ISO 22241-2:2019

INTERNATIONAL ISO
STANDARD 22241-2
Second edition
2019-02
Diesel engines — NOx reduction agent
AUS 32 —
Part 2:
Test methods
Moteurs diesel — Agent AUS 32 de réduction des NOx —
Partie 2: Méthodes d'essai
Reference number
©
ISO 2019
© ISO 2019
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Published in Switzerland
ii © ISO 2019 – All rights reserved

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Specifications . 1
5 Sampling . 2
6 Precision and dispute . 2
Annex A (normative) Sampling . 3
Annex B (normative) Determination of urea content by total nitrogen . 5
Annex C (normative) Refractive index and determination of urea content by refractive index .9
Annex D (normative) Determination of alkalinity .12
Annex E (normative) Determination of biuret content .15
Annex F (normative) Determination of aldehyde content .20
Annex G (normative) Determination of insoluble matter content by gravimetric method .24
Annex H (normative) Determination of phosphate content by photometric method .27
Annex I (normative) Determination of trace element content (Al, Ca, Cr, Cu, Fe, K, Mg, Na, Ni,
P and Zn) by ICP-OES method .33
Annex J (informative) Determination of identity by FTIR spectrometry method .41
Annex K (informative) Precision of test method .43
Bibliography .44
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
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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
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on the ISO list of patent declarations received (see www .iso .org/patents).
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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 22, Road vehicles, Subcommittee SC 34,
Propulsion, powertrain and powertrain fluids.
This second edition cancels and replaces the first edition (ISO 22241-2:2006), which has been
technically revised. It also incorporates the Technical Corrigendum ISO 22241-2:2006/Cor. 1:2008. The
main changes compared to the previous edition are as follows:
— Major revisions to test methods of Annex C and Annex I,
— Precision values for all test methods were revised,
— Annex K was updated.
A list of all parts in the ISO 22241 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2019 – All rights reserved

INTERNATIONAL STANDARD ISO 22241-2:2019(E)
Diesel engines — NOx reduction agent AUS 32 —
Part 2:
Test methods
WARNING — The use of this document can involve hazardous materials, operations and
equipment. This document does not purport to address all the safety issues associated with its
use. It is the responsibility of users of this document to respond appropriately to ensure the
safety and health of personnel prior to application of the document.
1 Scope
This document specifies test methods required for determination of the quality and chemical
characteristics of NOx reduction agent AUS 32 (aqueous urea solution) as specified in ISO 22241-1.
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
ISO 4259 (all parts), Petroleum and related products — Precision of measurement methods and results
ISO 12185, Crude petroleum and petroleum products — Determination of density — Oscillating U-tube
method
ISO 17034, General requirements for the competence of reference material producers
ISO 22241-1, Diesel engines — NOx reduction agent AUS 32 — Part 1: Quality requirements
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 22241-1 and the following apply.
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/
3.1
certified reference material
CRM
substance or material used to check the quality and metrological traceability of products, to validate
analytical measurement methods or for the calibration of instruments
4 Specifications
Conformance with the limits specified in ISO 22241-1 shall be determined by the test methods specified
in Annexes B through I of this document. If necessary, the identity of the product can be determined as
specified in Annex J.
5 Sampling
Samples shall be taken in accordance with Annex A.
6 Precision and dispute
Each of the test methods specified in Annex B through Annex I include a precision statement using
guidance from ISO 4259 (all parts). In cases of dispute, the procedures described in ISO 4259 (all parts)
shall be used for resolving the dispute, and interpretation of the results based on the test method
precision shall be used.
For the convenience of the user, the respective precision data are summarized in Table K.1.
2 © ISO 2019 – All rights reserved

Annex A
(normative)
Sampling
A.1 General
The sampling method specified in this annex is valid for each sampling of AUS 32 throughout the supply
chain after the shipment from the manufacturer’s site to the AUS 32 containers of the vehicles.
A.2 Principle
The limits for the quality characteristics of AUS 32, which are specified in ISO 22241-1, are the
representative analytical results that can only be obtained when the sample is protected from any
contamination before the analysis.
Therefore, suitable bottles shall be used for sampling, which do not contaminate the sample, especially
regarding the trace elements.
NOTE The sampling method specified in this annex is based on ISO 5667-3.
A.3 Possible contaminants
During the sampling process, foreign matter can lead to contamination of the sample. Under realistic
conditions, the following sources of contamination will pose a major hazard:
— residues of process aids used for the production of the sampling bottles;
— contaminants which have been deposited in the empty bottles during the time they are stored empty;
— contaminants from the air, i.e. dust or any foreign matter from the surrounding, during the sampling;
— residues of cleaning agents, which have been used for cleaning the sampling equipment and the
bottles as well;
— fuel.
A.4 Apparatus
A.4.1 Sampling bottles.
1 000-ml wide neck bottles shall be used. Suited materials for these bottles are high density polyethylene
(HDPE), high density polypropylene (HDPP), polyvinylidene fluoride (PVDF) and perfluoroalkoxy
alkane (PFA).
Prior to the first use with AUS 32, the bottles shall be cleaned and finally rinsed with deionized water
followed by AUS 32.
A.4.2 Labels.
Each bottle shall be labelled using labels of approximately 10 cm by 5 cm. The labels and the writing on
these labels shall be resistant to water and to AUS 32.
A.5 Sampling
The locked wide-neck bottle shall be opened and the cap shall be placed on a clean surface with the
opening turned downward. After flushing the sampling pipe, the bottle shall be filled completely with
AUS 32 from the container. The first filling shall be discarded, and the bottle shall immediately be re-
filled with AUS 32 and closed tightly. The label shall be attached to the bottle (see A.4.2). During the filling
of the sample, maximum care shall be taken that neither dust nor liquid pollutants get into the bottle.
The filled bottle should reach the laboratory as soon as possible. During transportation and storage, the
sample should be kept at the lowest possible temperature, preferably between 0 °C and 15 °C.
It is recommended to conduct the analysis within three weeks in order to take into account possible
changes in the ammonia content.
A.6 Sample quantity
The minimum quantity of sample material depends on the type of analysis conducted. Whenever
possible, make sure that a sufficient volume of sample material is available (recommendation: 1 litre),
and at least double that which is required for complete verification of AUS 32 specifications. In case of
dispute, a sufficient number of samples shall be taken according to ISO 4259 (all parts).
A.7 Labelling
The label should contain the following information:
— product name;
1)
— name of the company which owns the sample product ;
1)
— address where the sample was taken from ;
1)
— manufacturer of the sample product ;
— batch or lot number;
1)
— container from which the sample was taken ;
1)
— part of the container where the sample was taken from (sampling point) ;
1)
— date and time of sampling ;
1)
— sample shipment date ;
1)
— name and signature of the person who took the sample .
1) Mandatory only in cases of dispute.
4 © ISO 2019 – All rights reserved

Annex B
(normative)
Determination of urea content by total nitrogen
B.1 General
This annex specifies the procedure for determining the urea content of AUS 32.
The method is applicable for the determination of the urea content in the range of 30 to 35 % (mass
fraction).
B.2 Principle
The sample is combusted at high temperatures in a stream of oxygen. Following the reduction of formed
nitrogen oxides to elemental nitrogen and removal of any interfering products of combustion, nitrogen
is measured with a thermal-conductivity detector. The urea content is calculated from the determined
total nitrogen minus the nitrogen content of biuret.
B.3 Apparatus
B.3.1 Automatic nitrogen analyser, based on combustion methods.
B.3.2 Analytical balance.
The accuracy of the balance is a function of the analyser used and the required weighed portions.
Resolution should be 0,1 % of the weighed portion or better.
B.3.3 Auxiliary devices for sample preparation, for example:
— tweezers with a blunt tip;
— micro-spatula with a flattened tip;
— pipette.
The pipette is recommended for weighing in and thus does not need to be calibrated. It is important,
however, to obtain a good droplet size (small droplets). Fixed-volume pipettes or pipettes with an
adjustable volume in the range from 10 µl to 1 000 µl or single-trip Pasteur pipettes with a fine tip may
also be used.
B.3.4 Customary chemically resistant glass.
B.4 Chemicals
B.4.1 Distilled or deionized water, conductivity less than 0,1 mS/m, according to ISO 3696 grade 2.
B.4.2 Auxiliary combustion agent and other equipment, appropriate for use with the selected
nitrogen analyser.
The following materials are merely examples. Other or similar materials may be used as required,
depending on the system that is available:
— tin capsule or similar sample containers;
— auxiliary combustion agent, non-nitrogenous, such as saccharose, cellulose;
— absorbing agent for liquids, non-nitrogenous, such as magnesium oxide.
B.4.3 Standard substances for nitrogen determination, preferably with certified nitrogen content.
EXAMPLE Suitable standard substances include ethylenediamine tetraacetic acid (EDTA), nicotinic acid amide.
Low-biuret urea of adequate purity (for example crystalline ultra pure or analytical) or other such
standard substances recommended by and available from the equipment manufacturer may also be
used. Certified standard substances should be preferred.
NOTE Liquid standard substances (e.g. urea solutions) are not suited for calibration purposes.
B.4.4 Oxygen, min. 99,995 % O .
B.4.5 Other ultrapure gases, if required to operate the nitrogen analyser, such as helium,
min. 99,996 % He.
B.4.6 Other reagents or auxiliary agents, as required by the equipment.
B.5 Procedure
B.5.1 General
The sample should be fully dissolved and free from urea crystals. It may be heated to max. 40 °C as
required prior to further processing.
NOTE Different types of apparatus are available on the market. The resulting various resources and modes
of operation are not an object of this document. Rather, operation is based on the respective operation manuals.
B.5.2 Reference curve
Perform calibration as required for the specific type of analyser and according to the respective
operation manuals (for example, after replacement of the combustion tube, reagent or similar)
by performing measurements as described in B.5.4. Weigh in an appropriate amount of standard
substances repeatedly as appropriate for the respective types of apparatus to obtain a reference curve.
B.5.3 Inspecting the apparatus for good working order and the reference curve
Use an appropriate standard substance to review the good working order of the apparatus and the
reference curve. Preferably, a certified urea standard solution should be used.
Frequency of inspection is a function of the analyser used.
B.5.4 Measurement
Weigh a portion of the sample in a suitable holder (such as a tin capsule) as specified for the type of
nitrogen analyser used. The amount should be such that the absolute amount of nitrogen is in the
middle range of the reference curve.
6 © ISO 2019 – All rights reserved

Use approximately the three-fold amount of combustion agent (for example, non-nitrogenous cellulose),
and additional binders (for example, magnesium oxide) as required.
When using liquid feeder systems, the volume used should be no less than 100 µl. The sample mass
density shall be determined according to ISO 12185.
Enter the required data (weighed portion, sample identification) into the analyser or a control computer,
depending on the type of apparatus. Feed the weighed-in sample to the analyser and start combustion.
Perform at least three (3) single determinations.
B.6 Results
B.6.1 Calculation
Prior to calculating the reference curve, drift of the baseline or samples, determine the blank reading
value by means of blank samples and use this value to correct the respective analytical sequences.
Use the apparatus-specific programme to calculate the reference curve or the drift correction for the
samples.
Calculate the mean value for the samples. If there is a strong dispersion of single values (relative
standard deviation RSD > 1,0 %), repeat the affected sample. After that, determine the mean value for
this sample from all single values.
Determine the urea content from the mean value from at least three nitrogen determinations:
ww=×2,1438 ()−×Fw
UN Bi
where
w is the urea content (mass fraction, in %);
U
w is the mean value of the nitrogen content (mass fraction, in %) to the nearest 0,01 %;
N
w is the mean value of the biuret content (mass fraction, in %), determined according to
Bi
Annex E;
F is the factor for converting the biuret content to nitrogen (0,407 6).
B.6.2 Expression of results
The result is the arithmetic mean value from at least three (3) single determinations (nitrogen
determinations).
Round off the result of the urea content calculation to the nearest 0,1 %.
B.7 Precision
B.7.1 General
The precision evaluation programme with a matrix of only four samples of AUS 32 solutions with
urea content in the range 31,09 % (mass fraction) to 35,12 % (mass fraction) did not conform to the
requirements of ISO 4259 (all parts), and thus only an estimate of precision based upon inter-laboratory
test results is given in B.7.2, B.7.3 and Table B.1.
B.7.2 Repeatability, r
The difference between two test results, obtained by the same operator with the same apparatus
under constant operating conditions on identical test material would, in the long run, in the normal and
correct operation of the test method, exceed the following in only one case in twenty.
r = 0,466 % (mass fraction)
B.7.3 Reproducibility, R
The difference between two single and independent results, obtained by different operators working
in different laboratories on identical test material would, in the long run, in the normal and correct
operation of the test method, exceed the following value in only one case in twenty.
R = 1,053 % (mass fraction)
Table B.1 — Precision (estimated)
Urea content Repeatability Reproducibility
w r R
U
% (mass fraction) % (mass fraction) % (mass fraction)
31,09 to 35,12 0,466 1,053
B.8 Test report
The test report shall contain at least the following information:
a) type and description of tested product;
b) reference to this document, i.e. ISO 22241-2:2019;
c) sampling method used;
d) test result (see B.6);
e) deviations from the specified mode of operation;
f) any unusual features observed; and
g) test date.
8 © ISO 2019 – All rights reserved

Annex C
(normative)
Refractive index and determination of urea content by
refractive index
C.1 General
This annex specifies the procedure for the determination of the refractive index, relative to air, for
AUS 32 at 20 °C, and at a reference wavelength at 589,3 nm ± 5 nm.
Based on the measurement of refractive index, the method shall be used for determining the
concentration of urea in the range of a mass fraction of 30 % to 35 % using existing data.
NOTE The method specified in this annex is based on ISO 5661.
C.2 Principle
Measurement is based on the dependence of refractive index on the concentration of urea in an aqueous
solution at a definite temperature and wavelength.
The content is determined by comparison to the agreed upon mathematical relationship between AUS
concentration and refraction index.
C.3 Apparatus
C.3.1 Refractometer, capable of measuring refractive index at a reference wavelength of 589,3 nm,
with a measuring range of at least 1,330 00 to 1,390 00, and a resolution of 0,000 01, with means of
controlling temperature to (20 ± 0,1) °C.
NOTE Different types of apparatus are available on the market. The resulting various resources and modes
of operation are not an object of this document. Rather, operation is based on the respective operation manuals.
C.4 Chemicals
C.4.1 Distilled or deionized water, conductivity less than 0,5 mS/m according to ISO 3696 grade 3.
C.4.2 Certified reference material, fluid of known refractive index, prepared in accordance with
ISO 17034 and traceable through an unbroken chain of calibrations to a national measurement institute
(NMI). For purposes of this standard, CRMs should have an uncertainty of ±0,000 05 nD or better.
C.5 Procedure
C.5.1 General
Each test sample should be fully dissolved and free from urea crystals. They may be heated to <40 °C
as required prior to further processing. Care should be taken to avoid heating solutions above 40 °C for
any time longer than is required to bring the crystals into solution.
C.5.2 Refractometer calibration and verification
Daily, the refractometer shall be zero-set to distilled or deionized water at (20 ± 0,1) °C in accordance
with the instructions provided by the refractometer manufacturer.
Weekly, the refractometer shall be verified using distilled or deionized water and at least one CRM
with a value greater than or equal to 1,382 7 nD20. If the refractive index of any CRM, as read on the
refractometer, deviates by more than 0,000 05 nD20 from the certified value, then the refractometer
shall be recalibrated according to the manufacturer’s instructions, using a series of CRMs.
Monthly, the refractometer calibration shall be verified using distilled or deionized water and a series
of CRMs (a minimum of three) spanning beyond the measuring range (1,381 4 to 1,384 3 nD20). If
the refractive index of any CRM, as read on the refractometer, deviates by more than 0,000 05 nD20
from the certified value, then the refractometer shall be recalibrated according to the manufacturer’s
instructions, using a series of CRMs.
After completion of each verification/calibration, the refractometer shall be marked or tagged with the
date and type of that verification/calibration.
If, after the calibration, the certified value for any CRM cannot be verified within ±0,000 05 nD20, then
the instrument shall be tagged as “Out of service” until instrument calibration can be properly verified.
C.5.3 Sample preparation and measuring
If using a circulating water bath to maintain a constant temperature in the refractometer, adjust the
thermostat to the desired temperature, reading this temperature on the refractometer thermometer
on the discharge side. Maintain the flow of water so that the desired temperature is maintained at
(20 ± 0,1) °C.
If the refractometer is equipped with a solid-state Peltier temperature control device, adjust the
controls so that the refractometer is controlled to a temperature of (20 ± 0,1) °C.
Measure the sample refractive index three times and then determine the arithmetic mean of the three
refractive index values. If any two of the three measurements deviate by more than 0,000 05 nD20, the
measurements shall be repeated.
C.6 Calculation and expression of results
C.6.1 Calculation
Urea content, w , shall be calculated from the refractive index determined in C.5.3 using the following
U
formula, which has a correlation of R2 = 1,00, less the Biuret content as determined according to
Annex E.
w = (−742,747 88 × (nD20) + 2 669,653 61 × (nD20) − 2 238,799 1) − B
U
where
nD20 represents the refractive index of the sample at 20 °C as determined in C.5.3;
B is the Biuret mass fraction (%) according to Annex E.
NOTE Biuret has the same refractive index per unit of mass as urea.
C.6.2 Expression of results
The result is defined as the arithmetic mean of the three refractive index measurements rounded to
the nearest 0,000 1 nD20. For the urea content, the result shall be rounded to the nearest 0,1 % (mass
fraction).
10 © ISO 2019 – All rights reserved

C.7 Precision
C.7.1 General
The precision, as determined by statistical examination in accordance with ISO 4259 (all parts) of inter-
laboratory study results on AUS 32 blends with test results in the range 31,76 % (mass fraction) to
34,75 % (mass fraction) (and refractive index nD20 from 1,379 to 1,385), is given in C.7.2, C.7.3 and
Table C.1.
C.7.2 Repeatability, r
The difference between two test results, obtained by the same operator with the same apparatus
under constant operating conditions on identical test material would, in the long run, in the normal and
correct operation of the test method, exceed the value given in Table C.1 in only one case in twenty.
r (Urea content) = 0,154 % (mass fraction)
r (refractive index nD20) = 0,000 25
C.7.3 Reproducibility, R
The difference between two single and independent results, obtained by different operators working
in different laboratories on identical test material would, in the long run, in the normal and correct
operation of the test method, exceed the value given in Table C.1 only in one case in twenty.
R (Urea content) = 0,211 % (mass fraction)
R (refractive index nD20) = 0,000 33
Table C.1 — Precision
Repeatability Reproducibility
Property
r R
Urea content (mass frac-
0,154 0,211
tion, in %)
Refractive index nD20 0,000 25 0,000 33
C.8 Test report
The test report shall contain at least the following information:
a) type and description of tested product;
b) reference to this document, i.e. ISO 22241-2:2019;
c) sampling method used;
d) test result (see C.6);
e) deviations from the specified mode of operation;
f) any unusual features observed; and
g) test date.
Annex D
(normative)
Determination of alkalinity
D.1 General
This annex specifies the procedure for the determination of the alkalinity of AUS 32, calculated as
ammonia, in the range of 0,1 % to 0,5 %.
D.2 Principle
The measurement is based on potentiometric titration of free ammonia of a test portion with a standard
volumetric hydrochloric acid solution to the endpoint at pH = 5,7.
D.3 Apparatus
D.3.1 Analytical balance, resolution 0,1 mg or better.
D.3.2 Automatic burette.
D.3.3 Potentiometer, capable of measuring with a precision of 0,01 pH units, equipped with glass
combined pH-electrode.
D.3.4 Magnetic stirrer.
D.3.5 Beaker, 150 ml, tall shaped.
D.3.6 Measuring cylinder, 100 ml.
D.4 Chemicals
D.4.1 General.
During the analysis, use only reagents of recognized analytical grade and only distilled or deionized
water of an electric conductivity lower than 0,5 mS/m, according to ISO 3696 grade 3.
D.4.2 Hydrochloric acid, 0,01 mol/l standard solution.
D.4.3 Buffer solutions.
The following standard buffer solutions shall be used for the determination of alkalinity:
— standard buffer solution, pH = 4,008;
— standard buffer solution, pH = 9,184;
— standard buffer solution, pH = 8,00 or 6,86.
NOTE Such solutions are commercially available.
12 © ISO 2019 – All rights reserved

D.5 Procedure
D.5.1 Interferences
The samples of AUS 32 taken shall be stored and shipped at a temperature not higher than 25 °C in
order to avoid ammonia formation.
The containers shall be closed tightly and the analysis time shall not be protracted by interruption to
avoid evaporation of ammonia.
D.5.2 Check of potentiometric system
The correct function of the potentiometric system shall be checked by use of the standard buffer
solutions at pH = 4,008 and pH = 9,180.
The standard buffer solution at pH = 8,00 or 6,86 shall be used for daily check of the potentiometric
system.
D.5.3 Preliminary test
Weigh about 1 g of the homogenous sample to 0,05 g (sample mass m ) and put it into a 150 ml beaker
S
filled with about 100 ml distilled or deionized water.
Titrate with the hydrochloric acid solution (0,01 mol/l) under stirring to the endpoint at pH = 5,7.
Calculate the content of ammonia.
Depending on the content of alkalinity found, weigh the following sample portions for the determination:
— alkalinity content found by the preliminary test (%) 0,02 0,05 0,1 0,2 to 0,5
— mass of test portion for the determination (g): 10 5 2 1
— see D.6.1 for an example.
D.5.4 Determination
Weigh the mass of the homogenous sample to 0,05 g found by the preliminary test (sample mass m )
S
and put it into a 150 ml beaker filled with about 100 ml distilled or deionized water.
Titrate with the hydrochloric acid solution (0,01 mol/l) under stirring at first to pH = 7,5 with normal
speed, then titrate to the endpoint at pH = 5,7 with reduced speed.
Perform two measurements.
D.6 Results
D.6.1 Calculation
The alkalinity, expressed as a percentage by mass of ammonia (NH ), is given by the formula
wVNH =×0,/017 m
() ()
3 S
where
w(NH ) is the alkalinity, calculated as ammonia (mass fraction, in %);
V is the volume of the hydrochloric acid solution used for the titration (ml);
m is the mass of the test portion (g).
S
D.6.2 Expression of results
Calculate the mean value of the two measurements. Express the result to the nearest 0,01 % (mass
fraction).
D.7 Precision
D.7.1 General
The precision evaluation programme with a matrix of only four samples of AUS 32 solutions with
alkalinity in the range 0,084 % (mass fraction) to 0,529 % (mass fraction) did not conform to the
requirements of ISO 4259 (all parts), and thus only an estimate of precision based upon inter-laboratory
test results is given in D.7.2, D.7.3 and Table D.1.
D.7.2 Repeatability, r
The difference between two test results, obtained by the same operator with the same apparatus
under constant operating conditions on identical test material would, in the long run, in the normal and
correct operation of the test method, exceed the following in only one case in twenty.
r = 0,077 % (mass fraction)
D.7.3 Reproducibility, R
The difference between two single and independent results, obtained by different operators working
in different laboratories on identical test material would, in the long run, in the normal and correct
operation of the test method, exceed the following value in only one case in twenty.
R = 0,124 % (mass fraction)
Table D.1 — Precision (estimated)
Alkalinity content Repeatability Reproducibility
w(NH ) r R
% (mass fraction) % (mass fraction) % (mass fraction)
0,084 to 0,529 0,077 0,124
D.8 Test report
The test report shall contain at least the following information:
a) type and description of tested product;
b) reference to this document, i.e. ISO 22241-2:2019;
c) sampling test method;
d) test result (see D.6);
e) deviations from the specified mode of operation;
f) any unusual features observed; and
g) test date.
14 © ISO 2019 – All rights reserved

Annex E
(normative)
Determination of biuret content
E.1 General
This annex specifies the procedure for the determination of the biuret content of AUS 32 with contents
of biuret with 0,1 % (mass fraction) to 0,5 % (mass fraction) by photometric method.
E.2 Principle
Biuret forms in alkaline solution in the presence of sodium-potassium-tartrate with bivalent copper
a violet-coloured complex with an absorption maximum at 550 nm. The colour complex is read
spectrophotometrically at 550 nm and the biuret concentration is determined by reference to a
calibration curve prepared from standard biuret solutions.
E.3 Apparatus
E.3.1 Laboratory balance, resolution in reading 0,001 g.
E.3.2 Vacuum filtration unit, applicable for filter with 0,45 µm pore size.
E.3.3 Spectrophotometer, for use at 550 nm with 10-50-mm-cell.
E.3.4 Volumetric flasks, 1 000 ml, 250 ml, 100 ml, 50 ml.
E.3.5 Pipettes.
E.3.6 Rotary evaporator.
E.3.7 Constant-temperature bath, capable of maintaining a temperature of 30 °C ± 1 °C.
E.4 Chemicals
E.4.1 Chemicals of analytical grade.
These shall be used in all tests. The water shall be distilled or deionized, conductivity less than 0,5 mS/m
according to ISO 3696 grade 3.
E.4.2 Copper sulphate-solution.
Dissolve 15 g copper sulphate (CuSO ⋅5H O) in CO -free water and dilute to 1 000 ml.
4 2 2
E.4.3 Alkaline potassium sodium tartrate-solution.
Dissolve 40 g sodium hydroxide in 500 ml water in a 1 000 mI volumetric flask. After cooling, add 50 g
potassium sodium tartrate (KNaC H O ⋅4H O) and fill up the flask with water to the mark. Let the
4 4 6 2
flasks stand 1 day before use.
E.4.4 Biuret-standard-solution, of 0,8 mg biuret/ml.
Dissolve 800 mg pure biuret in CO -free water and dilute to 1 000 ml. Dry the biuret for 3 h at 105 °C
before use.
Biuret may be purified as follows:
— add 50 g biuret to 500 ml ammonia solution of 25 % (mass fraction) concentration and stir for
15 minutes;
— filter, rinse with ammonia-free water and dry the biuret;
— dissolve in ethanol (1 litre/10 g), filter, and concentrate by gentle heating to one-fourth the volume;
— cool to 5 °C and filter;
— dry the biuret in vacuum oven at 80 °C;
— check the purity by photometrical measurements according to E.5.5.
The step of re-crystallizing from ethanol shall be repeated until there is no more noticeable
improvement of purity.
E.4.5 Standard acid, 0,1 N hydrochloric or sulfuric acid.
E.5 Procedure
E.5.1 Interferences
Spectrophotometric measurements are only suitable for clear solutions. If the sample is not clear, filter
through a 0,45 µm filter to get a clear solution.
Ammonia forms with bivalent copper a coloured complex, which absorbs light energy at 550 nm. The
method is applicable only if the ammonia-content of the sample is less than 500 mg/kg.
To eliminate ammonia content greater than 500 mg/kg, neutralize pre-weighed sample to less than
7,0 pH with standard acid.
E.5.2 Preparation of the calibration curve
Into a series of six 50 ml volumetric flasks, transfer 2 ml, 5 ml, 10 ml, 15 ml, 20 ml and 25 ml of the biuret
standard solution and add water (to each of the six flasks) to a total of mixture volume of approximately
25 ml. Add, while stirring after each addition, 10 ml of the alkaline potassium sodium tartrate-solution
and 10 ml of the copper sulphate-solution. Immerse the flasks in the constant-temperature bath,
regulated at 30 °C ± 1 °C and leave them there for about 15 minutes.
Carry out a blank test in parallel with the determination, following the same procedure and using the
same quantities of all the reagents used for the measurement (see E.5.5).
After cooling to room temperature, fill up the flasks with water to the mark and mix well. Carry out the
photometric measurements with the spectrophotometer at a wavelength of about 550 nm using a 10-
50 mm cell against water as the reference.
Subtract the extinction of the blank test from the extinction of the measured values and set up the
calibration curve. In the concentration range, the curve shall be strictly linear.
16 © ISO 2019 – All rights reserved

E.5.3 Calculation of the calibration factor
Calculate the calibration factor according to the following formula:
m
Bi,i
∑ 61,6
i=1
F = =
C
6 6
EE− EE−
() ()
12,,i 12i
∑∑
i==1 i 1
where
F is the calibration factor (mg);
C
m is the mass of biuret of the i-th sample (mg);
Bi,i
E is the extinction of the i-th sample;
1,i
E is the extinction of the blank test.
The determination of the calibration curve and the calibration factor shall be repeated on a yearly basis
and shall be documented.
E.5.4 Day-factor
The day-factor shall be determined weekly.
Perform a measurement of 10 ml of the biuret standard solution (8 mg biuret) as described in E.5.5.
Calculate as follows:
F =
D
EE−
()
12,i
where
F is the day-factor (mg);
D
E is the extinction of the standard solution (average from 2 measures);
E is the extinction of the blank test.
The deviation of the day-factor shall be within ±5 % to the calibration factor. For measuring of samples,
the day-factor shall be used.
E.5.5 Measurement
Weigh 100 g of the test sample, to the nearest 0,01 g, in a 250 ml beaker. Neutralize to below 7,0 pH with
standard acid. Quantitatively transfer the sample to a 250 ml volumetric flask. Fill the flask to the mark
with demineralized water and mix thoroughly.
Transfer an aliquot of 10 ml from the test solution into a 50 ml volumetric flask and add water to
approximately 25 ml. Add, with stirring after each addition, 10 ml of the alkaline potassium sodium
tartrate-solution and 10 ml of the copper sulphate-solution. Immerse the flask in the constant-
temperature bath, regulated at 30 °C ± 1 °C and leave it there for about 15 minutes.
Carry out a blank test in parallel with the determination, following the same procedure and using the
same quantities of all the reagents used for the determination.
After cooling to room temperature, fill up the flask with water to the mark and mix well. Carry out
the photometric measurements with the spectrophotometer at a wavelength of about 550 nm using a
50 mm cell against water as the reference.
To determine non-specific absorptions, put another 10 ml of the test solution into a 50 ml volumetric
flask, fill the flask up to the mark with water and measure the absorption in the same order.
Duplicate determinations shall be carried out.
E.6 Results
E.6.1 Calculation
The biuret content is given, as a percentage by mass, by the formula:
EE− ×F ×250
()
SB D
w = ×100
Bi
m ×10×1000
S
where
w is the biuret content (mass fraction, in %);
Bi
E is the extinction of the sample;
S
E is the extinction of the blank test (reagent blank + sample blank);
B
m is the mass of sample used to prepare the test solution (g);
S
F is the day-factor (mg).
D
E.6.2 Expression of results
Express the result to the nearest 0,01 % (mass fraction).
E.7 Precision
E.7.1 General
The precision evaluation programme with a matrix of only four samples of AUS 32 solutions with biuret
in the range 0,115 % (mass fraction) to 0,461 % (mass fraction) did not conform to the requirements of
ISO 4259 (all parts), and thus only an estimate of precision based upon inter-laboratory test results is
given in E.7.2, E.7.3 and Table E.1.
E.7.2 Repeatability, r
The difference between two test results, obtained by the same operator with the same apparatus
under constant operating conditions on identical test material would, in the long run, in the normal and
correct operation of the test method, exceed the following in only one case in twenty.
r = 0,008 % (mass fraction)
E.7.3 Reproducibility, R
The difference between two single and independent results, obtained by different operators working
in different laboratories on identical test material would, in the long run, in the normal and correct
operation of the test method, exceed the following value in only one case in twenty.
R = 0,044 % (mass fraction)
18 © ISO 2019 – All rights reserved

Table E.1 — Precision (estimated)
Biuret content Repeatability Reproducibility
w
r R
Bi
% (mass fraction) % (mass fraction) % (mass fraction)
0,115 to 0,461 0,008 0,044
E.8 Test report
The test report shall contain at least the following information:
a) type and description of tested product;
b) reference to this document, i.e. ISO 22241-2:2019;
c) sampling method used;
d) test result (see E.6);
e) deviations from the specified mode of operation;
f) any unusual features observed; and
g) test date.
Annex F
(normative)
Determination of aldehyde content
F.1 General
This annex specifies the procedure for the determination of the content of free and bound aldehyde,
calculated as formaldehyde, of AUS 32 with contents of aldehyde from 0,5 mg/kg to 10 mg/kg.
F.2 Principle
Formaldehyde forms in strong sulphuric acid solution with chromotropic acid a purple colour with
absorption maximum at 565 nm. The colour complex is read spectrophotometrically at 565 nm and
the aldehyde concentration is determined by reference to a calibration curve prepared from standard
formaldehyde solutions.
NOTE The method specified in this annex is based on Reference [4].
F.3 Apparatus
F.3.1 Laboratory balance, resolution in reading 0,001 g.
F.3.2 Spectrophotometer, for use at 565 nm with 10-mm-cell.
F.3.3 Volumetric flasks.
F.3.4 Pipettes.
F.4 Chemicals
F.4.1 Chemicals of analytical grade, which shall be used in all tests.
F.4.2 Sulphuric acid, a mass fraction of 96 %.
F.4.3 Chromotropic acid (4,5-dihydroxynaphthalene-2,7-disulphonic acid sodium salt or
4,5-dihydroxy- naphthalene-2,7-disulphonic acid disodium salt dihydrate), with a mass fraction of 3 % in
a mass fr
...