ISO 22241-2:2006

Diesel engines - NOx reduction agent AUS 32 - Part 2: Test methods

ISO 22241-2:2006 specifies test methods required for determination of the quality characteristics of the NOx reduction agent AUS 32 (aqueous urea solution) specified in ISO 22241-1.

Moteurs diesel — Agent AUS 32 de réduction des NOx — Partie 2: Méthodes d'essai

General Information

• Status: Withdrawn
• Publication Date: 10-Oct-2006
• Withdrawal Date: 10-Oct-2006
• ICS: 43.060.40 - Fuel systems
• Technical Committee: ISO/TC 22/SC 34 - Propulsion, powertrain and powertrain fluids
• Drafting Committee: ISO/TC 22/SC 34 - Propulsion, powertrain and powertrain fluids
• Current Stage: 9599 - Withdrawal of International Standard
• Start Date: 12-Feb-2019
• Completion Date: 13-Dec-2025

Relations

• Revised: ISO 22241-2:2019 - Diesel engines - NOx reduction agent AUS 32 - Part 2: Test methods
• Effective Date: 19-Apr-2014
• Revises: ISO/PAS 22241-2:2005 - Diesel engines - NOx reduction agent AUS 32 - Part 2: Test methods
• Effective Date: 15-Apr-2008

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 22241-2
First edition
2006-10-15
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 2006
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©  ISO 2006
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ii © ISO 2006 – All rights reserved

Contents Page
Foreword. iv
1 Scope . 1
2 Normative references . 1
3 Specifications. 1
4 Sampling. 1
5 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. 13
Annex E (normative) Determination of biuret content . 16
Annex F (normative) Determination of aldehyde content. 21
Annex G (normative) Determination of insoluble matter content by gravimetric method . 25
Annex H (normative) Determination of phosphate content by photometric method. 28
Annex I (normative) Determination of trace element content (Al, Ca, Cr, Cu, Fe, K, Mg, Na, Ni, Zn)
by ICP-OES method. 34
Annex J (normative) Determination of identity by FTIR spectrometry method. 40
Annex K (informative) Precision of test methods . 42
Bibliography . 43

Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 22241-2 was prepared by Technical Committee ISO/TC 22, Road vehicles, Subcommittee SC 5, Engine
tests.
This first edition cancels and replaces ISO/PAS 22241-2:2005, which has been technically revised.
ISO 22241 consists of the following parts, under the general title Diesel engines — NOx reduction agent
AUS 32:
⎯ Part 1: Quality requirements
⎯ Part 2: Test methods
The following parts are under preparation:
⎯ Part 3: Packaging, transportation and storage
⎯ Part 4: Refilling interface
Annexes A to J form a normative part of this International Standard. Annex K is for information only.

iv © ISO 2006 – All rights reserved

INTERNATIONAL STANDARD ISO 22241-2:2006(E)

Diesel engines — NOx reduction agent AUS 32 —
Part 2:
Test methods
1 Scope
This part of ISO 22241 specifies test methods required for determination of the quality characteristics of the
NOx reduction agent AUS 32 (aqueous urea solution) specified in ISO 22241-1.
In the remaining parts of ISO 22241, the term “NOx reduction agent AUS 32” will be abbreviated to “AUS 32”.
2 Normative references
The following referenced documents are indispensable for the application 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 22241-1, Diesel engines — NOx reduction agent AUS 32 — Part 1: Quality requirements
ISO 3675, Crude petroleum and liquid petroleum products — Laboratory determination of density —
Hydrometer method
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 4259, Petroleum products — Determination and application of precision data in relation to methods of test
ISO 12185, Crude petroleum and petroleum products — Determination of density — Oscillating U-tube
method
3 Specifications
Compliance with the limits specified in Table 1 of ISO 22241-1 shall be determined by the test methods
specified in Annexes B through J of this part of ISO 22241.
Determination of the density shall be conducted in accordance with ISO 3675 or ISO 12185.
NOTE For the purposes of this International Standard, the terms “% (m/m)” and “% (V/V)” are used to represent the
mass fraction and the volume fraction of a material respectively.
4 Sampling
Samples shall be taken in accordance with Annex A.
5 Precision and dispute
5.1 General
All test methods referred to in this part of ISO 22241 include a precision statement according to ISO 4259. In
cases of dispute, the procedures described in ISO 4259 shall be used for resolving the dispute, and
interpretation of the results based on the test method precision shall be used.
The precision of the test method, as determined by statistical examination in accordance with ISO 4259, is
specified in each annex. Additionally, this information is summarized in Annex K for all test methods for the
convenience of the user of this part of ISO 22241.
The statistical significance of the precision quoted in this part of ISO 22241 is generically defined in 5.2 and
5.3, in which the “xx (unit)” stands for the repeatability and reproducibility in question.
5.2 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 xx (unit) in only one case in 20.
5.3 Reproducibility, R
The difference between two single and independent test 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 xx (unit) in only one case in 20.
2 © ISO 2006 – 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, and which minimize the risk of algae or bacteria growth.
NOTE The sampling method specified in this annex is based on ISO 5667-2 and ISO 5667-3.
A.3 Possible contaminants
During the sampling process, foreign matter may 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
1000-ml wide neck bottles shall be used. Suited materials for these bottles are HD-polyethylene,
HD-polypropylene, polyfluorethylene, polyvinylidenedifluoride and tetrafluoroethylene-perfluoroalky vinyl ether
copolymer (PFA). In case of dispute, PFA bottles should be used.
Prior to the first use with AUS 32, the bottles shall be cleaned and finally rinsed with de-ionized water followed
by AUS 32.
A.4.2 Labels
Each bottle shall be labelled using labels of approximately 10 cm × 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, the cap shall be put down 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, and kept away
from daylight to prevent growth of algae.
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.
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 2006 – 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 from 30 % to 35 % (m/m).
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 % or better of the weighed portion.
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 De-ionized water, conductivity less than 0,1 mS/m, according to ISO 3696 grade 2.
B.4.2 Auxiliary combustion agent and other equipments, 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 part of ISO 22241. Rather, operation should be 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 2006 – 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 is
calculated using the density that was calculated 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 −F×w
( )
UN Bi
where
w is the urea content [% (m/m)];
U
w is the mean value of the nitrogen content [% (m/m)] (to the nearest 0,01%);
N
w is the mean value of the biuret content (%), determined according to Annex E;
Bi
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
See 5.2, 5.3 and Table B.1.
Table B.1 — Precision
Urea content Repeatability Reproducibility
w r R
U
% (m/m) % (m/m) % (m/m)
30 to 35 0,4 1,0
B.8 Test report
The report shall include the following data as a minimum requirement:
a) type and description of tested product;
b) reference to this part of ISO 22241;
c) sampling method used;
d) test result (see B.6);
e) deviations from the specified mode of operation, if any; and
f) test date.
8 © ISO 2006 – 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 of AUS 32. The test method is
applicable to liquids having refractive indices in the range of 1,33 to 1,39 and at temperatures of 20 °C to
30 °C.
Based on the measurement of refractive index, the method shall be used for determining the content of urea in
the range of 30 % to 35 % (m/m).
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.
The content is determined by means of a reference curve.
NOTE The method specified in this annex is based on ISO 5661.
C.3 Apparatus
C.3.1 Refractometer, measuring range 1,330 00 to 1,390 00, resolution 0,000 01.
C.3.2 Analytical balance, resolution 0,1 mg or better.
C.3.3 Thermostat, temperature-control precision 0,02 °C.
C.3.4 Drying oven.
C.3.5 150 ml beaker, tall form.
C.3.6 Typical laboratory glass.
C.4 Chemicals
C.4.1 De-ionized water, conductivity less than 0,5 mS/m according to ISO 3696 grade 3.
C.4.2 Urea, crystalline, with biuret content less than 0,1 % (m/m).
Prior to weighing the urea to draw the reference curve, it shall be dried for 2 h at 105 °C.
C.4.3 Urea test solution, 32,5 % (m/m).
The test solution shall be made by exactly weighing urea and water. The desired value and the permissible
dispersion shall be established through 10 measurements.
The solution shall be kept air-tight in the refrigerator and should be used within 4 weeks maximum.
C.5 Procedure
C.5.1 General
The sample should be fully dissolved and free from urea crystals. It may be heated to u 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 part of ISO 22241. Rather, operation should be based on the respective operation manuals.
C.5.2 Drawing the reference curve and determining the evaluation factor
The following urea solutions shall be prepared by weighing urea in glass beakers and then adding the
corresponding quantity of de-ionized water: 30,0 % (m/m) / 31,5 % (m/m) / 32,5 % (m/m) / 33,5 % (m/m) /
35,0 % (m/m).
The refractive index of these solutions shall be determined at 20 °C ± 0,02 °C.
The diagram shall show a stringent linear relationship between the refractive index and concentration.
An evaluation factor shall be calculated from the urea concentrations and the refractive indices:
w
∑ U,i
i=1
F=
nn−
()
∑ U,i W
i=1
where
F is the evaluation factor (%);
w is the urea content of the i-th reference solution [%(m/m)];
U,i
n is the refractive index of the i-th reference solution;
U,i
n is the refractive index of water and is 1,332 96 when measured with a refractometer of five-decimal
W
resolution.
C.5.3 Checking the instrument function and the reference curve
The instrument function shall be checked weekly using water or a reference standard. If a greater deviation
from desired value than 0,000 02 occurs, adjust the instrument according to the instructions provided by the
manufacturer. If afterwards the desired value is not attained, then the instrument shall be disabled for further
measurements and the manufacturer’s service should be called for.
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 shall be reached and
maintained within ± 0,02 °C.
Furthermore, the reference curve shall be verified weekly with urea solution [32,5 % (m/m)]. In the process, the
refractive index shall be determined and the concentration shall be calculated with the help of the factor
according to C.6. If the concentration determined deviates from the desired value by more than 0,1 % (m/m), a
new test solution shall used. If the deviation persists, the reference curve shall be created anew.
10 © ISO 2006 – All rights reserved

C.5.4 Sample preparation and measuring
The original sample shall be measured at 20 °C ± 0,02 °C without further preparation.
Measure the urea content two times with different test portions. Should the difference between the separate
values be more than 0,000 05, the measurements shall be repeated.
C.6 Results
C.6.1 Calculation
Urea content shall be calculated according to the following equation:
wn=−n ×F−w
( )
UP W Bi
where
w is the urea content [% (m/m)];
U
n is the refractive index of sample (with 5 decimals);
P
n is the refractive index of water (with 5 decimals);
W
F is the evaluation factor (%);
w is the biuret content of the solution [% (m/m)];
Bi
(determined according to Annex E; biuret has the same refractive index as urea).
C.6.2 Expression of results
The result is defined as the arithmetic mean of two single measurements. Round off the result of the refractive
index to four decimals. Round off the result of the urea content calculation to the nearest 0,1 % (m/m).
C.7 Precision
See 5.2, 5.3 and Table C.1.
Table C.1 — Precision
Characteristics Repeatability Reproducibility
r R
Refractive index n
P
0,000 1 0,001 0
1,33 to 1,39
Urea content w
U
0,1 % (m/m) 1,0 % (m/m)
30 % to 35 % (m/m)
C.8 Test report
The report shall include the following data as a minimum requirement:
a) type and description of tested product;
b) reference to this part of ISO 22241;
c) sampling method used;
d) test result (see C.6);
e) deviations from the specified mode of operation, if any; and
f) test date.
12 © ISO 2006 – All rights reserved

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 0,1 % to 0,5 % (m/m).
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 de-ionized water of
an electric conductivity lower than 0,5 mS/m, according to ISO 3696 grade 3.
D.4.2 Hydrochloric acid.
This is 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.
NOTE Such solutions are commercially available.
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 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 filled
S
with about 100 ml distilled or de-ionized 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 [% (m/m)]: 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 example.
D.5.4 Determination
Weigh the mass of the homogenous sample to 0,05 g found by the preliminary test (sample mass m ) and put
S
it into a 150 ml beaker filled with about 100 ml distilled or de-ionized water.
Titrate with the hydrochlorid acid solution (0,1 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
w(NH ) = (V × 0,017) / m
3 S
where
w(NH ) is the alkalinity, calculated as ammonia [% (m/m)];
V is the volume of the hydrochloric acid solution used for the titration (ml);
m is the mass of the test portion (g).
S
14 © ISO 2006 – All rights reserved

D.6.2 Expression of results
Calculate the mean value of the two measurements. Express the result to the nearest 0,01 % (m/m).
D.7 Precision
See 5.2, 5.3 and Table D.1.
Table D.1 — Precision
Alkalinity content Repeatability Reproducibility
w(NH ) r R
% (m/m) % (m/m) % (m/m)
0,1 to 0,5 0,01 0,2 × x
x is mean value.
D.8 Test report
The report shall include the following data as a minimum requirement:
a) type and description of tested product;
b) reference to this part of ISO 22241;
c) sampling test method;
d) test result (see D.6);
e) deviations from the specified mode of operation, if any; and
f) test date.
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 from 0,1 % to 0,5 % (m/m) by photometric method. The method is also applicable to contents up to
1,5 % (m/m); however, precision data have not been determined.
E.2 Principle
Biuret forms in alkaline solution in the presence of sodium-potassium-tartrat 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 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 de-ionized and boiled out to remove carbon dioxide before
use.
E.4.2 Saturated potassium carbonate-solution.
E.4.3 Copper sulphate-solution.
Dissolve 15 g copper sulphate (CuSO⋅5H O) in CO -free water and dilute to 1 000 ml.
4 2 2
16 © ISO 2006 – All rights reserved

E.4.4 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 flasks stand
4 4 6 2
1 day before use.
E.4.5 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 % (m/m) 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.5 Procedure
E.5.1 Interferences
Spectrophotometric measuring is only allowed with clear solutions. Pass the sample 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 remove ammonia-contents greater than 500 mg/kg, put 50,0 g of the sample in a 1-litre-flask of a rotary
evaporator, add 15 ml potassium-carbonate-solution and evaporate it for 1 h at 40 °C with a rotating speed of
60 r/min and under a vacuum of 2 kPa to 3 kPa to an end volume of approximately 20 ml. Transfer this
volume into a 250-ml-volumetric flask.
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 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.
E.5.3 Calculation of the calibration factor
Calculate the calibration factor according to the following equation:
m
∑ Bi,i
61,6
i=1
F = =
6 6
E − E E − E
() ()
1,i 2 1,i 2
∑ ∑
i=1 i=1
where
F is the calibration factor (mg);
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
E − E
()
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, to the nearest 0,01 g, 100 g of the test sample in a 250 mI volumetric flask. Fill the flask to the mark
with water and mix well.
18 © ISO 2006 – All rights reserved

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 content of biuret [% (m/m)];
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 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 % (m/m).
E.7 Precision
See 5.2, 5.3 and Table E.1.
Table E.1 — Precision
Biuret content Repeatability Reproducibility
w r R
Bi
% (m/m) % (m/m) % (m/m)
0,1 to 0,5 0,01 0,04
E.8 Test report
The report shall include the following data as a minimum requirement:
a) type and description of tested product;
b) reference to this part of ISO 24441;
c) sampling method used;
d) test result (see E.6);
e) deviations from the specified mode of operation, if any; and
f) test date.
20 © ISO 2006 – All rights reserved

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 method Nr. 20062 from the Official Methods of Analysis of the
Association of Official Analytical Chemists (AOAC) International.
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, shall be used in all tests.
F.4.2 Sulphuric acid, 96 % (m/m).
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), 3 % (m/m) in sulphuric acid of 15 % (m/m).
In order to make this solution, add 41 ml sulphuric acid to 410 ml of water while cooling the mixture and then
add 15 g of chromotropic acid and mix them well.
NOTE If stored in a brown glass bottle, this solution is usable for at least 3 months.
F.4.4 Formaldehyde standard solution prepared as follows:
⎯ put 6,5 g to 7 g of formaldehyde solution having a concentration of 37 % (m/m) into a 500 ml volumetric
flask, fill up the flask with water and mix it well;
⎯ determine the formaldehyde content of the solution, for example in accordance with ISO 9020 method;
⎯ dilute the solution to 1:1 000. Mark the exact value of the formaldehyde content to the flask (the
formaldehyde content as determined in the previous step divided by 1 000).
F.5 Procedure
F.5.1 Preparation of the calibration curve
Into a series of six 50 ml volumetric flasks, transfer 0,2 ml, 0,5 ml, 1 ml, 2 ml, 5 ml, and 10 ml of the
formaldehyde standard solution and add water to a total mixture volume of approx. 10 ml. Add, while stirring,
1 ml of chromotropic acid solution followed by gradual addition of 20 ml sulphuric acid during the course of
5 minutes. The temperature rise during the addition of sulphuric acid shall exceed 100 °C, which is necessary
for the reaction to come to completion. Let the flask stand at ambient air for 15 minutes without any further
cooling.
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 F.5.4).
After cooling to room temperature, fill the flask with water up to the mark and mix well. Carry out the
photometric measurements with the spectrophotometer at a wavelength of about 565 nm using a 10 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.
F.5.2 Calculation of the calibration factor
Calculate the calibration factor according to the following equation:
m
HCHO,i
∑
i=1
F =
E − E
()
∑ 1,i 2
i=1
where
F is the calibration factor (ug);
m is the mass of the formaldehyde of the i-th sample (ug);
HCHO,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.
F.5.3 Check of the method
Every 3 months, the method shall be checked as follows.
Into a series of three 50 ml volumetric flasks, transfer 2 ml of the formaldehyde standard solution and add
water to a total volume of approx. 10 ml. Follow the procedure described in F.5.4 and calculate the aldehyde
content as shown in F.6.
Compare the findings with the content of the standard solution. If the deviation is less than or equal to 2 %, the
method is ready to use. If the deviation is more than 2 %, repeat the check. If the deviation is more than 2 %
again, the method shall not be used unless a new calibration curve is prepared.
22 © ISO 2006 – All rights reserved

F.5.4 Measuring of samples
Weigh, to the nearest 0,01 g, 5 g to 10 g of the test sample in a 50 mI volumetric flask and dilute it with water
to a total mixture volume of approximately 10 ml. Add, with stirring, 1 ml of chromotropic acid solution followed
by gradual addition of 20 ml sulphuric acid during the course of 5 minutes. The temperature rise during the
addition of sulphuric acid shall exceed 100 °C, which is necessary for the reaction to come to completion. Let
the flask stand at ambient air for 15 minutes without any further cooling.
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 the flask with water up to the mark and mix well. Carry out the
photometric measurements with th
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