ASTM D7304-22a

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.

General Information

• Status: Published
• Publication Date: 31-Aug-2022
• ICS: 71.100.45 - Refrigerants and antifreezes
• Technical Committee: D15 - Engine Coolants and Related Fluids
• Drafting Committee: D15.04 - Chemical Properties

Relations

• Refers: ASTM D1176-14(2019) - Standard Practice for Sampling and Preparing Aqueous Solutions of Engine Coolants or Antirusts for Testing Purposes
• Effective Date: 01-Oct-2019
• Refers: ASTM E177-14 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-May-2014
• Refers: ASTM D1176-14 - Standard Practice for Sampling and Preparing Aqueous Solutions of Engine Coolants or Antirusts for Testing Purposes
• Effective Date: 01-Apr-2014
• Refers: ASTM E691-13 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-May-2013
• Refers: ASTM E177-13 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-May-2013
• Refers: ASTM E691-11 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Nov-2011
• Refers: ASTM E177-10 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-Oct-2010
• Refers: ASTM E691-08 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Oct-2008
• Refers: ASTM E177-08 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-Oct-2008
• Refers: ASTM D1176-98(2008) - Standard Practice for Sampling and Preparing Aqueous Solutions of Engine Coolants or Antirusts for Testing Purposes
• Effective Date: 01-Apr-2008
• Refers: ASTM E177-06b - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 15-Nov-2006
• Refers: ASTM E177-06a - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-Nov-2006
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM E691-05 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Nov-2005
• Refers: ASTM E177-04 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-Nov-2004

Overview

ASTM D7304-22a is a standardized test method established by ASTM International to determine the concentration of denatonium benzoate (DNB) in engine coolant using high-performance liquid chromatography (HPLC). Denatonium benzoate is a widely used bitterant added to engine coolants to deter accidental ingestion by humans and animals. This analytical method is designed specifically for denatonium benzoate and is not suitable for other bitter agents.

This test method allows for both qualitative and quantitative determination of DNB, with results typically available in less than 10 minutes. A very small volume (approximately 1 mL) of engine coolant is required for each test, making it efficient and resource-conserving. The method can be applied to both new and used coolant samples.

Key Topics

• Purpose: Detecting and quantifying denatonium benzoate in engine coolant to ensure product safety and compliance.
• Analytical Technology: Uses HPLC with a C-18 reverse phase column and an ultraviolet (UV) detector for precise measurement.
• Sample Preparation: Coolant samples are diluted in a buffered dilute solution (pH ~4.6) to shift equilibrium for optimal detection of the denatonium cation and benzoate anion.
• Interference Management: Coelution (overlapping peaks) of other ions can cause interference. Sample fortification and dilution are recommended for unfamiliar coolant formulations.
• Reporting: Results are reported in milligrams per liter (mg/L), and laboratories must document sample data and analysis conditions.
• Precision and Bias: The method includes guidance on repeatability and reproducibility based on interlaboratory statistical studies.

Applications

• Quality Control in Manufacturing: Automotive coolant manufacturers use this method to verify the denatonium benzoate content for regulatory and safety compliance.
• Product Safety Assurance: Ensuring that engine coolants contain sufficient DNB to meet regulations, reducing risks of accidental ingestion.
• In-service Coolant Testing: Service centers and laboratories apply this standard to test both new and used coolant to confirm continued presence and concentration levels of bitterant.
• Regulatory Testing: Helps demonstrate compliance with local, national, or international requirements for hazardous substances in automotive products.
• Research and Development: Used by R&D teams to develop and validate new engine coolants or formulations that require precise DNB measurement.

Related Standards

• ASTM D1193 - Specification for Reagent Water: Ensures water purity for analytical procedures.
• ASTM D1176 - Practice for Sampling and Preparing Aqueous Solutions of Engine Coolants or Antirusts for Testing Purposes: Covers best practices for coolant sample collection and preparation.
• ASTM E177 - Practice for Use of the Terms Precision and Bias in ASTM Test Methods: Defines terminology and statistical practices.
• ASTM E691 - Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method: Provides statistical evaluation guidelines for repeatability and reproducibility.

Practical Value

Implementing ASTM D7304-22a ensures accurate, consistent, and defensible measurement of denatonium benzoate in engine coolants. The method’s speed, low sample volume, and high sensitivity make it highly practical for both industrial laboratories and independent testing facilities. Routine use of this standard supports safety in automotive products, helps prevent accidental poisoning, and underpins compliance with consumer safety and environmental regulations.

Keywords: denatonium benzoate, engine coolant, HPLC, ASTM D7304, bitterant, antifreeze analysis, analytical method, UV detection, product safety, automotive fluids testing, quality control, chemical analysis.

Standards Content (Sample)

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.
Designation: D7304 − 22a
Standard Test Method for
Determination of Denatonium Benzoate in Engine 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.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
2.1 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 components (denatonium and benzo-
standard. The values given in parentheses are for information
ate) in the engine coolant after they have been separated in the
only.
reversephasecolumn.Thedenatoniumandbenzoateresponses
1.5 This standard does not purport to address all of the
are compared to responses of known concentrations and the
safety concerns, if any, associated with its use. It is the
HPLC’scomputercalculatestheamountofDNBpresentinthe
responsibility of the user of this standard to establish appro-
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.
This test method is under the jurisdiction ofASTM Committee D15 on Engine
Coolants and Related Fluids and is the direct responsibility of Subcommittee
D15.04 on Chemical Properties. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved Sept. 1, 2022. Published September 2022. Originally contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
approved in 2006. Last previous edition approved in 2022 as D7304–22. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/D7304-22A. the ASTM website.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7304 − 22a
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 high concentration
through a 0.45 µm filter. For eluant preparation, degas the
compared to the analyte of interest, denatonium ion. Known
water by sparging with helium or vacuum degassing and
chromatographic interferences have been determined and the
sonication.
analysis modified to minimize any co-elution of interfering
peaks. The eluent strength and flow rate can be used to reduce 7.9 Potassium Phosphate Solution KH PO (0.5M):
2 4
or solve most interference problems.
7.9.1 Weigh 34.022 g of the KH PO into a 500 mL
2 4
volumetric flask.
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.
be taken to ensure that contamination is kept at the lowest 2 4
7.9.3 Dilute to the mark with de-ionized water and thor-
possible level.
oughly mix.
6. Apparatus
7.10 Sample Dilution Solution (pH 4.6): Potassium Phos-
phate Solution KH PO (0.1M):
6.1 HPLC System—High Performance Liquid Chromato- 2 4
7.10.1 Transfer 200 mL of Potassium Phosphate Solution
graph system equipped with appropriate computer and soft-
KH PO (0.5M) to 1000 mL volumetric flask.
ware.
2 4
7.10.2 Dilute to mark with de-ionized water.
6.1.1 Gradient Pump.
7.10.3 Measure the pH of the de-ionized water with a pH
6.1.2 UV/VIS-Mulitple Wavelength Detector.
meter.
6.1.3 Eluant Degas System.
6.1.4 Analytical Column, Kinetex C-18, 2.6 µm packing,
NOTE 1—The pH of the sample dilution water should be checked
75mm × 4.6 mm or equivalent column.
bi-weekly and if the pH has changed the dilution water should be
re-prepared.
6.1.5 Guard Column, Ultra Cartridge UPLC C-18 for
4.6mm or equivalent.
7.11 pH 4.3 Mobile Phase, 30 % Acetonitrile 70 % of
Potassium Phosphate Solution KH PO (0.1M):
6.2 Analytical Balance, 0.0001 g precision. 2 4
7.11.1 Add 300 mLof acetonitrile to a 1000 mLvolumetric
6.3 Volumetric Flasks, 50 mL, 100 mL, 200 mL, 500 mL,
flask.
1L.
7.11.2 Dilute to the mark with Potassium Phosphate Solu-
6.4 pH Meter, capable of pH/mV/temperature measure-
tion KH PO (0.1M).
2 4
ments.
7.11.3 Stopper and mix.
7.11.4 Degas the solution for 10 min in an ultra-sonic bath
6.5 Glassware, class "A" laboratory glassware and plastic
as a large amount of dissolved gas will be present in the
ware.
solution.
6.6 Eppendorf Series 2100 Pipetter, capable of delivering
7.11.5 Place solution in a 1000 mLbeaker and measure the
1000 µL.
solution’s pH.
6.7 Electronic Pipetter,capableofdeliveringbetween30 →
7.11.6 Adjust the pH of the mobile phase solution with
300 µL.
0.5MKH PO or85%phosphoricaciduntilitreachespH4.3.
2 4
7.11.7 Transfer the solution to the HPLC reagent bottle.
7. Reagents and Materials
NOTE2—ThefinalpHoftheMobilePhaseshouldbeapproximately4.3
7.1 Acetonitrile, (ACN), Reagent Grade.
pH units.
7.2 Ortho Phosphoric acid, H PO , 85 %, Reagent Grade.
3 4
8. Hazards
7.3 Potassium Phosphate, Monobasic, KH PO , 99.995 %
2 4
8.1 Personnel protective equipment (such as eye
purity.
protections, gloves, laboratory coat, etc.) should be used in the
7.4 Denatonium Benzoate, 98 % purity.
handling of all chemicals. Special care should be taken when
7.5 Ethylene Glycol, Reagent Grade. handling solutions around electrical equipment. All solution
should be prepared in a hood.
7.6 De-ionized water, Type II water.
8.2 Read all equipment manuals before attempting to oper-
7.7 Purity of Reagents—Reagent grade chemicals shall be
ateHPLCinstrumentation.Specialattentionshouldbegivento
used in all tests. Unless otherwise indicated, it is intended that
all warnings.
all reagents shall conform to the specifications of the Commit-
tee onAnalytical Reagents of theAmerican Chemical Society, 8.3 Be familiar with the safety data sheet (SDS) for all
where such specifications are available. Other grades may be chemicalsusedinthisprocedure.Adustmaskisrecommended
used, provided it is first ascertained that the reagent is of for the handling of Denatonium Benzoate. Review your com-
sufficiently high purity to permit its use without lessening the pany’s policy concerning the use of a dust mask. Prepare
accuracy of the determination. standards in a hood.
D7304 − 22a
9. Sampling, Test Specimens and Test Units 12. Gradient Program
9.1 Collect the sample in a scrupulously clean glass or 12.1 See Table 2.
polyethylenebottleinaccordancewithPracticeD1176.Collect
NOTE 3—If the coolant contains 4-Tert-Butylbenzoic acid (TBBA) the
at least 100 mL of sample.
analysis time will be 9 min.
10. Calibration Solutions
13. Sample Preparation
10.1 Stock Denatonium Benzoate Calibration Solution
13.1 Place a 2000 µL auto-sampler vial on an analytical
(500 mg ⁄L DNB):
balance and zero the balance.
10.1.1 Weigh 0.0500g 6 0.0001 g of denatonium benzoate
13.2 Pipette 50 µLof sample into the auto-sampler vial and
into a 100 mL volumetric flask and record the weight.
record the sample weight in milligrams (mg).
10.1.2 Add de-ionized water, stopper and mix until solid
dissolves.
13.3 Pipette 1950 µL of pH 4.6 Sample Dilution Solution
10.1.3 Dilute to mark with de-ionized water and m
...

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 − 22 D7304 − 22a
Standard Test Method for
Determination of Denatonium Benzoate in Engine 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 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.
2. Referenced Documents
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
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 1, 2022Sept. 1, 2022. Published April 2022September 2022. Originally approved in 2006. Last previous edition approved in 20162022
as D7304–16.–22. DOI: 10.1520/D7304-22.10.1520/D7304-22A.
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’sstandard’s Document Summary page on the ASTM website.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7304 − 22a
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 components (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.
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 minimize any co-elution of interfering peaks. The eluent strength and flow rate can be used to reduce or solve most interference
problems.
5.2 Method interferences can also be caused by the contamination of glassware, eluant, reagents, etc. Great care must be taken
to ensure that contamination is kept at the lowest possible level.
6. Apparatus
6.1 HPLC System—High Performance Liquid Chromatograph system equipped with appropriate computer and software.
6.1.1 Gradient Pump.
6.1.2 UV/VIS-Mulitple Wavelength Detector.
6.1.3 Eluant Degas System.
6.1.4 Analytical Column, Kinetex C-18, 2.6 μm packing, 75 mm × 4.6 mm or equivalent column.
6.1.5 Guard Column, Ultra Cartridge UPLC C-18 for 4.6 mm or equivalent.
6.2 Analytical Balance, 0.0001 g precision.
6.3 Volumetric Flasks, 50 mL, 100 mL, 200 mL, 500 mL, 1 L.
6.4 pH Meter, capable of pH/mV/temperature measurements.
6.5 Glassware, class "A" laboratory glassware and plastic ware.
6.6 Eppendorf Series 2100 Pipetter, capable of delivering 1000 μL.
6.7 Electronic Pipetter, capable of delivering between 30 → 300 μL.
7. Reagents and Materials
7.1 Acetonitrile, (ACN), Reagent Grade.
7.2 Ortho Phosphoric acid, H PO , 85 %, Reagent Grade.
3 4
D7304 − 22a
7.3 Potassium Phosphate, Monobasic, KH PO , 99.995 % purity.
2 4
7.4 Denatonium Benzoate, 98 % purity.
7.5 Ethylene Glycol, Reagent Grade.
7.6 De-ionized water, Type II water.
7.7 Purity of Reagents—Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where
such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high
purity to permit its use without lessening the accuracy of the determination.
7.8 Unless otherwise indicated, references to water shall be understood to mean reagent water conforming to Specification D1193,
Type II. It is recommended that all water be filtered through a 0.45 μm filter. For eluant preparation, degas the water by sparging
with helium or vacuum degassing and sonication.
7.9 Potassium Phosphate Solution KH PO (0.5M):
2 4
7.9.1 Weigh 34.022 g of the KH PO into a 500 mL volumetric flask.
2 4
7.9.2 Add 250 mL of de-ionized water and mix until the KH PO has dissolved.
2 4
7.9.3 Dilute to the mark with de-ionized water and thoroughly mix.
7.10 Sample Dilution Solution (pH 4.6): Potassium Phosphate Solution KH PO (0.1M):
2 4
7.10.1 Transfer 200 mL of Potassium Phosphate Solution KH PO (0.5M) to 1000 mL volumetric flask.
2 4
7.10.2 Dilute to mark with de-ionized water.
7.10.3 Measure the pH of the de-ionized water with a pH meter.
NOTE 1—The pH of the sample dilution water should be checked bi-weekly and if the pH has changed the dilution water should be re-prepared.
7.11 pH 4.3 Mobile Phase, 30 % Acetonitrile 70 % of Potassium Phosphate Solution KH PO (0.1M):
2 4
7.11.1 Add 300 mL of acetonitrile to a 1000 mL volumetric flask.
7.11.2 Dilute to the mark with Potassium Phosphate Solution KH PO (0.1M).
2 4
7.11.3 Stopper and mix.
7.11.4 Degas the solution for 10 min in an ultra-sonic bath as a large amount of dissolved gas will be present in the solution.
7.11.5 Place solution in a 1000 mL beaker and measure the solution’s pH.
7.11.6 Adjust the pH of the mobile phase solution with 0.5M KH PO or 85 % phosphoric acid until it reaches pH 4.3.
2 4
7.11.7 Transfer the solution to the HPLC reagent bottle.
NOTE 2—The final pH of the Mobile Phase should be approximately 4.3 pH units.
D7304 − 22a
8. Hazards
8.1 Personnel protective equipment (such as eye protections, gloves, laboratory coat, etc.) should be used in the handling of all
chemicals. Special care should be taken when handling solutions around electrical equipment. All solution should be prepared in
a hood.
8.2 Read all equipment manuals before attempting to operate HPLC instrumentation. Special attention should be given to all
warnings.
8.3 Be familiar with the safety data sheet (SDS) for all chemicals used in this procedure. A dust mask is recommended for the
handling of Denatonium Benzoate. Review your company’s policy concerning the use of a dust mask. Prepare standards in a hood.
9. Sampling, Test Specimens and Test Units
9.1 Collect the sample in a scrupulously clean glass or polyethylene bottle in accordance with Practice D1176. Collect at least 100
mL of sample.
10. Calibration Solutions
10.1 Stock Denatonium Benzoate Calibration Solution (500 mg ⁄L DNB):
10.1.1 Weigh 0.05000.0500 g 6 0.0001 g of denatonium benzoate into a 100 mL volumetric flask and record the weight.
10.1.2 Add de-ionized water, stopper and mix until solid dissolves.
10.1.3 Dilute to mark with de-ionized water and mix.
10.2 Working Standard (20 mg/L DNB):
10.2.1 Pipette 4 mL of the Stock Calibration Solution in a 100 mL volumetric flask.
10.2.2 Dilute to mark with de-ionized water.
10.2.3 Stopper and mix.
10.3 Calibration Standards:
10.3.1 Prepare a series of calibration standards by placing the appropriate amount of Stock Solution (see Table 1) into a 2000 μL
auto-sampler vial.
10.3.2 Dilute to
...