ASTM D7304-22
(Test Method)Standard Test Method for Determination of Denatonium Benzoate in Engine Coolant by HPLC
Standard Test Method for Determination of Denatonium Benzoate in Engine Coolant by HPLC
SIGNIFICANCE AND USE
4.1 This test method provides for the qualitative and quantitative determination of denatonium benzoate in engine coolant in milligrams per litre to low percent range and requires approximately 1 mL per test, with results available in less than 10 min. Denatonium benzoate is a compound composed of a quaternary ammonium cation, denatonium and an inert anion, benzoate. In solution the denatonium benzoate exists in equilibrium between the denatonium benzoate compound, the denatonium cation and benzoate anion. By slightly adjusting the pH of the solution to be more acidic (≈ pH 4.6) the equilibrium will be shifted to the direction of forming more denatonium and benzoate ions in the solution.
SCOPE
1.1 This test method covers the chemical analysis of engine coolant for denatonium benzoate (DNB) by high-performance liquid chromatography (HPLC). DNB is added to potentially render engine coolant unpalatable to animals and humans. This analytical method was designed for the analysis of DNB and is not valid for any other bittering agents such as denatonium saccharide.
1.2 This test method is applicable to both new and used coolants.
1.3 Coelution of other ions may cause interferences in the detection of the denatonium cation. In the case of unfamiliar formulations, identification verification should be performed by either or both fortification and dilution of the sample matrix with denatonium ion.
1.4 The values stated in SI units are to be regarded as standard. The values given in parentheses are for information only.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
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Designation: D7304 − 22
Standard Test Method for
Determination of Denatonium Benzoate in Engine Coolant
1
by HPLC
This standard is issued under the fixed designation D7304; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
2
2.1 ASTM Standards:
1.1 This test method covers the chemical analysis of engine
D1193Specification for Reagent Water
coolant for denatonium benzoate (DNB) by high-performance
D1176Practice for Sampling and Preparing Aqueous Solu-
liquid chromatography (HPLC). DNB is added to potentially
tionsofEngineCoolantsorAntirustsforTestingPurposes
renderenginecoolantunpalatabletoanimalsandhumans.This
E177Practice for Use of the Terms Precision and Bias in
analyticalmethodwasdesignedfortheanalysisofDNBandis
ASTM Test Methods
not valid for any other bittering agents such as denatonium
E691Practice for Conducting an Interlaboratory Study to
saccharide.
Determine the Precision of a Test Method
1.2 This test method is applicable to both new and used
3. Summary of Test Method
coolants.
3.1 The denatonium benzoate analysis is achieved by an
1.3 Coelution of other ions may cause interferences in the
HPLC method, where a weight of engine coolant is placed in
detection of the denatonium cation. In the case of unfamiliar
an auto-sampler vial and mixed with a known volume of
formulations, identification verification should be performed
de-ionized water. The auto-sampler vial is placed in a HPLC
byeitherorbothfortificationanddilutionofthesamplematrix
autosampler and the measurement of denatonium benzoate is
with denatonium ion.
performed using a C-18 reverse phase column attached to an
ultraviolet detector.The ultraviolet detector is used to measure
1.4 The values stated in SI units are to be regarded as
the response of the DNB active ingredients (denatonium and
standard. The values given in parentheses are for information
benzoate) in the engine coolant after they have been separated
only.
in the reverse phase column. The denatonium and benzoate
1.5 This standard does not purport to address all of the
responses are compared to responses of known concentrations
safety concerns, if any, associated with its use. It is the
and the HPLC’s computer calculates the amount of DNB
responsibility of the user of this standard to establish appro-
present in the coolant.
priate safety, health, and environmental practices and deter-
4. Significance and Use
mine the applicability of regulatory limitations prior to use.
4.1 This test method provides for the qualitative and quan-
1.6 This international standard was developed in accor-
dance with internationally recognized principles on standard- titative determination of denatonium benzoate in engine cool-
ant in milligrams per litre to low percent range and requires
ization established in the Decision on Principles for the
approximately 1 mLper test, with results available in less than
Development of International Standards, Guides and Recom-
10 min. Denatonium benzoate is a compound composed of a
mendations issued by the World Trade Organization Technical
quaternary ammonium cation, denatonium and an inert anion,
Barriers to Trade (TBT) Committee.
benzoate. In solution the denatonium benzoate exists in equi-
librium between the denatonium benzoate compound, the
denatonium cation and benzoate anion. By slightly adjusting
the pH of the solution to be more acidic (≈ pH 4.6) the
equilibrium will be shifted to the direction of forming more
denatonium and benzoate ions in the solution.
1
This test method is under the jurisdiction ofASTM Committee D15 on Engine
Coolants and Related Fluids and is the direct responsibility of Subcommittee
2
D15.04 on Chemical Properties. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved March 1, 2022. Published April 2022. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved in 2006. Last previous edition approved in 2016 as D7304–16. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/D7304-22. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
D7304 − 22
5. Interferences 7.8 Unless otherwise indicated, references to water shall be
understood to mean reagent water conforming to Specification
5.1 Interferences can be caused by substances with similar
D1193, Type II. It is recommended that all water be filtered
retention times, especially if they are in h
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D7304 − 16 D7304 − 22
Standard Test Method for
Determination of Denatonium IonBenzoate in Engine
1
Coolant by HPLC
This standard is issued under the fixed designation D7304; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This test method covers the chemical analysis of engine coolant for denatonium benzoate (DNB) by high-performance liquid
chromatography (HPLC). DNB is added to potentially render engine coolant unpalatable to animals and humans. This analytical
method was designed for the analysis of DNB and is not valid for any other bittering agents such as denatonium saccharide.
1.2 This test method is applicable to both new and used coolants.
1.3 Coelution of other ions may cause interferences in the detection of the denatonium cation. In the case of unfamiliar
formulations, identification verification should be performed by either or both fortification and dilution of the sample matrix with
denatonium ion.
1.4 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.6 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2
2.1 ASTM Standards:
D1193 Specification for Reagent Water
D1176 Practice for Sampling and Preparing Aqueous Solutions of Engine Coolants or Antirusts for Testing Purposes
E177 Practice for Use of the Terms Precision and Bias in ASTM Test Methods
E691 Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
3. Summary of Test Method
3.1 The denatonium benzoate analysis is achieved by an HPLC method, where a weight of engine coolant is placed in an
1
This test method is under the jurisdiction of ASTM Committee D15 on Engine Coolants and Related Fluids and is the direct responsibility of Subcommittee D15.04 on
Chemical Properties.
Current edition approved May 15, 2016March 1, 2022. Published July 2016April 2022. Originally approved in 2006. Last previous edition approved in 20142016 as
D7304–14.–16. DOI: 10.1520/D7304-16.10.1520/D7304-22.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
D7304 − 22
auto-sampler vial and mixed with a known volume of de-ionized water. The auto-sampler vial is placed in a HPLC autosampler
and the measurement of denatonium benzoate is performed using a C-18 reverse phase column attached to an ultraviolet detector.
The ultraviolet detector is used to measure the response of the DNB active ingredients (denatonium and benzoate) in the engine
coolant after they have been separated in the reverse phase column. The denatonium and benzoate responses are compared to
responses of known concentrations and the HPLC’s computer calculates the amount of DNB present in the coolant.
4. Significance and Use
4.1 This test method provides for the qualitative and quantitative determination of denatonium benzoate in engine coolant in
milligrams per litre to low percent range and requires approximately 1 mL per test, with results available in less than 10 min.
Denatonium benzoate is a compound composed of a quaternary ammonium cation, denatonium and an inert anion, benzoate. In
solution the denatonium benzoate exists in equilibrium between the denatonium benzoate compound, the denatonium cation and
benzoate anion. By slightly adjusting the pH of the solution to be more acidic (≈ pH 4.6) the equilibrium will be shif
...
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