1、Designation: D 7304 06Standard Test Method forDetermination of Denatonium Ion in Engine Coolant byHPLC1This standard is issued under the fixed designation D 7304; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last rev
2、ision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers the chemical analysis of enginecoolant for denatonium ion by high-performance liquid chro-matography
3、(HPLC).1.2 This test method is applicable to both new and usedcoolants.1.3 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 t
4、he sample matrixwith denatonium ion1.4 The values stated in SI units are 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 use
5、r of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 1193 Specification for Reagent WaterD 1176 Practice for Sampling and Preparing Aqueous So-lutions of Engine Co
6、olants or Antirusts for Testing Pur-poses3. Summary of Test Method3.1 A sample volume of working sample is prepared bydilution of the sample with water. A high-pressure pump forcesthe mobile phase, eluant, through the HPLC columns (a guardand analytical column) at typical flow rates of 0.1- 2 mL/min
7、.A sample to be separated is introduced in the mobile phase byan injection device prior to the column. The analytes areseparated as they pass through the column. An optical sensordetects the changes in characteristics of the eluant stream andconverts the signal into an absorbance spectrum. The datas
8、ystem compares this response with an external calibrationcurve and the results of the concentration of analyte reported asppm or milligrams per litre (mg/L). Refer to Appendix X1 fora HPLC flow diagram4. Significance and Use4.1 This test method provides for the qualitative and quan-titative determin
9、ation of denatonium ion in engine coolant inmilligrams per liter to low percent range and requires approxi-mately 100 mL per test, with results available in less than 30min. Acceptable levels of denatonium vary with manufactur-ers blending specifications and applicable minimum or maxi-mum industry a
10、nd state specifications. Typically specificationranges from 30 to 50 ppm denatonium ion.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. Sampledilution and optim
11、ized gradient elution can be used to mini-mize or solve most interference problems.5.2 A water dip (solvent system peak) can cause interfer-ence with some integrators. This is eliminated by dilution withthe eluant if the sample dilution factor is 4+1 (v/v) or greater.5.3 Method interferences can be
12、caused by the contamina-tion of glassware, eluant, reagents, etc. Great care must betaken to ensure that contamination is kept at the lowest possiblelevel6. Apparatus6.1 HPLC SystemAnalytical system equipped with allrequired accessories including syringes, gasses, columns,pumps, detectors and softwa
13、re.6.1.1 Gradient Pump.6.1.2 UV/VIS-Mulitple Wavelength Detector.1This test method is under the jurisdiction of ASTM Committee D15 on EngineCoolants and is the direct responsibility of Subcommittee D15.04 on ChemicalProperties.Current edition approved Nov. 1, 2006. Published January 2007.2For refere
14、nced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West
15、Conshohocken, PA 19428-2959, United States.6.1.3 Eluant Degas System.6.1.4 Analytical Column ZORBAX RX-C8 or equivalentcolumn, capable of producing analyte separation equivalent toor better than that shown in Fig. 1.6.1.5 Guard Column for ZORBAX RX-C8 or equivalent,for protection of the analytical c
16、olumn from strongly retainedconstituents.6.1.6 Integrator or Chromatography Data System Softwarecapable of obtaining approximately the same detection limitsas listed in Table 1.6.2 Analytical Balance, 0.0001 g precision.6.3 Volumetric Flasks, 50 mL, 100 mL, 200 mL.7. Reagents and Materials7.1 Aceton
17、itrile, (ACN), Reagent Grade.7.2 Phosphoric acid, H3PO4, 85 %, Reagent Grade.7.3 Potassium Phosphate, Monobasic, KH2PO4, 99.995 %purity.7.4 Denatonium Benzoate, Reagent Grade.7.5 Ethylene Glycol, Reagent Grade.7.6 Deionized water, Typee II water.7.7 Purity of ReagentsReagent grade chemicals shall be
18、used 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 first ascertained that the rea
19、gent 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 SpecificationD 1193, Type II. It is recommended that all water be filteredthrough a 0.45
20、m filter. For eluant preparation, degas thewater by sparging with helium or vacuum degassing andsonication.7.9 Phosphate Buffer Stock SolutionWeigh 650 mg ofMonobasic Potassium Phosphate and transfer it intoa1Lvolumetric flask, add 0.5 mL Phosphoric acid and fill to themark with Type II water. Mix t
21、his solution thoroughly. Filterthe solution with 0.45 m filter before use.7.10 Denatonium Ion Standard Stock SolutionWeigh0.1372 g of Denatonium Benzoate into 100-mL volumetricflask and dilute with ethylene glycol to 100 g. This stocksolution is 1000 ppm of denatonium and is stable for onemonth. Mak
22、e-up working standard solutions in 25% v/vethylene glycol/Type II water to desired concentrations. Ex-ample: 5 ppm denatonium standard solution - weigh 0.5000 gof stock denatonium standard solution into a 100 mL volumet-ric flask and fill to 100 mL mark with 25 % v/v ethylene glycolwater solution.8.
23、 Hazards8.1 Personnel protective equipment (such as eye protec-tions, 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
24、 before attempting to oper-ate HPLC instrumentation. Special attention should be given toall warnings.8.3 Be familiar with the MSDS 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 ma
25、sk. 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 D 1176.Collect at least 100 mL of sample.FIG. 1 (a) Denatonium Benzoate Standard (b) Denatonium Benzoate in an Engine Co
26、olantTABLE 1 Minimum Detection LimitsAnalyte Detection Limits, ppmADenatonium ion 1.0ADetermination using 100-L sample volume. Sample diluted 1+3 (wt/v) withtype II water, full scale, UV/Vis detector set at 210 nm wavelength, Zorbax RX-C8analytical column with Zorbax RX-C8 guard columns. Other syste
27、ms will requireMDL determination using chosen dilution factors, eluants, columns and detectors.D730406210. Preparation of Apparatus10.1 Over the course of the HPLC run, the concentration ofthe eluant will change to improve the separation of theanalytes. The HPLC system is programmed to handle thecha
28、nging mix of eluant. Two different eluant gradient programsare given below, for a borate-silicate based engine coolant anda silicate free, organic acid based engine coolant. The program-ming of the eluant gradient will vary with the HPLC systemused and the inhibitor package utilized in the engine co
29、olant.10.2 Program Gradient 1: (for a borate, silicate based enginecoolant)Time Flow, mL Acetonitrile, Vol % Phosphate Buffer, Vol % CurveAInitial 1.9 20 80 0.1 1.9 20 80 110.0 1.9 45 55 620.0 1.9 90 10 623.0 1.9 20 80 627.0 1.9 20 80 1AThe Curve Number entered here specifies the manner in which suc
30、cessivegradient steps are connected.Curve 1 Go to end condition immediately2-5 Convex6 Linear7-10 Noncave11 Maintain start conditions until end of segment10.3 Program Gradient 2: (for a silicate free, organic acidbased engine coolant)Time Flow, mL Acetonitrile, Vol % Phosphate Buffer, Vol % CurveAIn
31、itial 1.9 20 80 0.1 1.9 20 80 110.0 1.9 40 60 615.0 1.9 80 20 623.0 1.9 20 80 627.0 1.9 20 80 1AThe Curve Number entered here specifies the manner in which successivegradient steps are connected.Curve 1 Go to end condition immediately2-5 Convex6 Linear7-10 Noncave11 Maintain start conditions until e
32、nd of segment11. Calibration and Standardization11.1 Analyze each standard solution separately to determinethe analytes retention time.11.2 Set the chromatograph up in accordance with theconditions specified in 11.9. The use of other equipment,eluants, or flows requires calculation of suitable dilut
33、ionfactors and instrument settings that permit the analyst to obtainthe resolution and detection limits given in Fig. 1 and Table 1,respectively.11.3 Prepare concentrations of denatonium ion at 0.005,0.025 and 0.055 g/L (5, 25 and 55 ppm) from the standardstock solution. Additional concentrations ca
34、n be prepared for amore comprehensive calibration curve. All final solutionsshould be made with Denatonium Ion Standard Stock solutionas described in 7.10. Calibrate chromatograph with a minimumof at least three levels of the analyte, starting near but above theminimum detection limit (MDL) and furt
35、her defining the workrange in samples subsequent to dilution. These denatoniumanalyte examples reflect a dilution of 3+1 (wt/v%) with Type IIwater.NOTE 1HPLC equipment other than that described in this test methodmay require that standards be prepared at higher or lower levels.11.4 Analyze a blank c
36、ontaining only the eluant as de-scribed in Section 12.11.5 A sample of known concentration should be run every10 injections to make sure the instrument is working correctly.11.6 The analytical calibration curve must be established atonly one detector scale setting in order to prevent a change ofslop
37、e affecting the analytical curve.11.7 The analytical calibration curve and an eluant blankshall be verified daily prior to the analysis of samples to verifythe system resolution, calibration and sensitivity.11.8 The analytical calibration curve, analyte retentiontimes and resolution, and an eluant b
38、lank shall be verifiedsubsequent to a change of the system eluant.11.9 Conditions (or equivalent conditions to meet require-ments of Table 1 and Fig. 1):(a) Detector Setting: 210 nm wave length(b) Analytical Column: Zorbax RX-C8 with Guard Col-umn: Zorbax RX-C8(c) Pump: High Pressure limits 3000 psi
39、; Low Pressurelimits 100 psi(d) Flow Rate: 1.9 mL/min(e) Gradient Program: See Section 10(f) Retention Time: 10 to 12 min depending on test sampleand gradient programNOTE 2These conditions may vary depending on the dimensions ofthe column used.12. Procedure12.1 Set up the HPLC (Waters Chromatographi
40、c System) inaccordance with the manufacturers instructions.12.2 Check the instruments setting: see 11.9.12.3 Fill eluant containers (acetonitrile and phosphatebuffer), sparge and pressurize with helium to 5 psi.12.4 Prime the pump, initiate flow and equilibrate thecolumn for 20-25 min with initial s
41、ettings in program gradient,Section 10.12.5 Turn on HPLC and detector. Allow the instrument toequilibrate fro 20-25 min before starting a run.12.6 At least one duplicate and one spiked coolant samplemust be analyzed with each batch of ten or fewer samples. It isrecommended that all test samples be f
42、iltered through a 0.45m filter prior to injection. Results must be recorded in thelaboratory manual or be electronically maintained. The spikedsample is to be prepared by addition of a mixed analytemid-range standard.NOTE 3Once the phosphate buffer has run through the system, do notpurge with 100 %
43、acetonitrile. This will precipitate buffer in the pump,column or detector and result in total blockage and pressure overload.12.7 The column and entire system should be flushed with50-50 %(v/v) water/acetonitrile solution after an analysis tofree from phosphate salts.13. Calculation or Interpretatio
44、n of Results13.1 Integrate the peak area for determination of the con-centrations. Plot the peak area against concentration. A linearD7304063calibration curve is generated for the denatonium ion. Calibra-tion is in the concentration range of interest (5 to 60 ppm) andmust have a linear least squares
45、 correlation coefficient of0.9990 or greater.13.2 The concentration of analyte is given by the concen-tration read from the calibration curve multiplied by thedilution factor used.DB concentration 5 DB concentration from curve dilution factorDB 5 denatonium ion (1)13.3 Relative percent difference (R
46、PD) of duplicate runs:RPD 5 Absolute Value A B!/A! 100 (2)where:A = concentration found in Analysis Run 1, andB = concentration found in Analysis Run 2.Results must be within 20 %.Spike recovery (SR) as percent:SR 5 C 1 B!/A! 100 (3)where:A = concentration found in analysis of spiked sample,B = conc
47、entration found in analysis of sample, andC = concentration of analyte (denatonium ion) added tosample.Spike recovery must be within a 80 to 120 % range.14. Report14.1 Convert and report the concentration of denatoniumion, ppm into the concentration of the denatonium benzoate inppm units found in th
48、e sample.Denatonium Benzoate, ppm 5 denationium, ppm 0.7287 (4)14.2 Include in the report: the sample analysis date, analyst,and the lot number or other identification number for thesample.15. Precision and Bias15.1 The precision and bias of this test method have notbeen determined.16. Keywords16.1
49、analytical method; antifreeze; Bitterant; chromatogra-phy; denatonium ion; denatonium benzoate; engine coolant;HPLCAPPENDIXX1. HPLC SystemNonmandatory InformationX1.1 See Fig. X1.1.3Solvent Delivery Systempushes the solvent(s) stream through the instrument at a controlled flowrate.X1.2 Autosamplerintroduces the sample into the liquidstream of the instrument.3http:/ X1.1 HPLC Flow DiagramD7304064X1.3 Columna stainless steel tube packed with siliconbeads that separates the denato
