1、Designation: D7304 06D7304 14Standard 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 la
2、st revision. 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 engine coolant for denatonium ion benzoate (DNB) by high-performanceliq
3、uid chromatography (HPLC). DNB is added to potentially render engine coolant unpalatable to animals and humans. Thisanalytical method was designed for the analysis of DNB and is not valid for any other bittering agents such as denatoniumsaccharide.1.2 This test method is applicable to both new and u
4、sed coolants.1.3 Coelution of other ions may cause interferences in the detection of the denatonium cation. In the case of unfamiliarformulations, identification verification should be performed by either or both fortification and dilution of the sample matrix withdenatonium ionion.1.4 The values st
5、ated 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 responsibilityof the user of this standard to establish appropriate safet
6、y and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterD1176 Practice for Sampling and Preparing Aqueous Solutions of Engine Coolants or Antirusts for Testing Purposes3. Summary of T
7、est Method3.1 A sample volume of working sample is prepared by dilution of the sample with water. A high-pressure pump forces themobile phase, eluant, through the HPLC columns (a guard and analytical column) at typical flow rates of 0.1- 2 mL/min.Asampleto be separated is introduced in the mobile ph
8、ase by an injection device prior to the column. The analytes are separated as theypass through the column. An optical sensor detects the changes in characteristics of the eluant stream and converts the signal intoan absorbance spectrum. The data system compares this response with an external calibra
9、tion curve and the results of theconcentration of analyte reported as ppm or milligrams per litre (mg/L). Refer to The denatonium benzoate analysis is achievedby an HPLC method, where a weight of engine coolant is placed in an auto-sampler vial and mixed with a known volume ofde-ionized water. The a
10、uto-sampler vial is placed in a HPLC autosampler and the measurement of denatonium benzoate isperformed using a C-18 reverse phase column attached to an ultraviolet detector. The ultraviolet detector is used to measure theresponse of the DNB active ingredients (denatonium and benzoate) in the engine
11、 coolant after they have been separated in thereverse phase column. The denatonium and benzoate responses are compared to responses of known concentrations and theHPLCs computer calculates the amount of DNB present in the coolant.Appendix X1 for a HPLC flow diagram4. Significance and Use4.1 This tes
12、t method provides for the qualitative and quantitative determination of denatonium ionbenzoate in engine coolantin milligrams per literlitre to low percent range and requires approximately 1001 mL per test, with results available in less than1 This test method is under the jurisdiction of ASTM Commi
13、ttee D15 on Engine Coolants and Related Fluids and is the direct responsibility of Subcommittee D15.04 onChemical Properties.Current edition approved Nov. 1, 2006March 15, 2014. Published January 2007May 2014. Originally approved in 2006. Last previous edition approved in 2006 asD730406. DOI: 10.152
14、0/D7304-06.10.1520/D7304-14.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.This document is not an ASTM stan
15、dard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all case
16、s only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States130 min. Acceptable levels of denatonium vary with manufacturers blending specific
17、ations and applicable minimum or maximumindustry and state specifications. Typically specification ranges from 30 to 50 ppm denatonium ion.10 min. Denatonium benzoateis a compound composed of a quaternary ammonium cation, denatonium and an inert anion, benzoate. In solution the denatoniumbenzoate ex
18、ists in equilibrium between the denatonium benzoate compound, the denatonium cation and benzoate anion. By slightlyadjusting the pH of the solution to be more acidic ( pH 4.6) the equilibrium will be shifted to the direction of forming moredenatonium and benzoate ions in the solution.5. Interference
19、s5.1 Interferences can be caused by substances with similar retention times, especially if they are in high concentration comparedto the analyte of interest, denatonium ion. Sample dilution and optimized gradient elution Known chromatographic interferenceshave been determined and the analysis modifi
20、ed to minimize any co-elution of interfering peaks. The eluent strength and flow ratecan be used to minimizereduce or solve most interference problems.5.2 A water dip (solvent system peak) can cause interference with some integrators. This is eliminated by dilution with theeluant if the sample dilut
21、ion factor is 4+1 (v/v) or greater.5.2 Method interferences can also be caused by the contamination of glassware, eluant, reagents, etc. Great care must be takento ensure that contamination is kept at the lowest possible levellevel.6. Apparatus6.1 HPLC SystemAnalytical High Performance Liquid Chroma
22、tograph system equipped with all required accessoriesincluding syringes, gasses, columns, pumps, detectors 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, ZORBAX RX-C8 or equivalent column, capable of
23、producing analyte separation equivalent to orbetter than that shown in Kinetex C-18, 2.6 m packing, 75 mm x 4.6 mm or equivalent column.Fig. 1.6.1.5 Guard Column, for ZORBAX RX-C8 or equivalent, for protection of the analytical column from strongly retainedconstituents.Ultra Cartridge UPLC C-18 for
24、4.6 mm or equivalent.6.1.6 Integrator or Chromatography Data System Software capable of obtaining approximately the same detection limits aslisted in Table 1.6.2 Analytical Balance, 0.0001 g precision.6.3 Volumetric Flasks, 50 mL, 100 mL, 200 mL. mL, 500 mL, 1 L.6.4 pH Meter, capable of pH/mV/temper
25、ature measurements.6.5 Glassware, class “A“ laboratory glassware and plastic ware.6.6 Eppendorf Series 2100 Pipetter, capable of delivering 1000 L.6.7 Electronic Pipetter, capable of delivering between 30 300 L.TABLE 1 Minimum Detection Limitscolwidth=“1.91in“colwidth=“1.09in“Analyte Detection Limit
26、s, ppmADenatonium ion 1.0TABLE 1 Denatonium Benzoate Calibration StandardsStandard Concentrationof 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
27、 1875DNB Std 4 1.00 100 50 1850DNB Std 5 1.25 125 50 1825DNB Std 6 1.50 150 50 1800A Determination using 100-L sample volume. Sample diluted 1+3 (wt/v) with typeII water, full scale, UV/Vis detector set at 210 nm wavelength, Zorbax RX-C8analytical column with Zorbax RX-C8 guard columns. Other system
28、s will requireMDL determination using chosen dilution factors, eluants, columns and detectors.D7304 1427. Reagents 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 Benz
29、oate, Reagent Grade.98 % purity.7.5 Ethylene Glycol, Reagent Grade.7.6 DeionizedDe-ionized water, TypeeType II water.7.7 Purity of ReagentsReagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that allreagents shall conform to the specifications of the Commit
30、tee on Analytical Reagents of the American Chemical Society, wheresuch specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently highpurity to permit its use without lessening the accuracy of the determination.7.8 Unless otherwise in
31、dicated, references to water shall be understood to mean reagent water conforming to SpecificationD1193, Type II. It is recommended that all water be filtered through a 0.45 m filter. For eluant preparation, degas the water bysparging with helium or vacuum degassing and sonication.7.9 Potassium Phos
32、phate Buffer StockSolution KH2PO4 Solution(0.5M): Weigh 650 mg of Monobasic Potassium Phosphateand transfer it into a 1 L volumetric flask, add 0.5 mL Phosphoric acid and fill to the mark with Type II water. Mix this solutionthoroughly. Filter the solution with 0.45 m filter before use.7.9.1 Weigh 3
33、4.022 g of the KH2PO4 into a 500 mL volumetric flask.7.9.2 Add 250 mL of de-ionized water and mix until the KH2PO4 has dissolved.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 KH2PO4 (0.1M):7.10.1 Transfer 200 mL
34、 of Potassium Phosphate Solution KH2PO4 (0.5M) to 1000 mL volumetric flask.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 1The pH of the sample dilution water should be checked bi-weekly and if the pH has changed the dilution water shou
35、ld be re-prepared.7.11 Denatonium Ion Standard Stock SolutionpH 4.3 Mobile Phase, 30 % Acetonitrile 70 % of Potassium Phosphate SolutionKH2PO4 (0.1M): Weigh 0.1372 g of Denatonium Benzoate into 100-mL volumetric flask and dilute with ethylene glycol to 100g. This stock solution is 1000 ppm of denato
36、nium and is stable for one month. Make-up working standard solutions in 25% v/vethylene glycol/Type II water to desired concentrations. Example: 5 ppm denatonium standard solution - weigh 0.5000 g of stockdenatonium standard solution into a 100 mLvolumetric flask and fill to 100 mLmark with 25 % v/v
37、 ethylene glycol water solution.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 KH2PO4 (0.1M).7.11.3 Stopper and mix.7.11.4 Degas the solution for 10 minutes in an ultra-sonic bath as a large amount of dissolved gas will be
38、present in the solution.7.11.5 Place solution in a 1000 mL beaker and measure the solutions pH.7.11.6 Adjust the pH of the mobile phase solution with 0.5M KH2PO4 or 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 P
39、hase should be approximately 4.3 pH units.8. Hazards8.1 Personnel protective equipment (such as eye protections, gloves, laboratory coat, etc)etc.) should be used in the handlingof all chemicals. Special care should be taken when handling solutions around electrical equipment.All solution should be
40、preparedin a hood.8.2 Read all equipment manuals before attempting to operate HPLC instrumentation. Special attention should be given to allwarnings.8.3 Be familiar with the MSDS for all chemicals used in this procedure. A dust mask is recommended for the handling ofDenatonium Benzoate. Review your
41、companys policy concerning the use of a dust mask. Prepare standards in a hood.9. Sampling, Test Specimens and Test Units9.1 Collect the sample in a scrupulously clean glass or polyethylene bottle in accordance with Test Method D1176. Collect atleast 100 mL of sample.D7304 14310. Preparation of Appa
42、ratus10.1 Over the course of the HPLC run, the concentration of the eluant will change to improve the separation of the analytes.The HPLC system is programmed to handle the changing mix of eluant. Two different eluant gradient programs are given below,for a borate-silicate based engine coolant and a
43、 silicate free, organic acid based engine coolant. The programming of the eluantgradient will vary with the HPLC system used and the inhibitor package utilized in the engine coolant.10.2 Program Gradient 1: (for a borate, silicate based engine coolant)Time Flow, mL Acetonitrile, Vol % Phosphate Buff
44、er, 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 1A The Curve Number entered here specifies the manner in which successive gradient steps are connected.Curve 1 Go to end condition immediately2-5 Convex6 Linear7-10 Noncave11 Maintain start
45、conditions until end of segment10.3 Program Gradient 2: (for a silicate free, organic acid based engine coolant)Time Flow, mL Acetonitrile, Vol % Phosphate Buffer, Vol%CurveAInitial 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 1A The Curve Number entered he
46、re specifies the manner in which successive gradient steps are connected.Curve 1 Go to end condition immediately2-5 Convex6 Linear7-10 Noncave11 Maintain start conditions until end of segment10. Calibration Solutions:10.1 Stock Denatonium Benzoate Calibration Solution (500 mg/L DNB):10.1.1 Weigh 0.0
47、500 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
48、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 2000L auto-sampler vial.10.3.2 Dilute to volume with Sa
49、mple Dilution Solution (pH 4.6).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 the methods external calibration function.11. Calibration and Standardization11.1 Analyze each standard solution separately to determine the analytes retention time.11.2 Set the chromatograph up in accordance with the conditions specified in 11.9. The use of other equipment, eluants, or flowsrequires calculation of suitable dilution factors and instrument settings that per
