST EN 17294:2019
EN 17294:2019 (E)
5.3 Alternative preparation of stock solution based single standard substances
NOTE Differentiation between the enantiomers (D, L, DL) is not of interest.
5.3.1 Citric acid, minimum 99 % purity.
5.3.2 Malic acid, minimum 99 % purity.
5.3.3 Formic Acid, minimum 98 % purity.
5.3.4 Lactic acid, minimum 85 % purity.
NOTE Lithium Lactate, minimum 95 % purity, can also be used.
5.3.5 Acetic acid, minimum 99 % purity.
5.3.6 Fumaric acid, minimum 99 % purity.
5.3.7 Propionic acid, minimum 99 % purity.
5.3.8 Single standard stock solutions, app. 2 500 [mg/l].
Weigh 250 mg acid (5.3.1 to 5.3.7) each into a 100 ml volumetric flask. Dissolve with approximately 80 ml
water (5.1), mix and fill up to the mark with water (5.1).
For fumaric acid weigh 250 mg (5.3.6) into a 100 ml volumetric flask. Add 80 ml water (5.1) and dissolve
10 min in an ultrasonic bath at 60 °C. After cooling to room temperature fill up to the mark with water.
The maximum storage time is 6 month at 4 °C.
NOTE Addition of 1 % Isopropanol has a positive effect on the stability of this solution.
Determine the exact concentration of the stock solution using the reference standard purity value
provided by the supplier according to Formula (1):
mP×
C = (1)
s
V
where
C is the experimental concentration of the organic acid in the standard stock solution, in mg/ml;
S
P is the purity of the organic acid standard given by the supplier in percent divided by 100;
NOTE For example 0,98.
m is the weighed mass of the organic acid, in mg;
V is the volume of the volumetric flask, in ml.
5.4 Mixed standard solution, (app.) 100 [mg/l]
Accurately pipette 4,0 ml of each single standard stock solution (5.3.8) into a 100 ml volumetric flask and
fill up to the mark with water (5.1). The exact concentration of each organic acid shall be calculated
according to the real concentration of the single standard stock solutions.
In the case of ready-to-use solutions (5.2) pipette 10,0 ml each into a 100 ml volumetric flask and fill up
to the mark with water (5.1).
The maximum storage time is 2 months at 4 °C.
7

---------------------- Page: 9 ----------------------
SIST EN 17294:2019
EN 17294:2019 (E)
5.5 IC Mobile phase
Degas all water used for eluent preparation.
The choice of eluent depends on the chosen column and detector (examples in Annex B, Table B.1). The
chosen combination of ion-chromatography column (IC-column) and eluent shall meet the resolution
requirements stated in 6.11.
A selection of reagents for common eluents is given in Annex B.
6 Apparatus
Usual laboratory apparatus, in particular, the following.
6.1 Laboratory grinder, capable of grinding to a particle size of less than or equal to 1,0 mm.
6.2 Analytical balance, suitable to accurately weigh between 0 g and 10 g with an accuracy of 0,1 mg.
6.3 Magnteic stirrer, with Polytetrafluoroethylene (PTFE)-coated stirring bar.
6.4 Ultrasonic bath
6.5 Pipettes (electronic or manual), in the range 100 μl to 5 000 μl.
6.6 Centrifuge, approx. 5 000 × g.
6.7 Folded filter, pore size 4 µm to 7 µm (ash free paper filter).
6.8 Membrane filter, for HPLC use, 0,45 µm (for example Ready-to-use filter unit with a hydrophilic,
low protein-binding membrane made of regenerated cellulose).
6.9 Reversed phase solid phase extract (RP SPE)
Optional for protecting the IC column, e.g. OnGuard II RP (Dionex).
6.10 Ion chromatograph (pump, autosampler) with suppressed conductivity detection
6.10.1 IC column, with specified separation performance (6.11).
6.10.2 Precolumn
6.11 Quality requirements for the separator column
In chromatograms of samples and standard solutions (see Figure 1), the peak resolution, R, between the
acid of interest and its nearest peak, shall not fall below 1,3 [see Formula (2) or Formula (3) and Figure 2].
Separation conditions shall be such that possible interfering organic acids or substances will not interfere
with the organic acids of interest.
8

---------------------- Page: 10 ----------------------
SIST EN 17294:2019
EN 17294:2019 (E)

Key
X = retention time t (s) 1 to 10 = peak 1 to 10
R
Y = signal
Figure 1 — Example chromatogram of organic acids with sufficient peak resolution
9

---------------------- Page: 11 ----------------------
SIST EN 17294:2019
EN 17294:2019 (E)

Key
X retention time tR (s) 1 peak 1
Y signal 2 peak 2
W peak width (s)
Figure 2 — Graphical representation of the parameters to calculate the peak resolution R
Base peak widths, w and w , are obtained by constructing isosceles triangles over the Gaussian peaks.
1 2
Calculate the peak resolution R using Formula (2).
2×()tt−
R2 R1
R =
(2)
2,1
w + w
21
where
R is the peak resolution;
2,1
t is the retention time of the first peak, in s;
R1
tR2 is the retention time of the second peak, in s;
W is the peak width on the time axis of the first peak, in s;
1
W is the peak width on the time axis of the second peak, in s.
2
NOTE Mostly the chromatography software can calculated R by itself.
10

---------------------- Page: 12 ----------------------
SIST EN 17294:2019
EN 17294:2019 (E)
If R shall be calculated manually follow the described procedure: The width at the base of a peak is the
segment of the peak base intercept by the tangents drawn to the inflection points on either side of the
peak. Alternatively the peaks width can be measured at half the peak height using Formula (3).
18,(× tt− )
R2 R1
R =
2,1
ww+
h2 h1
(3)
where
R is the peak resolution;
2,1
t is the retention time of the first peak, in s;
R1
t is the retention time of the second peak, in s;
R2
W is the peak width on the time axis of the first peak at half the peak height, in s;
h1
W is the peak width on the time axis of the second peak at half the peak height, in s.
h2
7 Sampling
It is important that the laboratory receives a sample which is truly representative and has not been
damaged or changed during transport or storage.
Sampling is not part of the method specified in this European Standard. A recommended sampling
method is given in EN ISO 6497 [3].
Store the sample in such a way that deterioration and change in its composition are prevented.
8 Preparation of test sample
Prepare the test sample in accordance with EN ISO 6498.
Grinding (≤ 1 mm) shall be done in such conditions that the substance is not appreciably heated.
The whole ground product is placed in a flask made of e.g. polypropylene, which can be stoppered and
stored in such way to prevent any change in composition.
Before any weighing is carried out for the analysis, the whole test sample shall be thoroughly mixed for
reasons of homogeneity.
9 Procedure
9.1 General
An overview of the procedure is given in Annex A.
9.1.1 Extraction of formic, lactic, propionic, citric, acetic and malic acid
Accurately weigh 5,00 g ± 10 mg of prepared sample into a 250 ml conical flask. Add 100,0 ml water (5.1)
and mix for 60 min on a magnetic stirrer at ambient temperature (20 °C to 25 °C).
NOTE Samples can also be extracted 30 min by ultrasonic bath at ambient temperature (20 °C to 25 °C).
Let the particles settle down. Filter through folded filter (6.7), discarding the first 3 ml of the filtrate, and
collect the rest. Alternatively centrifuge the sample extract for 3 min (appr. 5 000 × g).
11

---------------------- Page: 13 ----------------------
SIST EN 17294:2019
EN 17294:2019 (E)
Dilute the filtered/centrifuged solution with water (5.1) to a final concentration according to the working
range of the calibration (10 µg/ml to 100 µg/ml) – see below for a dilution example. For different
concentration levels of organic acids more than one dilution can be done.
Before IC analysis filter through a 0,45 µm membrane filter (6.8).
9.1.2 Extraction of fumaric acid
Accurately weigh 5,00 g ± 10 mg of prepared sample into a 250 ml conical flask. Add 100,0 ml water (5.1),
stopper the flask, heat up to 60 °C for complete solution and mix for 60 min on a magnetic stirrer.
NOTE Samples can also be extracted 30 min by ultrasonic bath at 60 °C.
The flask should be stoppered in order to avoid evaporation of extract solution.
Cool to room temperature and let the particles settle down. Filter through folded filter (6.7), discarding
the first 3 ml of the filtrate, and collect the rest. Alternatively centrifuge the sample extract for 3 min
(appr. 5 000 × g).
Dilute the filtered/centrifuged solution with water (5.1) to a final concentration according to the working
range of the calibration (10 µg/ml to 100 µg/ml) – see below for a dilution example.
Before IC analysis filter through a 0,45 µm membrane filter (6.8).
9.1.3 Extraction of coated acids
Accurately weigh 5,00 g ± 10 mg of prepared sample into a 250 ml conical flask. Add 100,0 ml water (5.1),
stopper the flask and extract 30 min by ultrasonic bath at 60 °C.
The flask shall be stoppered in order to avoid evaporation of extract solution
Cool to room temperature and let the particles settle down. Filter through folded filter (6.7), discarding
the first 3 ml of the filtrate, and collect the rest. Alternatively centrifuge the sample extract for 3 min
(appr. 5 000 × g).
Dilute the filtered/centrifuged solution with water (5.1) to a final concentration according to the working
range of the calibration (10 µg/ml to 100 µg/ml) – see below for a dilution example.
Before IC analysis filter through a 0,45 µm membrane filter (6.8).
For protecting the IC column the sample solution can be purified through an RP SPE (6.9). Follow the
description of the supplier. Blank solution and recoveries of organic acids shall be checked.
EXAMPLE Calculation of dilution:
Expected concentration of the sample 0,8 % (8 000 µg/g) formic acid.
Desired final concentration: 80 µg/ml.
5 g sample weight dissolved in 100 ml.
5g× 8000µg / g
Dilution factor 5

100ml× 80µg / ml
Therefore the sample extract solution shall be diluted 1:5 to meet the calibration range.
12
==

---------------------- Page: 14 ----------------------
SIST EN 17294:2019
EN 17294:2019 (E)
9.2 Calibration
The following calibration solutions are recommended.
Prepare the calibration standards with water (5.1) according to Table 2. The exact concentration of each
organic acid shall be calculated according to the concentration of the mixed standard solution (5.4).
Table 2 — Preparation of calibration standards
a
Concentration of organic
ml standard
acids each in the calibration
Calibration standard solution (5.4)
solution in µg/ml
in 20 ml
(working range)
1 2 10
2 5 25
3 10 50
4 15 75
5 20 100
Level 1: pipette 2,0 ml mixed standard solution (5.4) in a 20 ml volumetric flask and
fill to the mark with water (5.1)
Level 2: pipette 5,0 ml mixed standard solution (5.4) in a 20 ml volumetric flask and
fill to the mark with water (5.1)
Level 3: pipette 10,0 ml mixed standard solution (5.4) in a 20 ml volumetric flask
and fill to the mark with water (5.1)
Level 4: pipette 15,0 ml mixed standard solution (5.4) in a 20 ml volumetric flask
and fill to the mark with water (5.1)
Level 5: pipette 20,0 ml mixed standard solution (5.4) in a 20 ml volumetric flask
and fill to the mark with water (5.1)
a
The actual concentration shall be calculated.
The maximum storage time is 2 months at 4 °C.
9.3 Measurement of the calibration standards and sample solutions
Conditions in Annex B (informative) have been shown to provide examples for an adequate separation.
Due to the different laboratory equipment for ion chromatography the described specifications are given
for guidance. Other parameters should be applicable if they lead to comparable results.
Inject twice at least, randomly the calibration standards 1 to 5 prepared according to 9.2 into the
IC system (6.10) to calculate a linear standard calibration curve (R2 should be 0,99 at a minimum).
Inject the sample extract solution prepared according to 9.1. If the concentration of the analyte exceeds
the calibration range, dilute the sample according to 9.1 or establish a separate calibration function for a
higher working range and re-analyse it.
9.4 Confirmation of identity
The identity of the target analyte(s) can be confirmed by co-chromatography.
Prepare a spiked sample extract by adding an appropriate amount of calibration solution of the organic
acid in question to the sample extract. The amount of organic acid added shall be approximately equal to
the estimated amount of the corresponding organic acid detected in the sample extract. Inject the spiked
sample extract. If the signal detected in the chromatogram obtained in (9.3) was linked to the detection
13

---------------------- Page: 15 ----------------------
SIST EN 17294:2019
EN 17294:2019 (E)
of the organic acid, only this signal intensity should increase. The related peak height increase should be
proportional to the spiking level of the organic acid and the related peak width at half height should
increase by no more than ±10 % of the original width.
10 Calculation and expression of results
In general the calibration curve and the concentration of the test solution are calculated by the
measurement system itself. The quantification is carried out on the basis of the peak areas determined
according 9.2 by the calibration regression line (calibration curve) by the method of external standards.
y = bx+a
The concentration of organic acids in the sample, expressed in mg/kg, is determined using Formula (4):
C ××VD
Chr E
(4)
C =
A
m
where
C is the concentration of the organic acid in the sample, in mg/kg with 3 significant digits;
A
C is the concentration of the organic acid in the measurement solution, in µg/ml;
Chr
V is the extraction volume, in ml;
E
D is the dilution factor according 9.1 (extraction volume x dilution);
m is the mass of the sample, in g.
Especially the critical pair of peaks regarding the peak resolution R should be taken into account. If the
ratio of concentration of lactic acid to acetic acid exceeds factor 20, the determination of acetic acid is not
guaranteed.
If the acid is to be expressed as the corresponding salt, Formula (5) should be applied:
MW
S
C C×
(5)
s A
MW
A
where
C is the concentration of the salt of the organic acid in the sample, in mg/kg with 3 significant
S
digits;
C is the concentration of the organic acid in the sample, in mg/kg;
A
MW is the molecular weight of the salt of the organic acid;
S
MW is the molecular weight of the organic acid.
A
11 Precision
11.1 Interlaboratory test
An overview of an interlaboratory study on the precision of the method is shown in Table 3 to Table 9.
Details are shown in Annex C.
The values for performance characteristics derived from this interlaboratory study may not be applicable
to concentration ranges and/or matrices other than those given.
14
=

---------------------- Page: 16 ----------------------
SIST EN 17294:2019
EN 17294:2019 (E)
11.2 Repeatability
The absolute difference between two independent single test results or the ratio of the higher to the lower
of the two test results on the normal scale, obtained using the same method on identical test material in
the same laboratory by the same operator using the same apparatus within the shortest feasible time
...

SLOVENSKI STANDARD
oSIST prEN 17294:2018
01-oktober-2018
.UPDPHWRGHY]RUþHQMDLQDQDOL]H'RORþHYDQMHRUJDQVNLKNLVOLQ]LRQVNR
NURPDWRJUDILMRLQGHWHNFLMRQDRVQRYLSUHYRGQRVWL ,&&'
Animal feeding stuffs: Methods of sampling and analysis - Determination of organic acids
by Ion Chromatography with Conductivity Detection (IC-CD)
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung organischer
Säuren mittels Ionenchromatographie mit Leitfähigkeitsdetektion (IC-CD)
Aliments des animaux: Méthodes d’échantillonnage et d’analyse - Dosage des acides
organiques par chromatographie ionique avec détection conductimétrique (CI-DC)
Ta slovenski standard je istoveten z: prEN 17294
ICS:
65.120 Krmila Animal feeding stuffs
71.040.40 Kemijska analiza Chemical analysis
oSIST prEN 17294:2018 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN 17294:2018

---------------------- Page: 2 ----------------------
oSIST prEN 17294:2018


DRAFT
EUROPEAN STANDARD
prEN 17294
NORME EUROPÉENNE

EUROPÄISCHE NORM

August 2018
ICS 65.120; 71.040.40
English Version

Animal feeding stuffs: Methods of sampling and analysis -
Determination of organic acids by Ion Chromatography
with Conductivity Detection (IC-CD)
Aliments des animaux: Méthodes d'échantillonnage et Futtermittel - Probenahme- und
d'analyse - Dosage des acides organiques par Untersuchungsverfahren - Bestimmung organischer
chromatographie ionique avec détection Säuren mittels Ionenchromatographie mit
conductimétrique (CI-DC) Leitfähigkeitsdetektion (IC-CD)
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 327.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.

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 supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.


EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17294:2018 E
worldwide for CEN national Members.

---------------------- Page: 3 ----------------------
oSIST prEN 17294:2018
prEN 17294:2018 (E)
Contents
Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 7
4 Principle . 7
5 Reagents . 7
5.1 General . 7
5.2 Water. 7
5.3 Formic acids, lactic acid, propionic acid, citric acid, acetic acid, malic acid standard
solution, c = 1000 [mg/l] . 8
5.4 Alternative preparation of stock solution based single standard substances . 8
5.4.1 Citric acid, minimum 99 % purity . 8
5.4.2 Malic acid, minimum 99 % purity . 8
5.4.3 Formic Acid, minimum 98 % purity. 8
5.4.4 Lactic acid, minimum 85 % purity . 8
5.4.5 Acetic acid, minimum 99 % purity . 8
5.4.6 Fumaric acid, minimum 99 % purity . 8
5.4.7 Propionic acid, minimum 99 % purity. 8
5.4.8 Single standard stock solutions, app. 2500 [mg/l] . 8
5.5 Mixed standard solution, (app.) 100 [mg/l] . 9
5.6 IC Mobile phase . 9
6 Apparatus . 9
6.1 General . 9
6.2 Laboratory grinder . 9
6.3 Analytical balance . 9
6.4 Magnteic stirrer . 9
6.5 Ultrasonic bath . 9
6.6 Pipettes (electronic or manual) . 9
6.7 Centrifuge, approx. 5000 × g . 9
6.8 Folded filter, pore size 4-7 µm, (ash free paper filter) . 9
6.9 Membrane filter, for HPLC use . 9
2

---------------------- Page: 4 ----------------------
oSIST prEN 17294:2018
prEN 17294:2018 (E)
6.10 Reversed phase solid phase extract (RP SPE) .9
6.11 Ion chromatopgraph (pump, autosampler) with suppressed conductivity detection . 10
6.11.1 IC column, with specified separation performance (6.11) . 10
6.11.2 Precolumn. 10
6.12 Quality requirements for the separator column . 10
7 Sampling . 12
8 Preparation of test sample . 12
9 Procedure . 13
9.1 Extraction . 13
9.1.1 Extraction of formic, lactic, propionic, citric, acetic and malic acid . 13
9.1.2 Extraction of fumaric acid . 13
9.1.3 Extraction of coated acids . 13
9.2 Calibration . 14
9.3 Measurement of the calibration standards and sample solutions . 14
9.4 Confirmation of identity . 15
10 Calculation and expression of results . 15
11 Precision . 16
11.1 Interlaboratory test . 16
11.2 Repeatability. 16
11.3 Reproducibility . 16
12 Test report . 20
Annex A (informative) Examples of measuring equipment and appropriate eluents . 21
A.1 General . 21
A.2 Examples for mobile phases and for solutions to be used as regenerants for
suppression devices . 22
Annex B (informative) Precision data . 25
B.1 Results of the validation study . 25
B.2 Statistical paramaters for organic acids . 26
Annex C (informative) Flow chart . 33
Bibliography . 34

3

---------------------- Page: 5 ----------------------
oSIST prEN 17294:2018
prEN 17294:2018 (E)
European foreword
This document (prEN 17294:2018) has been prepared by Technical Committee CEN/TC 327 “Animal
feeding stuffs: Methods of sampling and analysis”, the secretariat of which is held by NEN.
This document is currently submitted to the CEN Enquiry.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
4

---------------------- Page: 6 ----------------------
oSIST prEN 17294:2018
prEN 17294:2018 (E)
Introduction
Organic acids and their salts such as citric acid, formic acid, lactic acid, acetic acid, propionic acid, fumaric
acid, benzoic acid and sorbic acid are animal feed additives which play an important role in the animal
feeding by improving the animals’ performance and decrease the development of (pathogenic)
microorganisms in the intestine especially in the pig production. Concerning the feed legislation the
substances can be used for different purposes depending on its functions and properties. According to
their functional principle or their function, the relevant organic acid could be allocated within one or
more of the functional groups mentioned in Annex I of Regulation (EC) no. 1831/2003 as preservative,
acidity regulators, flavouring compounds, silage additives or other zoo-technical additives.
5

---------------------- Page: 7 ----------------------
oSIST prEN 17294:2018
prEN 17294:2018 (E)
1 Scope
This document specifies a method for the determination of organic acids in animal feeding stuffs by Ion
Chromatography with conductivity detection (IC-CD).
The method is intended to be used for the determination of formic acid, lactic acid, propionic acid, citric
acid, fumaric acid and malic acid as active substances in feed additives, premixtures, compound feed and
water and for screening of acetic acid in the same matrices. This method determines the total extractable
concentration of the above mentioned organic acids and their salts.
It is advisable that the user of this standar determine the working range of the method for each organic
acid. The lower limit of the working range depends on the matrix and the interferences encountered. A
working range between 10 [mg/l] and 100 [mg/l] should be achievable.
The method was successfully tested in an inter-laboratory study in concentrations between 0,02 % up to
27 % of the above mentioned organic acids.
NOTE Limitation occurs during simultaneous determination of high concentration of lactic acid and low
concentration of acetic acid. If the ratio of concentration of lactic acid to acetic acid exceeds factor 20, the
determination of acetic acid is not guaranteed.
On the basis of the referred working range, sample weight and extraction volume, limits of quantification
(LOQ), as calculated (Table 1) should be achievable.
Table 1 — Limits of quantification (LOQ)
Organic acid LOQ [mg/kg]
Formic acid 200
Lactic acid 200
Propionic acid 200
Citric acid 200
Fumaric acid 200
Acetic acid 200
Malic acid 200
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.
EN ISO 3696:1995, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987)
EN ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)
EN ISO 10304-1, Water quality - Determination of dissolved anions by liquid chromatography of ions - Part
1: Determination of bromide, chloride, fluoride, nitrate, nitrite, phosphate and sulfate (ISO 10304-1)
6

---------------------- Page: 8 ----------------------
oSIST prEN 17294:2018
prEN 17294:2018 (E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
3.1
feed additives
substances, micro-organisms or preparations, other than feed material and premixtures, which are
intentionally added to feed or water
[SOURCE: Regulation (EC) No 1831/2003/Article 2/2 a [1]]
3.2
animal feeding stuffs
any substance or product,including additives, whether processed, partially processed or unprocessed,
intended to be used for oral feeding to animals
[SOURCE: Regulation (EC) No 178/2002/Article 2/4 [2]]
4 Principle
The sample is extracted with water. The extract is filtrated or centrifuged and – if necessary – diluted.
The amount of organic acids extracted from the sample is determined with ion chromatography (IC) in
conjunction with conductivity detection (CD) using external calibration.
When using CD it is essential that the eluent shows a sufficiently low conductivity. For this reason, CD is
usually combined with a suppressor device (cation exchanger), which will reduce the conductivity of the
eluent and transform the sample acids into their respective salts.
The method description follows a proven approach where the chromatographic resolution R has to be
checked to ensure that it complies with the required separation conditions in accordance with
EN ISO 10304-1.
WARNING — The use of this European Standard can involve hazardous materials, operations and
equipment. This standard does not purport to address all the safety problems associated with its use. It
is the responsibility of the user of this European Standard to establish appropriate safety and health
practices and determine the applicability of regulatory limitations prior to use.
5 Reagents
5.1 General
Use only reagents of recognized analytical grade, unless otherwise specified.
5.2 Water
Complying with grade 1 in accordance with EN ISO 3696:1995.
7

---------------------- Page: 9 ----------------------
oSIST prEN 17294:2018
prEN 17294:2018 (E)
5.3 Formic acids, lactic acid, propionic acid, citric acid, acetic acid, malic acid standard
solution, c = 1000 [mg/l]
Single acids standard solutions with adequate and required specification are commercially available
(ready-to-use solutions).
5.4 Alternative preparation of stock solution based single standard substances
NOTE Differntiation between the enantiometers (D, L, DL) are not of interest.
5.4.1 Citric acid, minimum 99 % purity
5.4.2 Malic acid, minimum 99 % purity
5.4.3 Formic Acid, minimum 98 % purity
5.4.4 Lactic acid, minimum 85 % purity
NOTE Lithium Lactate, minimum 95 % purity, can also be used.
5.4.5 Acetic acid, minimum 99 % purity
5.4.6 Fumaric acid, minimum 99 % purity
5.4.7 Propionic acid, minimum 99 % purity
5.4.8 Single standard stock solutions, app. 2500 [mg/l]
Weigh 250 mg acid (5.4.1 to 5.4.7) each into a 100 ml volumetric flask. Dissolve with approximately 80 ml
water (5.2), mix and fill up to the mark with water (5.2).
For fumaric acid weigh 250 mg (5.4.6) into a 100 ml volumetric flask. Add 80 ml water (5.2) and dissolve
10 min in an ultrasonic bath at 60 °C. After cooling to room temperature fill up to the mark with water.
The maximum storage time is 6 month at 4 °C.
NOTE Addition of 1 % Isopropanol has a positive effect on the stability of this solution.
Determine the exact concentration of the stock solution using the reference standard purity value
provided by the supplier:
mP×
C = (1)
s
V
where
C is the experimental concentration of the organic acid in the standard stock solution, in mg/ml;
S
P is the purity of the organic acid standard given by the supplier in percent divided by 100, e.g.
0,98;
m is the weighed mass of the organic acid, in mg;
V is the volume of the volumetric flask, in ml.
8

---------------------- Page: 10 ----------------------
oSIST prEN 17294:2018
prEN 17294:2018 (E)
5.5 Mixed standard solution, (app.) 100 [mg/l]
Accurately pipette 4,0 ml of each single standard stock solution (5.4.8) into a 100 ml volumetric flask and
fill up to the mark with water (5.2). The exact concentration of each organic acid has to be calculated
according to the real concentration of the single standard stock solutions.
In the case of ready-to-use solutions (5.3) pipette 10,0 ml each into a 100 ml volumetric flask and fill up
to the mark with water (5.2).
The maximum storage time is 2 month at 4 °C.
5.6 IC Mobile phase
Degas all water used for eluent preparation.
The choice of eluent depends on the chosen column and detector (examples in Table A.1). The chosen
combination of ion-chromatography column (IC-column) and eluent shall meet the resolution
requirements stated in 6.11.
A selection of reagents for common eluents is given in Annex A.
6 Apparatus
6.1 General
Usual laboratory apparatus, in particular, the following.
6.2 Laboratory grinder
Laboratory grinder capable of grinding to a particle size of less than or equal to 1,0 mm.
6.3 Analytical balance
Capable of weighing to an accurancy of 0,1 mg.
6.4 Magnteic stirrer
With Polytetrafluoroethylene (PTFE)-coated stirring bar.
6.5 Ultrasonic bath
6.6 Pipettes (electronic or manual)
In the range 100 μl to 5000 μl.
6.7 Centrifuge, approx. 5000 × g
6.8 Folded filter, pore size 4-7 µm, (ash free paper filter)
0,45 µm (Ready-to-use filter unit with a hydrophilic, low protein-binding membrane made of regenerated
cellulose)
6.9 Membrane filter, for HPLC use
6.10 Reversed phase solid phase extract (RP SPE)
Optional for protecting the IC column, e.g. OnGuard II RP (Dionex).
9

---------------------- Page: 11 ----------------------
oSIST prEN 17294:2018
prEN 17294:2018 (E)
6.11 Ion chromatopgraph (pump, autosampler) with suppressed conductivity detection
6.11.1 IC column, with specified separation performance (6.11)
6.11.2 Precolumn
6.12 Quality requirements for the separator column
In chromatograms of samples and standard solutions (see Figure 1), the peak resolution, R, between the
acid of interest and its nearest peak, shall not fall below 1.3 [see Formula (1) or Formula (2) and Figure 2].
Separation conditions shall be such that possible interfering organic acids or substances will not interfere
with the organic acids of interest.

Figure 1 — Example chromatogram of organic acids with sufficient peak resolution
10

---------------------- Page: 12 ----------------------
oSIST prEN 17294:2018
prEN 17294:2018 (E)

Key
X = retention time t (s) 1 = Peak 1
R
Y = signal 2 = Peak 2
W = peak width (s)
Figure 2 — Graphical representation of the parameters to calculate the peak resolution R
Base peak widths, w and w , are obtained by constructing isosceles triangles over the Gaussian peaks.
1 2
11

---------------------- Page: 13 ----------------------
oSIST prEN 17294:2018
prEN 17294:2018 (E)
Calculate the peak resolution R using Formula (2).
2×()tt−
R2 R1
R =
(2)
2,1
w + w
21
where
R is the peak resolution;
2,1
t is the retention time of the first peak, in s;
R1
t is the retention time of the second peak, in s;
R2
w
is the peak width on the time axis of the first peak, in s;
1
w is the peak width on the time axis of the second peak, in s.
2
NOTE Mostly the chromatography software can calculated R by itself.
If R has to be calculated manually follow the described procedure: The width at the base of a peak is the
segment of the peak base intercept by the tangents drawn to the inflection points on either side of the
peak. Alternatively the peaks width can be measured at half the peak height using Formula (3).
18,(× tt− )
R2 R1
R =
(3)
2,1
ww+
h2 h1
where
R is the peak resolution;
2,1
t
is the retention time of the first peak, in s;
R1
t is the retention time of the second peak, in s;
R2
w
is the peak width on the time axis of the first peak at half the peak height, in s;
h1
w is the peak width on the time axis of the second peak at half the peak height, in s.
h2
7 Sampling
It is important that the laboratory receives a sample which is truly representative and has not been
damaged or changed during transport or storage.
Sampling is not part of the method specified in this International Standard. A recommended sampling
method is given in EN ISO 6497 [3].
Store the sample in such a way that deterioration and change in its composition are prevented.
8 Preparation of test sample
Prepare the test sample in accordance with EN ISO 6498.
Grinding (≤ 1 mm) shall be done in conditions such that the substance is not appreciably heated.
The whole ground product is placed in a flask made of e.g. polypropylene, which can be stoppered and
stored in such way to prevent any change in composition.
Before any weighing is carried out for the analysis, the whole test sample shall be thoroughly mixed for
reasons of homogeneity.
12

---------------------- Page: 14 ----------------------
oSIST prEN 17294:2018
prEN 17294:2018 (E)
9 Procedure
9.1 Extraction
9.1.1 Extraction of formic, lactic, propionic, citric, acetic and malic acid
Accurately weigh 5,00 g ± 10 mg of prepared sample into a 250 ml conical flask. Add 100,0 ml water (5.2)
and mix for 60 min on a magnetic stirrer at ambient temperature (20 °C to 25 °C).
NOTE Samples can also be extracted 30 min by ultrasonic bath at ambient temperature (20 C to 25 C).
Let the particles settle down. Filter through folded filter (6.7), discarding the first 3 ml of the filtrate, and
collect the rest. Alternatively centrifuge the sample extract for 3 min (appr. 5000 × g).
Dilute the filtrated/centrifuged solution with water (5.2) to a final concentration according to the
working range of the calibration (10 µg/ml – 100 µg/ml). – see below for a dilution example. For different
concentration levels of organic acids more than one dilution can be done.
Before IC analysis filter through a 0,45 µm membrane filter (6.8).
9.1.2 Extraction of fumaric acid
Accurately weigh 5,00 g ± 10 mg of prepared sample into a 250 ml conical flask. Add 100,0 ml water (5.2),
stopper the flask, heat up to 60 °C for complete solution and mix for 60 min on a magnetic stirrer.
NOTE Samples can also be extracted 30 min by ultrasonic bath at 60 °C.
The flask should be stoppered in order to avoid evaporation of extraction solution.
Cool to room temperature and let the particles settle down. Filter through folded filter (6.7), discarding
the first 3 ml of the filtrate, and collect the rest. Alternatively centrifuge the sample extract for 3 min
(appr. 5000 × g).
Dilute the filtrated/centrifuged solution with water (5.2) to a final concentration according to the
working range of the calibration (10 – 100 µg/ml). – see below for a dilution example.
Before IC analysis filter through a 0,45 µm membrane filter (6.8).
9.1.3 Extraction of coated acids
Accurately weigh 5,00 g ± 10 mg of prepared sample into a 250 ml conical flask. Add 100,0 ml water (5.2),
stopper the flask and extract 30 min by ultrasonic bath at 60 °C.
The flask shall be stoppered in order to avoid evaporation of extraction solution
Cool to room temperature and let the particles settle down. Filter through folded filter (6.7), discarding
the first 3 ml of the filtrate, and collect the rest. Alternatively centrifuge the sample extract for 3 min
(appr. 5000 × g).
Dilute the filtrated/centrifuged solution with water (5.2) to a final concentration according to the
working range of the calibration (10 – 100 µg/ml). – see below for a dilution example.
Before IC analysis filter through a 0,45 µm membrane filter (6.8).
For protecting the IC column the sample solution can be purified through an RP SPE (6.9). Follow the
description of the supplier. Blank solution and recoveries of organic acids have to be checked.
EXAMPLE Example of dilution:
Expected concentration of the sample 0,8 % (8000 µg/g) formic acid.
Desired final concentration: 80 µg/ml.
13

---------------------- Page: 15 ----------------------
oSIST prEN 17294:2018
prEN 17294:2018 (E)
5 g sample weight dissolved in 100 ml.
5g× 8000µg / g
Dilution factor 5

100ml× 80µg / ml
Therefore the sample extraction solution has to be diluted 1:5 to meet the calibration range.
9.2 Calibration
The following calibration solutions are recommended:
The maximum storage time is 2 months at 4 °C.
Prepare the calibration standards with water (5.2) according Table 3. The exact concentration of each
organic acid has to be calculated according to the concentration of the mixed standard solution (5.5).
Table 2 — Preparation of calibration standards
a
Concentration of organic
ml standard
acids each in the calibratio
...

Questions, Comments and Discussion

Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.