1、Designation:D730414Standard 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 revision.A
2、 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 chromatography(HPLC).
3、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 Coelution of o
4、ther 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 to be regarded as
5、 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 and determine the app
6、lica-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 Purposes3.Summary of Test Method3.1 The denatonium benzoate analys
7、is is achieved by anHPLC method,where a weight of engine coolant is placed inan auto-sampler vial and mixed 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
8、 anultraviolet detector.The ultraviolet detector is used to measurethe response of the DNB active ingredients(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 concentrationsa
9、nd the HPLCs computer calculates the amount of DNBpresent in the coolant.4.Significance and Use4.1 This 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 t
10、est,with results available in less than10 min.Denatonium benzoate is a compound composed of aquaternary 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.B
11、y slightly adjustingthe pH of the solution to be more acidic(pH 4.6)theequilibrium will be shifted 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 conc
12、entrationcompared to the analyte of interest,denatonium ion.Knownchromatographic interferences 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.5.2 Method interferen
13、ces 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.1This test m
14、ethod is under the jurisdiction of ASTM Committee D15 on EngineCoolants and Related Fluids and is the direct responsibility of SubcommitteeD15.04 on Chemical Properties.Current edition approved March 15,2014.Published May 2014.Originallyapproved in 2006.Last previous edition approved in 2006 as D730
15、406.DOI:10.1520/D7304-14.2For referenced 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.Copyright ASTM International,100 Barr Har
16、bor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 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,75mm x 4.6 mm or equivalent column.6.1.5 Guard Column,Ultra Cartridge UPLC C-18 for 4.6mm or equivalent.6.2 Analytical Balance,0.0001 g precision.6.3 Volumetric 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 glass