1、Designation: D6584 131Standard Test Method forDetermination of Total Monoglycerides, Total Diglycerides,Total Triglycerides, and Free and Total Glycerin in B-100Biodiesel Methyl Esters by Gas Chromatography1This standard is issued under the fixed designation D6584; the number immediately following t
2、he designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1NOTEThe formats of Eq 11 and Eq 16
3、 were editorially corrected in April 2014.1. Scope*1.1 This test method covers the quantitative determinationof total monoglyceride, total diglyceride, total triglyceride, andfree and total glycerin in B-100 methyl esters by gas chroma-tography. The range of quantitation for monoglyceride is 0.009to
4、 0.77860 mass %, for diglyceride is 0.092353 to 0.54475mass %, and for triglyceride is 0.00092857 to 1.3881 mass %.The range of quantitation for free glycerin is 0.0005714 to0.019533 mass % and for total glycerin from 0.0090714 to0.42767 mass %. This procedure is not applicable to vegetableoil methy
5、l esters obtained from lauric oils, such as coconut oiland palm kernel oil.1.2 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.3 This standard does not purport to address all of thesafety concerns, if any, associated with its
6、use. It is theresponsibility of the user 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:2D4307 Practice for Preparation of Liquid Blends for Use asAnalytical Stan
7、dardsE355 Practice for Gas Chromatography Terms and Relation-shipsE594 Practice for Testing Flame Ionization Detectors Usedin Gas or Supercritical Fluid Chromatography3. Terminology3.1 Definitions:3.1.1 biodiesel (B-100), nfuel comprised of mono-alkylesters of long chain fatty acids derived from veg
8、etable oils oranimal fats.3.1.2 bonded glycerin, nglycerin portion of the mono-,di-, and triglyceride molecules.3.2 Definitions of Terms Specific to This Standard:3.2.1 total glycerin, nsum of free and bonded glycerin.3.3 This test method makes reference to many common gaschromatographic procedures,
9、 terms, and relationships. Detaileddefinitions can be found in Practices E355 and E594.4. Summary of Test Method4.1 The sample is analyzed by gas chromatography, aftersilyating with N-methyl-N-trimethylsilyltrifluoracetamide(MSTFA). Calibration is achieved by the use of two internalstandards and fou
10、r reference materials. Mono-, di-, and triglyc-eride are determined by comparing to monoolein, diolein, andtriolein standards respectively. Average conversion factors areapplied to mono-, di-, and triglycerides to calculate the bondedglycerin content of the sample.5. Significance and Use5.1 Free and
11、 bonded glycerin content reflects the quality ofbiodiesel. A high content of free glycerin may cause problemsduring storage, or in the fuel system, due to separation of theglycerin. A high total glycerin content can lead to injectorfouling and may also contribute to the formation of deposits atinjec
12、tion nozzles, pistons, and valves.6. Apparatus6.1 Chromatographic SystemSee Practice E355 for spe-cific designations and definitions.6.1.1 Gas Chromatograph (GC)The system must be ca-pable of operating at the conditions given in Table 1.6.1.2 Column, open tubular column with a 5 % phenylpo-lydimethy
13、lsiloxane bonded and cross linked phase internal1This test method is under the jurisdiction of ASTM Committee D02 onPetroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility ofD02.04.0L on Gas Chromatography Methods.Current edition approved May 1, 2013. Published May 2013. Or
14、iginallyapproved in 2000. Last previous edition approved in 2010 as D658410a1. DOI:10.1520/D6584-13E01.2For 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
15、 Document Summary page onthe ASTM website.*A Summary of Changes section appears at the end of this standardCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1coating. The column should have an upper temperature limit ofat least 400C. Col
16、umns, either 10 m or 15 m in length, with a0.32 mm internal diameter, and a 0.1 m film thickness havebeen found satisfactory. Any column with better or equivalentchromatographic efficiency and selectivity can be used. It isrecommended thata2to5metre 0.53 mm high temperatureguard column be installed
17、from the injector to the analyticalcolumn. This allows the use of autoinjectors and also increasescolumn life.6.2 Electronic Data Acquisition System:6.2.1 Integrator or Computer, capable of providing real timegraphic and digital presentation of the chromatographic data isrecommended for use. Peak ar
18、eas and retention times shall bemeasured by computer or electronic integration.6.2.2 This device must be capable of performing multilevelinternal-standard-type calibrations and be able to calculate thecorrelation coefficient (r2) and internal standard calculationsfor each data set.7. Reagents and Ma
19、terials7.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.3Other grades may b
20、e usedprovided it is first ascertained that the reagent is of sufficientpurity to permit its use without lessening the accuracy of thedetermination.7.2 n-Heptane, reagent grade.7.3 N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA),reagent grade.7.4 Pyridine, reagent grade.7.5 Carrier Gas, hydrogen
21、 or helium of high purity. Addi-tional purification is recommended by the use of molecularsieves or other suitable agents to remove water, oxygen, andhydrocarbons.Available pressure must be sufficient to ensure aconstant carrier gas flow rate.7.6 Microlitre Syringes, 100 L and 250 L capacity.7.7 Scr
22、ew Cap Vials, with polytetrafluoroethylene (PTFE)-faced septa, 10 mL capacity.8. Preparation of Apparatus8.1 Install and condition the column in accordance withmanufacturer or suppliers instructions. After conditioning,attach column outlet to flame ionization detector inlet andcheck for leaks throug
23、hout the system. If leaks are found,tighten or replace fittings and recheck for leaks before proceed-ing.9. Calibration and Standardization9.1 Preparation of Calibration StandardsPrepare stan-dards using fresh compounds listed in Table 2 according toPractice D4307. Weigh the components directly into
24、 thevolumetric flasks specified and record the mass to the nearest0.1 mg. Dilute the volumetric flasks to mark with pyridine.Store the calibration standards in a refrigerator when not in use.9.2 Standard SolutionsPrepare the five standard solutionsin Table 3 by transferring the specified volumes by
25、means ofmicrolitre syringes to 10 mLsepta vials.Add to each of the fivestandard solutions 100 Lof MSTFA. Close the vial and shake.Allow the vial to stand for 15 to 20 min at room temperature.Add approximately 8 mL n-Heptane to the vial and shake.9.3 Chromatographic AnalysisIf using an automaticsampl
26、er, transfer an aliquot of the solution into a glass GC vialand seal with a TFE-fluorocarbonlined cap.9.4 StandardizationAnalyze the calibration standards un-der the same operating conditions as the sample solutions.Inject 1 L of the reaction mixture into the cool on-columninjection port and start t
27、he analysis. Obtain a chromatogramand peak integration report. For each reference substance,determine the response ratio (rspi) and amount ratio (amti) foreach component using Eq 1 and 2.rspi5 Ai/As! (1)where:Ai= area of reference substance, andAs= area of internal standard.3Reagent Chemicals, Ameri
28、can Chemical Society Specifications, AmericanChemical Society, Washington, DC. For Suggestions on the testing of reagents notlisted by the American Chemical Society, see Annual Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary,
29、 U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.TABLE 1 Operating ConditionsInjectorCool on column injectionSample size 1 LColumn Temperature ProgramInitial temperature 50C hold 1 minRate 1 15C / min to 180CRate 2 7C / min to 230CRate 3 30C / min 380C hold 10 minDefectorType Flame ionizati
30、onTemperature 380CCarrier GasType Hydrogen or helium measured at 50CFlow rate 3 mL/minTABLE 2 Stock SolutionsCompound CAS No.ApproximateMass (mg)VolumetricFlask Size(mL)Glycerin 56-81-5 25 501-Mono cis-9-octadecenoyl-rac-glycerol (monoolein)111-03-5 50 101,3-Di cis-octadecenoylglycerol(diolein)2465-
31、32-9 50 101,2,3-Tri cis-octadecenoylglycerol(triolein)122-32-7 50 10(S) - (-) -1,2,4-Butanetriol - (InternalStandard 1)42890-76-6 25 251,2,3-Tridecanolylglycerol (tricaprin) -(Internal Standard 2)621-71-6 80 10D6584 1312amti5 Wi/Ws! (2)where:Wi= mass of reference substance, andWs= mass of internal s
32、tandard.9.4.1 Prepare a calibration curve for each reference compo-nent by plotting the response ratios (rspi), as the y-axis, versusthe amount ratios (amti), as the x-axis.9.5 Calculate the correlation coefficient r2value for eachreference component in the calibration set using Eq 3. The r2value sh
33、ould be at least 0.99 or greater. If the above criteria forr2are not met, rerun the calibration or check instrumentparameters and hardware.r25(xy!2(x2!(y2!(3)where:x 5 Xi2 x (4)y 5 Yi2 y (5)and:Xi= amtiratio data point,x = average values for all amtidata pointsYi= corresponding rspidata points,y = a
34、verage values for all rspidata points.9.6 Calibration FunctionsFor each reference calibrationfunctions are calculated in the form:AxAis5FaxSWxWisDG1bx(6)where:Wx= mass of reference substance, mg,Wis= mass of internal standard, mg,Ax= peak area of reference substance,Ais= peak area of internal standa
35、rd,ax= slope of the calibration function, andbx= intercept of the calibration function.10. Procedure10.1 Set the instrument operating variables to the valuesspecified in Table 1.Weigh to the nearest 0.1 mg approximately100 mg of sample directly into a 10 mL septa vial. Usingmicrolitre syringes, add
36、exactly 100 L of each internalstandard and MSTFA. Shake the vials, and allow to set for 15to 20 min at room temperature. Add approximately 8 mL ofn-Heptane to the vial and shake.10.2 Inject 1 L of the reaction mixture into the coolon-column injection port and start the analysis. Obtain achromatogram
37、 and peak integration report.10.3 Peak IdentificationIdentify peaks by comparison ofretention times to the standards. For identification of additionalpeaks, use the relative retention times given in Table 4 and thereference chromatograms given in Fig. 1. Mono-, di-, andtriglycerides are separated ac
38、cording to carbon numbers (CN).10.4 Monoglyceride consists of the four overlapping peakswith relative retention times (RRT) of 0.76 and 0.83 to 0.86with respect to the internal standard tricaprin. A pair of peaks,methyl esters with a carbon number of 24, may appear withRRT of 0.80 to 0.82, and shoul
39、d not be included in thecalculation of monoglyceride.10.5 Diglyceride is also primarily separated according tocarbon number, but due to varying double bonds in themolecules, baseline resolution of the peaks does not occur. Thegrouping of 3 to 4 peaks with RRT of 1.05 to 1.09 (CN 34, 36,and 38) shall
40、 be attributed to diglyceride. Carbon number alsoseparates triglyceride. Peaks with RRT of 1.16 to 1.31 (CN 52,54, 56, and 58) should be included in the calculation.11. Calculation and Report11.1 After identifying the peaks, measure the areas of thepeaks identified as glycerin, mono-, di-, and trigl
41、yceride. Usingthe slope and y-intercept of the calibration functions, calculatethe mass of each as follows:11.1.1 Glycerin:G 5FWis1agGSFAgAisG2 bgDF100WG(7)where:G = mass percentage of glycerin in sample,Ag= peak area of glycerin,Ais1= peak area of Internal Standard 1,Wis1= weight of Internal Standa
42、rd 1, mg,W = weight of sample, mg,ag= slope of the calibration function,bg= intercept of the calibration function.11.1.2 Individual Glyceride:Glj5FWis2aolGSFAgljAis2G2 bo1DF100WG(8)where:Glj= mass percentage of individual glyceride in sample,Aglj= peak area of individual glyceride,Ais 2= peak area o
43、f Internal Standard 2,Wis2= weight of Internal Standard 2, mg,TABLE 3 Standard SolutionsStandard Solution Number 1 2 3 4 5L of glycerin stock solution 10 30 50 70 100L of monoolein stock solution 20 50 100 150 200L of diolein stock solution 10 20 40 70 100L of triolein stock solution 10 20 40 70 100
44、L of butanetriol stock solution 100 100 100 100 100L of tricaprin stock solution 100 100 100 100 100TABLE 4 Approximate Relative Retention TimesComponent Use InternalStandardRelative RetentionTimeGlycerin 1 0.851,2,4 Butanetriol 1.00Internal Standard 1Monopalmitin 2 0.76Monoolein, monolinolein 2 0.8
45、3-0.86monolinolenin, and monostearinTricaprin 1.00Internal Standard 2Diglycerides 2 1.05-1.09Triglycerides 2 1.16-1.31D6584 1313W = weight of sample, mg,aol= slope of the calibration function for mono, di-, ortriolein, andbol= intercept of the calibration function for mono, di, ortriolein.11.1.3 Cal
46、culation of Total Glycerin:total glycerin 5 free glycerin1bound glycerin (9)where:free glycerin = glycerin determined in Eq 7,bound glycerin = (GlM,GlD,GlT)where:GlM= 0.2591 monoglyceride, mass % determined inEq 8,GlD= 0.1488 diglyceride, mass % determined in Eq 8,andGlT= 0.1044 triglyceride, mass %
47、 determined in Eq 8.11.2 Report the total monoglyceride, total diglyceride, totaltriglyceride, and free and total glycerin to the nearest 0.001mass %.12. Precision and Bias12.1 The precision of this procedure, as determined bystatistical examination of the 2012 interlaboratory test results,4obtained
48、 from 11 laboratories on 14 B-100 biodiesel samplesfrom a variety of sources, is as follows:12.1.1 RepeatabilityThe difference between successiveresults obtained by the same operator with the same apparatusunder constant operating conditions on identical test material,would in the long run, in the n
49、ormal and correct operation ofthe test method, exceed the following values in on case intwenty.12.1.1.1 Total Glycerin Repeatability:r 5 0.76E201*TG0.73(10)where:TG = the calculated result for total glycerin expressed as amass % of the glycerin content in the sample, andr = repeatability.12.1.1.2 Free Glycerin Repeatability:r 5 0.195E201*FG10.0001!0.27(11)where:FG = the calculated result for free glycerin expressed as amass % of the glycerin content in the sample, andr = repeatability.12.1.1.3 Total Monoglyce
