ASTM D7304-06
(Test Method)Standard Test Method for Determination of Denatonium Ion in Engine Coolant by HPLC
Standard Test Method for Determination of Denatonium Ion in Engine Coolant by HPLC
SIGNIFICANCE AND USE
This test method provides for the qualitative and quantitative determination of denatonium ion in engine coolant in milligrams per liter to low percent range and requires approximately 100 mL per test, with results available in less than 30 min. Acceptable levels of denatonium vary with manufacturer’s blending specifications and applicable minimum or maximum industry and state specifications. Typically specification ranges from 30 to 50 ppm denatonium ion.
SCOPE
1.1 This test method covers the chemical analysis of engine coolant for denatonium ion by high-performance liquid chromatography (HPLC).
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.
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
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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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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 − 06
StandardTest Method for
Determination of Denatonium Ion 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 an injection device prior to the column. The analytes are
separated as they pass through the column. An optical sensor
1.1 This test method covers the chemical analysis of engine
detects the changes in characteristics of the eluant stream and
coolant for denatonium ion by high-performance liquid chro-
converts the signal into an absorbance spectrum. The data
matography (HPLC).
system compares this response with an external calibration
1.2 This test method is applicable to both new and used
curveandtheresultsoftheconcentrationofanalytereportedas
coolants.
ppm or milligrams per litre (mg/L). Refer to Appendix X1 for
a HPLC flow diagram.
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 4. Significance and Use
by either or both fortification and dilution of the sample matrix
4.1 This test method provides for the qualitative and quan-
with denatonium ion.
titative determination of denatonium ion in engine coolant in
1.4 The values stated in SI units are to be regarded as the
milligrams per liter to low percent range and requires approxi-
standard. The values given in parentheses are for information
mately 100 mL per test, with results available in less than 30
only.
min. Acceptable levels of denatonium vary with manufactur-
er’s blending specifications and applicable minimum or maxi-
1.5 This standard does not purport to address all of the
mum industry and state specifications. Typically specification
safety concerns, if any, associated with its use. It is the
ranges from 30 to 50 ppm denatonium ion.
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica-
5. Interferences
bility of regulatory limitations prior to use.
5.1 Interferences can be caused by substances with similar
2. Referenced Documents
retention times, especially if they are in high concentration
2.1 ASTM Standards:
compared to the analyte of interest, denatonium ion. Sample
D1193 Specification for Reagent Water
dilution and optimized gradient elution can be used to mini-
D1176 Practice for Sampling and Preparing Aqueous Solu-
mize or solve most interference problems.
tions of Engine Coolants orAntirusts forTesting Purposes
5.2 A water dip (solvent system peak) can cause interfer-
ence with some integrators. This is eliminated by dilution with
3. Summary of Test Method
the eluant if the sample dilution factor is 4+1 (v/v) or greater.
3.1 A sample volume of working sample is prepared by
dilution of the sample with water.Ahigh-pressure pump forces 5.3 Method interferences can be caused by the contamina-
the mobile phase, eluant, through the HPLC columns (a guard
tion of glassware, eluant, reagents, etc. Great care must be
and analytical column) at typical flow rates of 0.1- 2 mL/min. takentoensurethatcontaminationiskeptatthelowestpossible
Asample to be separated is introduced in the mobile phase by
level
6. Apparatus
This test method is under the jurisdiction ofASTM Committee D15 on Engine
Coolants and Related Fluids and is the direct responsibility of Subcommittee
6.1 HPLC System—Analytical system equipped with all
D15.04 on Chemical Properties.
required accessories including syringes, gasses, columns,
Current edition approved Nov. 1, 2006. Published January 2007. DOI: 10.1520/
pumps, detectors and software.
D7304-06.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
6.1.1 Gradient Pump.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
6.1.2 UV/VIS-Mulitple Wavelength Detector.
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. 6.1.3 Eluant Degas System.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7304 − 06
TABLE 1 Minimum Detection Limits
6.1.4 Analytical Column ZORBAX RX-C8 or equivalent
A
column, capable of producing analyte separation equivalent to Analyte Detection Limits, ppm
or better than that shown in Fig. 1. Denatonium ion 1.0
A
6.1.5 Guard Column for ZORBAX RX-C8 or equivalent,
Determination using 100-µL sample volume. Sample diluted 1+3 (wt/v) with type
II water, full scale, UV/Vis detector set at 210 nm wavelength, Zorbax RX-C8
for protection of the analytical column from strongly retained
analytical column with Zorbax RX-C8 guard columns. Other systems will require
constituents.
MDL determination using chosen dilution factors, eluants, columns and detectors.
6.1.6 Integrator or Chromatography Data System Software
capable of obtaining approximately the same detection limits
as listed in Table 1.
7.9 Phosphate Buffer Stock Solution—Weigh 650 mg of
6.2 Analytical Balance, 0.0001 g precision.
Monobasic Potassium Phosphate and transfer it intoa1L
6.3 Volumetric Flasks, 50 mL, 100 mL, 200 mL.
volumetric flask, add 0.5 mL Phosphoric acid and fill to the
mark with Type II water. Mix this solution thoroughly. Filter
7. Reagents and Materials
the solution with 0.45 µm filter before use.
7.1 Acetonitrile, (ACN), Reagent Grade.
7.10 Denatonium Ion Standard Stock Solution—Weigh
7.2 Phosphoric acid, H PO , 85 %, Reagent Grade.
3 4 0.1372 g of Denatonium Benzoate into 100-mL volumetric
flask and dilute with ethylene glycol to 100 g. This stock
7.3 Potassium Phosphate, Monobasic, KH PO , 99.995 %
2 4
solution is 1000 ppm of denatonium and is stable for one
purity.
month. Make-up working standard solutions in 25% v/v
7.4 Denatonium Benzoate, Reagent Grade.
ethylene glycol/Type II water to desired concentrations. Ex-
7.5 Ethylene Glycol, Reagent Grade.
ample: 5 ppm denatonium standard solution - weigh 0.5000 g
of stock denatonium standard solution into a 100 mLvolumet-
7.6 Deionized water, Typee II water.
ric flask and fill to 100 mLmark with 25 % v/v ethylene glycol
7.7 Purity of Reagents—Reagent grade chemicals shall be
water solution.
used in all tests. Unless otherwise indicated, it is intended that
all reagents shall conform to the specifications of the Commit-
8. Hazards
tee onAnalytical Reagents of theAmerican Chemical Society,
8.1 Personnel protective equipment (such as eye
where such specifications are available. Other grades may be
protections, gloves, laboratory coat, etc) should be used in the
used, provided it is first ascertained that the reagent is of
handling of all chemicals. Special care should be taken when
sufficiently high purity to permit its use without lessening the
handling solutions around electrical equipment. All solution
accuracy of the determination.
should be prepared in a hood.
7.8 Unless otherwise indicated, references to water shall be
8.2 Read all equipment manuals before attempting to oper-
understood to mean reagent water conforming to Specification
ateHPLCinstrumentation.Specialattentionshouldbegivento
D1193, Type II. It is recommended that all water be filtered
all warnings.
through a 0.45 µm filter. For eluant preparation, degas the
water by sparging with helium or vacuum degassing and 8.3 BefamiliarwiththeMSDSforallchemicalsusedinthis
sonication. procedure. A dust mask is recommended for the handling of
FIG. 1 (a) Denatonium Benzoate Standard (b) Denatonium Benzoate in an Engine Coolant
D7304 − 06
Denatonium Benzoate. Review your company’s policy con- stock solution.Additional concentrations can be prepared for a
cerning the use of a dust mask. Prepare standards in a hood. more comprehensive calibration curve. All final solutions
should be made with Denatonium Ion Standard Stock solution
9. Sampling, Test Specimens and Test Units
as described in 7.10. Calibrate chromatograph with a minimum
9.1 Collect the sample in a scrupulously clean glass or
ofatleastthreelevelsoftheanalyte,startingnearbutabovethe
polyethylene bottle in accordance with Test Method D1176.
minimum detection limit (MDL) and further defining the work
Collect at least 100 mL of sample.
range in samples subsequent to dilution. These denatonium
analyteexamplesreflectadilutionof3+1(wt/v%)withTypeII
10. Preparation of App
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