ISO/PAS 22241-2:2005

PUBLICLY ISO/PAS
AVAILABLE 22241-2
SPECIFICATION
First edition
2005-11-01
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 2005
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.

©  ISO 2005
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2005 – All rights reserved

Contents Page
Foreword. iv
1 Normative references .1
2 Specifications.1
3 Sampling.1
4 Precision and dispute .2
4.1 General.2
4.2 Repeatability, r .2
4.3 Reproducibility, R .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 .15
Annex F (normative) Determination of aldehyde .20
Annex G (normative) Determination of insoluble matter 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, Zn)
by ICP-OES method.32
Annex J (normative) Determination of identity by FTIR spectrometry method.37
Annex K (informative) Precision of test methods .39

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.
In other circumstances, particularly when there is an urgent market requirement for such documents, a
technical committee may decide to publish other types of normative document:
⎯ an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical
experts in an ISO working group and is accepted for publication if it is approved by more than 50 %
of the members of the parent committee casting a vote;
⎯ an ISO Technical Specification (ISO/TS) represents an agreement between the members of a
technical committee and is accepted for publication if it is approved by 2/3 of the members of the
committee casting a vote.
An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a
further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is
confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an
International Standard or be withdrawn.
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/PAS 22241-2 was prepared by Technical Committee ISO/TC 22, Road vehicles, Subcommittee SC 5,
Engine tests.
ISO/PAS 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
iv © ISO 2005 – All rights reserved

PUBLICLY AVAILABLE SPECIFICATION ISO/PAS 22241-2:2005(E)

Diesel engines — NOx reduction agent AUS 32 —
Part 2:
Test methods
This Publicly Available Specification 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”.
1 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 or relative
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
2 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 Publicly Available Specification.
Determination of the density shall be conducted in accordance with ISO 3675 or ISO 12185.
NOTE For the purposes of this Publicly Available Specification, the terms “% (m/m)” and “% (V/V)” are used to
represent the mass fraction and the volume fraction of a material respectively.
3 Sampling
Samples shall be taken in accordance with Annex A.
4 Precision and dispute
4.1 General
All test methods referred to in this Publicly Available Specification 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 methods, as determined by statistical examination in accordance with ISO 4259, is
summarized in Annex K for all test methods. Additionally, each test method specified in this part of
ISO PAS 22241 contains this information, too.
The statistical significance of the precision quoted in this Publicly Available Specification is generically defined
in 4.2 and 4.3, in which the “xx (unit)” stands for the repeatability and reproducibility in question.
4.2 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.
4.3 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 2005 – 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 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 area, 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 HD-Polyethylene,
HD-Polypropylene, Polyfluorethylene, Polyvinylidenedifluoride and Poly(perfluoroalkoxy) 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 is opened, the cap is put down on a clean surface with the opening turned
downward. After flushing the sampling pipe, the bottle is filled completely with AUS 32 from the container. The
first filling is discarded, and the bottle is immediately re-filled with AUS 32 and locked tightly. The label is
attached to the bottle (see A.4.2).
During the filling of the sample, maximum care shall be taken that neither dust nor liquid pollution gets 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.
NOTE 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, a
sufficient volume of sample material should be made available (recommendation: 1 litre), and at least double
that 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 that owns the sample product;
1)
⎯ address where the sample has been taken;
1)
⎯ manufacturer of the sample product;
⎯ batch or lot number;
1)
⎯ container from which the sample was taken;
1)
⎯ part of the container from where the sample has been taken;
⎯ 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 2005 – 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 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 analyzer, based on combustion methods.
B.3.2 Analytical balance. The accuracy of the balance is a function of the analyzer used and the required
weighed portions. Resolution should be 0,1 % or better of the weighed portion.
B.3.3 Auxiliary contrivances for sample preparation, for example:
⎯ tweezers with a blunt tip;
⎯ micro-spatula with a flattened tip;
⎯ pipette.
NOTE The pipette is required 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 < 0,1 mS/m, according to ISO 3696 grade 2.
B.4.2 Auxiliary combustion agent, appropriate for use with the selected nitrogen analyzer.
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 or 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 standards include: ethylenediamine tetraacetic acid (EDTA), nicotinic acid amide.
Low-biuret urea of adequate purity (for example, crystalline ultra pure or analytical) or other such standards
recommended by and available from the equipment manufacturer may also be used. Certified standards
should be preferred.
NOTE Liquid standards (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 analyzer, 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 Publicly Available Specification. Rather, operation should be based on the respective
operation manual.
B.5.2 Reference curve
Perform calibration as required for the specific type of analyzer and according to the respective operation
manuals (for example, after replacement of the combustion tube, reagent or similar). Weigh in an appropriate
amount of standard substance repeatedly as appropriate for the respective type of apparatus to obtain a
reference curve.
Perform measurement as described in B.5.4.
B.5.3 Inspecting the apparatus for good working order, and the reference curve
Use an appropriate standard to review the good working order of the apparatus, and the reference curve.
Preferably, a certified urea standard should be used.
Frequency of inspection is a function of the analyzer used.
Perform measurements as described in B.5.4.
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.
Use approximately the three-fold amount of combustion agent (for example, non-nitrogenous cellulose), and
additional binders (for example, magnesium oxide) as required.
NOTE When using liquid feeder systems, the volume used should be no less than 100 µl. The sample mass shall be
calculated using the density that was calculated according to ISO 12185.
6 © ISO 2005 – All rights reserved

Enter the required data (weighed portion, sample identification) into the analyzer (or a control computer)
depending on the type of apparatus. Feed the weighed-in sample to the analyzer, 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 sequence.
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 of the nitrogen determination:
ww=×2,143 8 (−F×w )
UBNi
where
w is the urea content [% (m/m)];
U
w is the nitrogen content [% (m/m)] (to the nearest 0,01 %);
N
w is 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 three (3) single determinations (nitrogen determination).
Round off the result of the urea content calculation to the nearest 0,1 %.
B.7 Precision
B.7.1 Repeatability, r
(see 4.2)
r = 0,4 % (m/m).
B.7.2 Reproducibility, R
(see 4.3)
R = 1,0 % (m/m).
B.8 Test report
The report shall include the following data as a minimum requirement:
⎯ type and description of tested product;
⎯ reference to this Publicly Available Specification;
⎯ sampling method used;
⎯ test result (see B.6);
⎯ deviations from the specified mode of operation, if any;
⎯ test date.
8 © ISO 2005 – 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 1,33 to 1,39 and at temperatures of 20 °C to 30 °C.
Based on the measurement of refractive index the method is used for determining the content of urea in the range
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 < 0,1 mS/m according to ISO 3696 grade 2.
C.4.2 Urea, crystalline, with biuret content < 0,1 % (m/m).
Prior to weighing the urea to draw the reference curve, it shall be dried for 2 hours 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 ten-fold measurement.
The solution shall be kept air-tight in the refrigerator and should be used within four (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 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 standard. Rather, operation should be based on the respective operation manual.
C.5.2 Drawing the reference curve and determining the evaluating factor
The following urea solutions are 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 is determined at 20 °C ± 0,02 °C.
The graphical diagram shows a stringent linear relationship between the refractive index and concentration.
An evaluation factor is calculated from the urea concentrations and the refractive indices:
()w
U
∑
F=
()nn−
UW
∑
where
F is the evaluation factor (%);
w is the urea content of the reference solution [% (m/m)];
U
n is the refractive index of reference solution;
U
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 is checked weekly using water or a reference standard. If a deviation from desired
value greater 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 is disabled for further
measurements and the manufacture’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 is reached and maintained
within ± 0,02 °C.
Furthermore, the reference curve is verified weekly with urea solution [32,5 % (m/m)]. In the process, the
refractive index is determined and the concentration is 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
is used. If the deviation persists, the reference curve is created anew.
C.5.4 Sample preparation and measuring
The original sample is measured at 20 °C ± 0,02 °C without further preparation.
Measure the urea content twice with different test portions. Should the difference between the separate values
be more than 0,000 05, the measurements shall be repeated.
10 © ISO 2005 – All rights reserved

C.6 Results
C.6.1 Calculation
Urea content is 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 factor (%);
w is the biuret content of the solution [% (m/m)] (determined according to Annex E; biuret has the same
Bi
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
C.7.1 Repeatability, r
(see 4.2)
Refractive index: r = 0,000 1;
Urea content: r = 0,1 % (m/m).
C.7.2 Reproducibility, R
(see 4.3)
Refractive index: R = 0,001 0;
Urea content: R = 1,0 % (m/m).
C.8 Test report
The report shall include the following data as a minimum requirement:
⎯ type and description of tested product;
⎯ reference to this Publicly Available Specification;
⎯ sampling method used;
⎯ test result (see C.6);
⎯ deviations from the specified mode of operation, if any;
⎯ 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.
D.2 Principle
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, long 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 should be 0,01 molar standard volumetric 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.
12 © ISO 2005 – 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, respectively.
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) under stirring to the endpoint at pH = 5,7.
Calculate the content of ammonia.
Dependent on the content of ammonia found, weigh the following sample portions for the determination:
⎯ content found by the preliminary test [% (m/m)]: 0,02 0,05 0,1 0,2
⎯ mass of test portion for the determination (g): 10 5 2 1
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 hydrochloric acid solution (0,01 mol) under stirring to the endpoint at pH = 5,7.
Titrate slowly from pH = 7,5 to pH = 5,7. Determine twice.
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
D.6.2 Expression of results
Calculate the mean value of the two results. Express the result to the nearest 0,01 % (m/m).
D.7 Precision
D.7.1 Repeatability, r
(see 4.2)
r = 0,01 % (m/m) in the range of 0,1 % to 0,5 % (m/m).
D.7.2 Reproducibility, R
(see 4.3)
R = 0,2 × x % (m/m) in the range of 0,1 % to 0,5 % (m/m) (x is the mean value).
D.8 Test report
The report shall include the following data as a minimum requirement:
⎯ the reference of the test method;
⎯ the test results according to D.6 and the method of expression used;
⎯ any unusual features noted during the determination;
⎯ any operation not included in this Publicly Available Specification, or regarded as optional.
14 © ISO 2005 – All rights reserved

Annex E
(normative)
Determination of biuret
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 1,5 % (m/m).
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 of 550 nm. The colour complex is read
spectrophotometrically at 550 nm and the biuret concentration is determined by reference to an analytical
curve prepared from standards.
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 Reagent grade chemicals 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 *5 H O) in CO -free water and
4 2 2
dilute to 1 000 ml.
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 down add 50 g
potassium sodium tartrate (KNaC H O *4 H O) and fill up to the mark. Let stand 1 day before use.
4 4 6 2
E.4.5 Biuret-standard-solution 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 gently heating to one quarter of the volume;
⎯ cool to 5 °C and filter;
⎯ dry the biuret in vacuum oven at 80 °C; and
⎯ check the purity by photometrical measure according to E.5.5.
The step of re-crystallizing from ethanol shall be repeated until there is no improvement of purity noticeable
anymore.
E.5 Procedure
E.5.1 Interferences
Spectrophotometric measuring is only allowed with clear solutions. To remove turbidities, pass the sample
through a 0,45 µm filter.
Ammonia forms with bivalent copper a coloured complex, which absorbs at 550 nm. The method is applicable
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 1 h at 40 °C with 60 rpm and a vacuum of
2 kPa to 3 kPa to an end volume of approx. 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 of approx. 25 ml to each of the six flasks. Add, 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 min.
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 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.
16 © ISO 2005 – All rights reserved

E.5.3 Calculation of the calibration factor
Calculate the calibration factor according to the following equation:
()m
∑ Bi 61,6
F==
()E ()E
∑∑
where
F is the calibration factor;
m is the mass of biuret;
Bi
E are the extinctions.
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.
Measure 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;
D
E is the extinction standard solution (average from 2 measures);
E is the extinction blank test.
The day-factor shall vary max ± 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.
Transfer an aliquot of 10 ml from the test solution into a 50 ml volumetric flask and add water to approx. 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 flasks in the constant-temperature bath, regulated at 30 °C ± 1 °C and
leave them there for about 15 min.
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 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 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 sample;
S
E is the extinction 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.
D
E.6.2 Expression of results
Express the result to the nearest 0,01 % (m/m).
E.7 Precision
E.7.1 Repeatability, r
See 4.2 and Table E.1.
E.7.2 Reproducibility, R
See 4.3 and Table E.1.
Table E.1 — Precision
Biuret content
Repeatability Reproducibility
w r R
Bi
% (m/m) % (m/m)
% (m/m)
0,1 – 0,5 0,01 0,04
18 © ISO 2005 – All rights reserved

E.8 Test report
The report shall include the following data as a minimum requirement:
⎯ type and description of tested product;
⎯ reference to this Publicly Available Specification;
⎯ sampling method used;
⎯ test result (see E.6);
⎯ deviations from the specified mode of operation, if any;
⎯ test date.
Annex F
(normative)
Determination of aldehyde
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 1 mg/kg to 1 000 mg/kg.
F.2 Principle
Formaldehyde forms in strong sulphuric acid solution with chromotropic acid a purple colour with absorption
maximum of 565 nm. The colour complex is read spectrophotometrically at 565 nm and the aldehyde
concentration is determined by reference to an analytical curve prepared from standards.
NOTE The method specified in this annex is based on method Nr. 20062 from the Official Methods of Analysis of the
Association Official Analytic Chemists (AOAC).
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 Reagent grade chemicals shall be used in all tests.
F.4.2 Sulphuric acid 96 % (m/m).
F.4.3 Chromotropic acid (4,5-dihydroxynaphthalene-2,7-disulfonic acid-sodium-salt) 3 % (m/m) in
sulphuric acid 15 % (m/m).
To 410 ml of water add 41 ml sulphuric acid (cooling) and 15 g chromotropic acid and dissolve.
NOTE If stored in a brown glass bottle, this solution is useable for at least 3 months.
F.4.4 Formaldehyde standard solution:
⎯ 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 well;
⎯ determine the formaldehyde content of the solution, for example in accordance with the ISO 9020
method;
⎯ dilute the solution to 1:1 000. Mark the exact value of formaldehyde to the flask (the formaldehyde content
as determined in the previous step divided by 1 000).
20 © ISO 2005 – All rights reserved

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 approx. 10 ml. Add, with stirring, 1 ml of chromotropic acid
solution and 20 ml of the sulphuric acid. The arising temperature during the addition of sulphuric acid is
necessary for the reaction.
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 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
F=
()E
∑
where
F is the calibration factor;
m is the mass of formaldehyde (µg);
HCHO
E is the extinction.
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.
Into a series of three 50 ml volumetric flasks, transfer 2 ml of the formaldehyde standard solution and add
water to 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 then or equal to 2 %, the
method is ready to use. If the deviation is above 2 %, repeat the check. If the deviation is above 2 % again,
the method shall be disabled unless a new calibration curve is prepared.
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 with water to
approximately 10 ml. Add, with stirring, 1 ml of chromotropic acid solution and 20 ml of the sulphuric acid.
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 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.
F.6 Results
F.6.1 Calculation
The aldehyde content is given by the formula:
E−×EF
()
SB
w =
A
m
S
where
w is the content of aldehyde (mg/kg);
A
E is the extinction sample;
S
E is the extinction blank test (reagent blank + sample blank);
B
m is the mass of sample used (g);
S
F is the calibration factor.
F.6.2 Expression of results
Express the result to the nearest 0,1 mg/kg.
F.7 Precision
F.7.1 Repeatability, r
See 4.2 and Table F.1.
F.7.2 Reproducibility, R
See 4.3 and Table F.1.
Table F.1 — Precision
Content of aldehyde Repeatability Reproducibility
w
r R
A
mg/kg mg/kg mg/kg
a
0,5 – 10 0,14
0,5 × x
a
x is mean value.
22 © ISO 2005 – All rights reserved

F.8 Test repo
...