1、Designation: D7304 16Standard Test Method forDetermination of Denatonium Ion in Engine Coolant byHPLC1This standard is issued under the fixed designation D7304; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revis
2、ion. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers the chemical analysis of enginecoolant for denatonium benzoate (DNB) by high-performanceliquid chromatog
3、raphy (HPLC). DNB is added to potentiallyrender engine coolant unpalatable to animals and humans. Thisanalytical method was designed for the analysis of DNB and isnot valid for any other bittering agents such as denatoniumsaccharide.1.2 This test method is applicable to both new and usedcoolants.1.3
4、 Coelution of other ions may cause interferences in thedetection of the denatonium cation. In the case of unfamiliarformulations, identification verification should be performedby either or both fortification and dilution of the sample matrixwith denatonium ion.1.4 The values stated in SI units are
5、to be regarded as thestandard. The values given in parentheses are for informationonly.1.5 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices
6、and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterD1176 Practice for Sampling and Preparing Aqueous Solu-tions of Engine Coolants orAntirusts for Testing PurposesE177 Practice for Use of the Terms P
7、recision and Bias inASTM Test MethodsE691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test Method3. Summary of Test Method3.1 The denatonium benzoate analysis is achieved by anHPLC method, where a weight of engine coolant is placed inan auto-sampler vial and mixed
8、 with a known volume ofde-ionized water. The auto-sampler vial is placed in a HPLCautosampler and the measurement of denatonium benzoate isperformed using a C-18 reverse phase column attached to anultraviolet detector. The ultraviolet detector is used to measurethe response of the DNB active ingredi
9、ents (denatonium andbenzoate) in the engine coolant after they have been separatedin the reverse phase column. The denatonium and benzoateresponses are compared to responses of known concentrationsand the HPLCs computer calculates the amount of DNBpresent in the coolant.4. Significance and Use4.1 Th
10、is test method provides for the qualitative and quan-titative determination of denatonium benzoate in engine cool-ant in milligrams per litre to low percent range and requiresapproximately 1 mL per test, with results available in less than10 min. Denatonium benzoate is a compound composed of aquater
11、nary ammonium cation, denatonium and an inert anion,benzoate. In solution the denatonium benzoate exists in equi-librium between the denatonium benzoate compound, thedenatonium cation and benzoate anion. By slightly adjustingthe pH of the solution to be more acidic ( pH 4.6) theequilibrium will be s
12、hifted to the direction of forming moredenatonium and benzoate ions in the solution.5. Interferences5.1 Interferences can be caused by substances with similarretention times, especially if they are in high concentrationcompared to the analyte of interest, denatonium ion. Knownchromatographic interfe
13、rences have been determined and theanalysis modified to minimize any co-elution of interferingpeaks. The eluent strength and flow rate can be used to reduceor solve most interference problems.1This test method is under the jurisdiction of ASTM Committee D15 on EngineCoolants and Related Fluids and i
14、s the direct responsibility of SubcommitteeD15.04 on Chemical Properties.Current edition approved May 15, 2016. Published July 2016. Originallyapproved in 2006. Last previous edition approved in 2014 as D730414. DOI:10.1520/D7304-16.2For referenced ASTM standards, visit the ASTM website, www.astm.or
15、g, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States15.2 Method int
16、erferences can also be caused by the con-tamination of glassware, eluant, reagents, etc. Great care mustbe taken to ensure that contamination is kept at the lowestpossible level.6. Apparatus6.1 HPLC SystemHigh Performance Liquid Chromato-graph system equipped with appropriate computer and soft-ware.
17、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 m packing,75 mm 4.6 mm or equivalent column.6.1.5 Guard Column, Ultra Cartridge UPLC C-18 for4.6 mm or equivalent.6.2 Analytical Balance, 0.0001 g precision.6.3 Volumetr
18、ic Flasks, 50 mL, 100 mL, 200 mL, 500 mL,1L.6.4 pH Meter, capable of pH/mV/temperature measure-ments.6.5 Glassware, class “A“ laboratory glassware and plasticware.6.6 Eppendorf Series 2100 Pipetter, capable of delivering1000 L.6.7 Electronic Pipetter, capable of delivering between 30 300 L.7. Reagen
19、ts and Materials7.1 Acetonitrile, (ACN), Reagent Grade.7.2 Ortho Phosphoric acid, H3PO4, 85 %, Reagent Grade.7.3 Potassium Phosphate, Monobasic, KH2PO4, 99.995 %purity.7.4 Denatonium Benzoate, 98 % purity.7.5 Ethylene Glycol, Reagent Grade.7.6 De-ionized water, Type II water.7.7 Purity of ReagentsRe
20、agent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents shall conform to the specifications of the Commit-tee on Analytical Reagents of the American Chemical Society,where such specifications are available. Other grades may beused, provided it is
21、first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the determination.7.8 Unless otherwise indicated, references to water shall beunderstood to mean reagent water conforming to SpecificationD1193, Type II. It is recommended that all wat
22、er be filteredthrough a 0.45 m filter. For eluant preparation, degas thewater by sparging with helium or vacuum degassing andsonication.7.9 Potassium Phosphate Solution KH2PO4(0.5M):7.9.1 Weigh 34.022 g of the KH2PO4into a 500 mLvolumetric flask.7.9.2 Add 250 mL of de-ionized water and mix until the
23、KH2PO4has dissolved.7.9.3 Dilute to the mark with de-ionized water and thor-oughly mix.7.10 Sample Dilution Solution (pH 4.6): Potassium Phos-phate Solution KH2PO4(0.1M):7.10.1 Transfer 200 mL of Potassium Phosphate SolutionKH2PO4(0.5M) to 1000 mL volumetric flask.7.10.2 Dilute to mark with de-ioniz
24、ed water.7.10.3 Measure the pH of the de-ionized water with a pHmeter.NOTE 1The pH of the sample dilution water should be checkedbi-weekly and if the pH has changed the dilution water should bere-prepared.7.11 pH 4.3 Mobile Phase, 30 % Acetonitrile 70 % ofPotassium Phosphate Solution KH2PO4(0.1M):7.
25、11.1 Add 300 mL of acetonitrile to a 1000 mL volumetricflask.7.11.2 Dilute to the mark with Potassium Phosphate Solu-tion KH2PO4(0.1M).7.11.3 Stopper and mix.7.11.4 Degas the solution for 10 min in an ultra-sonic bathas a large amount of dissolved gas will be present in thesolution.7.11.5 Place solu
26、tion in a 1000 mL beaker and measure thesolutions pH.7.11.6 Adjust the pH of the mobile phase solution with0.5M KH2PO4or 85 % phosphoric acid until it reaches pH 4.3.7.11.7 Transfer the solution to the HPLC reagent bottle.NOTE 2The final pH of the Mobile Phase should be approximately 4.3pH units.8.
27、Hazards8.1 Personnel protective equipment (such as eyeprotections, gloves, laboratory coat, etc.) should be used in thehandling of all chemicals. Special care should be taken whenhandling solutions around electrical equipment. All solutionshould be prepared in a hood.8.2 Read all equipment manuals b
28、efore attempting to oper-ate HPLC instrumentation. Special attention should be given toall warnings.8.3 Be familiar with the SDS for all chemicals used in thisprocedure. A dust mask is recommended for the handling ofDenatonium Benzoate. Review your companys policy con-cerning the use of a dust mask.
29、 Prepare standards in a hood.9. Sampling, Test Specimens and Test Units9.1 Collect the sample in a scrupulously clean glass orpolyethylene bottle in accordance with Test Method D1176.Collect at least 100 mL of sample.10. Calibration Solutions:10.1 Stock Denatonium Benzoate Calibration Solution(500 m
30、g L DNB):10.1.1 Weigh 0.0500 6 0.0001 g of denatonium benzoateinto a 100 mL volumetric flask and record the weight.D7304 16210.1.2 Add de-ionized water, stopper and mix until soliddissolves.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
31、 the Stock Calibration Solution in a100 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 placingthe appropriate amount of Stock Solution (see Table 1) into a2000 L auto-sampler vial.1
32、0.3.2 Dilute to volume with Sample Dilution Solution (pH4.6).10.3.3 Place vials in the instruments auto-sampler.10.3.4 Repeat for each calibration standard.10.3.5 Analyze standards and calibrate instrument using themethods external calibration function.11. Analytical Conditions11.1 Instrument:Column
33、: 75 mm 4.6 mmColumn Temperature: 40CEluent “A”: 30 % Acetonitrile70%ofKH2PO4(0.1M)Analysis Program: IsocraticFlow Rate: 1.95 mL/minSystem Pressure: 3100 psi or 214 barInjection Size: 5 L11.2 Ultra-Violet Detector:Signal: AbsorbanceWavelength: 210 mmResponse: 0.02 ABUCell Size: 12 LSignal Rate: 10.0
34、 pts/sec12. Gradient Program12.1 See Table 2.NOTE 3If the coolant contains 4-Tert-Butylbenzoic acid (TBBA) theanalysis time will be 9 min.13. Sample Preparation13.1 Place a 2000 L auto-sampler vial on an analyticalbalance and zero the balance.13.2 Pipette 50 L of sample into the auto-sampler vial an
35、drecord the sample weight in milligrams (mg).13.3 Pipette 1950 L of pH 4.6 Sample Dilution Solutioninto the auto-sampler vial.13.4 Cap the vial, shake and place vial in the auto-sampler.13.5 Repeat Steps 13.1 13.4 for each sample.13.6 Calculate the sample dilution factor using the formulain Eq 1.13.
36、7 Prepare an auto-sampler sequence and analyzesamples.14. Calculation14.1 Instrument Results:14.1.1 The instrument denatonium benzoate results arecalculated by comparing the sample denatonium peak arearesponse to the calibration curve for the denatonium benzoate.This calibration curve is part of the
37、 instrument method.14.2 Sample Denatonium Benzoate (DNB) Results:14.2.1 The concentration of the denatonium benzoate isdetermined by multiplying the denatonium benzoates instru-ment concentration by the samples dilution factor using thefollowing formula:DNB Concentration ppm wt./wt.!= Instr. concent
38、ration in mg/L 3 Dilution Factor! (1)where:Dilution Factor = 2000 L / weight of 50 L of sample (mg)14.3 Limit of Detection and Limit of Quantitation:14.3.1 The Limit of Detection (LOD) is the point at whichthe analyte cannot be determined.14.3.2 The Limit of Quantitation (LOQ) is the lowestconcentra
39、tion of analyte that can be reported with confidenceand is defined as 3.3 times the LOD.14.3.3 The instrument and method LOD and LOQ valuesare reported in Table 3.NOTE 4The Method LOD and LOQ are determined by multiplyingthe Instrument LOD and LOQ by the Sample Dilution Ratio.TABLE 1 Denatonium Benz
40、oate Calibration StandardsStandardConcentrationof StandardVolume ofWorkingStandard(20 mg/L)Volume ofMonoethyleneGlycolVolume ofpH 4.6Water Addedto Auto-sampler VialUnit (mg/L) L L LDNB Std 1 0.25 25 50 1925DNB Std 2 0.50 50 50 1900DNB Std 3 0.75 75 50 1875DNB Std 4 1.00 100 50 1850DNB Std 5 1.25 125
41、 50 1825DNB Std 6 1.50 150 50 1800TABLE 2 Gradient ProgramProgram Step Time (min) Flow (mL/min)30 % Acetonitrile70%ofKH2PO4(0.1M) (adjustedpH 4.3)Equilibration 0.5 1.95 100.01 5.0 1.95 100.0TABLE 3 Limit of Detection and Limit of Quantitation DataInstrumentLODppm (wt/vol)InstrumentLOQppm (wt/vol)Met
42、hod LODppm (wt/wt)Method LOQppm (wt/wt)BitteringAgent(DenatoniumPeak)0.050 0.165 2.0 6.6D7304 16315. Report15.1 Convert and report the concentration of denatoniumion, ppm found in the sample using the following formula:Denatonium Ion#5 DNB# * 0.7287 (2)15.2 Include in the report: the sample analysis
43、 date, analyst,and the lot number or other identification number for thesample.16. Precision and Bias316.1 PrecisionThe precision of this test method is basedon an interlaboratory study of ASTM D7304, Standard TestMethod for Determination of Denatonium Ion in EngineCoolant by HPLC, conducted in 2014
44、. Each of seven labora-tories tested fifteen different materials. Every “test result”represents an individual determination, and all participantsreported duplicate test results. Practice E691 was followed forthe design and analysis of the data; the details are given inASTM Research Report No. RR:D15
45、-1035.16.1.1 Repeatability (r)The difference between repetitiveresults obtained by the same operator in a given laboratoryapplying the same test method with the same apparatus underconstant operating conditions on identical test material withinshort intervals of time would, in the long run, in the n
46、ormal andcorrect operation of the test method, exceed the followingvalues only in one case in 20.16.1.1.1 Repeatability can be interpreted as maximum dif-ference between two results, obtained under repeatabilityconditions, that is accepted as plausible due to random causesunder normal and correct op
47、eration of the test method.16.1.1.2 Repeatability limits are listed in Table 4.16.1.2 Reproducibility (R)The difference between twosingle and independent results obtained by different operatorsapplying the same test method in different laboratories usingdifferent apparatus on identical test material
48、 would, in the longrun, in the normal and correct operation of the test method,exceed the following values only in one case in 20.16.1.2.1 Reproducibility can be interpreted as maximumdifference between two results, obtained under reproducibilityconditions, that is accepted as plausible due to rando
49、m causesunder normal and correct operation of the test method.16.1.2.2 Reproducibility limits are listed in Table 4.16.1.3 The above terms (repeatability limit and reproduc-ibility limit) are used as specified in Practice E177.16.1.4 Any judgment in accordance with statements 16.1.1and 16.1.2 would have an approximate 95 % probability ofbeing correct.16.2 BiasAt the time of the study, there was no acceptedreference material suitable for determining the bias for this testmethod, therefore no statement on bias is being made.1
