ASTM D7304-16

Standard Test Method for Determination of Denatonium Ion 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 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 and health practices and determine the applicability of regulatory limitations prior to use.

General Information

• Status: Historical
• Publication Date: 14-May-2016
• ICS: 71.100.45 - Refrigerants and antifreezes
• Technical Committee: D15 - Engine Coolants and Related Fluids
• Drafting Committee: D15.04 - Chemical Properties

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: D7304 − 16
Standard Test Method for
Determination of Denatonium Ion in Engine Coolant by
1
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 E691Practice for Conducting an Interlaboratory Study to
Determine the Precision of a Test Method
1.1 This test method covers the chemical analysis of engine
coolant for denatonium benzoate (DNB) by high-performance
3. Summary of Test Method
liquid chromatography (HPLC). DNB is added to potentially
renderenginecoolantunpalatabletoanimalsandhumans.This
3.1 The denatonium benzoate analysis is achieved by an
analyticalmethodwasdesignedfortheanalysisofDNBandis
HPLC method, where a weight of engine coolant is placed in
not valid for any other bittering agents such as denatonium
an auto-sampler vial and mixed with a known volume of
saccharide.
de-ionized water. The auto-sampler vial is placed in a HPLC
1.2 This test method is applicable to both new and used
autosampler and the measurement of denatonium benzoate is
coolants.
performed using a C-18 reverse phase column attached to an
ultraviolet detector.The ultraviolet detector is used to measure
1.3 Coelution of other ions may cause interferences in the
the response of the DNB active ingredients (denatonium and
detection of the denatonium cation. In the case of unfamiliar
benzoate) in the engine coolant after they have been separated
formulations, identification verification should be performed
in the reverse phase column. The denatonium and benzoate
byeitherorbothfortificationanddilutionofthesamplematrix
responses are compared to responses of known concentrations
with denatonium ion.
and the HPLC’s computer calculates the amount of DNB
1.4 The values stated in SI units are to be regarded as the
present in the coolant.
standard. The values given in parentheses are for information
only.
4. Significance and Use
1.5 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
4.1 This test method provides for the qualitative and quan-
responsibility of the user of this standard to establish appro-
titative determination of denatonium benzoate in engine cool-
priate safety and health practices and determine the applica-
ant in milligrams per litre to low percent range and requires
bility of regulatory limitations prior to use.
approximately 1 mLper test, with results available in less than
10 min. Denatonium benzoate is a compound composed of a
2. Referenced Documents
quaternary ammonium cation, denatonium and an inert anion,
2
2.1 ASTM Standards: benzoate. In solution the denatonium benzoate exists in equi-
D1193Specification for Reagent Water
librium between the denatonium benzoate compound, the
D1176Practice for Sampling and Preparing Aqueous Solu-
denatonium cation and benzoate anion. By slightly adjusting
tionsofEngineCoolantsorAntirustsforTestingPurposes
the pH of the solution to be more acidic (≈ pH 4.6) the
E177Practice for Use of the Terms Precision and Bias in
equilibrium will be shifted to the direction of forming more
ASTM Test Methods
denatonium and benzoate ions in the solution.
5. Interferences
1
This test method is under the jurisdiction ofASTM Committee D15 on Engine
Coolants and Related Fluids and is the direct responsibility of Subcommittee
5.1 Interferences can be caused by substances with similar
D15.04 on Chemical Properties.
retention times, especially if they are in high concentration
Current edition approved May 15, 2016. Published July 2016. Originally
compared to the analyte of interest, denatonium ion. Known
approved in 2006. Last previous edition approved in 2014 as D7304–14. DOI:
10.1520/D7304-16.
chromatographic interferences have been determined and the
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
analysis modified to minimize any co-elution of interfering
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
peaks. The eluent strength and flow rate can be used to reduce
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. or solve most interference problems.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
D7304 − 16
5.2 Method interferences can also be caused by the con- 7.9.2 Add 250 mL of de-ionized water and mix until the
tamination of glassware, eluant, reagents, etc. Great care must KH PO has dissolved.
2 4
be taken to ensure that contaminati
...

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 − 14 D7304 − 16
Standard Test Method for
Determination of Denatonium Ion in Engine Coolant by
1
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 and health practices and determine the applicability of regulatory
limitations prior to use.
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
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 shifted to the
direction of forming more denatonium and benzoate ions in the solution.
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 March 15, 2014May 15, 2016. Published May 2014July 2016. Originally approved in 2006. Last previous edition approved in 20062014 as
D7304–06.–14. DOI: 10.1520/D7304-14.10.1520/D7304-16.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. 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 − 16
5. Interferences
5.1 Interferences can be caused by substances with similar retention times, especially if they are in high concentration compared
to the analyte of interest, denatonium ion. Known chromatographic interferences have been determined and the analysis modified
to mi
...