1、Designation: D6584 10Standard Test Method forDetermination of Total Monoglyceride, Total Diglyceride,Total Triglyceride, 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 the d
2、esignation 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.1. Scope*1.1 This test method covers th
3、e 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 0.100to 1.000 mass %, for diglyceride is 0.050 to 0.500 mass %, andfor triglyceride is 0
4、.050 to 0.500 mass %. The range ofquantitation for free glycerin is 0.005 to 0.05 mass % and fortotal glycerin from 0.05 to 0.5 mass %. This procedure is notapplicable to vegetable oil methyl esters obtained from lauricoils, such as coconut oil and palm kernel oil.1.2 The values stated in SI units a
5、re 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 use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practice
6、s 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 StandardsE355 Practice for Gas Chromatography Terms and Rela-tionshipsE594 Practice for Testing Flame Ionization D
7、etectors 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 vegetable oils oranimal fats.3.1.2 bonded glycerin, nglycerin portion of the mono-,di-, and triglyceride molecule
8、s.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, terms, and relationships. Detaileddefinitions can be found in Practices E355 and E594.4. Summary of Test Meth
9、od4.1 The sample is analyzed by gas chromatography, aftersilyating with N-methyl-N-trimethylsilyltrifluoracetamide(MSTFA). Calibration is achieved by the use of two internalstandards and four reference materials. Mono-, di-, and trig-lyceride are determined by comparing to monoolein, diolein,and tri
10、olein standards respectively. Average conversion factorsare applied to mono-, di-, and triglycerides to calculate thebonded glycerin content of the sample.5. Significance and Use5.1 Free and bonded glycerin content reflects the quality ofbiodiesel. A high content of free glycerin may cause problemsd
11、uring 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 atinjection nozzles, pistons, and valves.6. Apparatus6.1 Chromatographic SystemSee Practice E355 for spe-cific design
12、ations 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 % phenylpoly-dimethylsiloxane bonded and cross linked phase internal coat-ing. The column should have an upper temperature limit o
13、f atleast 400C. Columns, 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 is1This test method is under the jurisdiction of ASTM Commit
14、teeD02 onPetroleum Products and Lubricants and is the direct responsibility of D02.04.0L onGas Chromatography Methods.Current edition approved Aug. 1, 2010. Published August 2010. Originallyapproved in 2000. Last previous edition approved in 2008 as D658408. DOI:10.1520/D6584-10.2For referenced ASTM
15、 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.1*A Summary of Changes section appears at the end of this standard.Copyright ASTM
16、 International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.recommended thata2to5metre 0.53 mm high temperatureguard column be installed from the injector to the analyticalcolumn. This allows the use of autoinjectors and also increasescolumn life.6.2 Electroni
17、c Data Acquisition System:6.2.1 Integrator or Computer, capable of providing realtime graphic and digital presentation of the chromatographicdata is recommended for use. Peak areas and retention timesshall be measured by computer or electronic integration.6.2.2 This device must be capable of perform
18、ing multilevelinternal-standard-type calibrations and be able to calculate thecorrelation coefficient (r2) and internal standard calculationsfor each data set.7. Reagents and Materials7.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended
19、thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.3Other grades may be usedprovided it is first ascertained that the reagent is of sufficientpurity to permit its use without lessening the accu
20、racy 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 or helium of high purity. Addi-tional purification is recommended by the use of molecularsieves or other suitable agents t
21、o 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 Screw Cap Vials, with polytetrafluoroethylene (PTFE)-faced septa, 10 mL capacity.8. Preparation of Apparatus8.1 Install and c
22、ondition the column in accordance withmanufacturer or suppliers instructions. After conditioning,attach column outlet to flame ionization detector inlet andcheck for leaks throughout the system. If leaks are found,tighten or replace fittings and recheck for leaks before proceed-ing.9. Calibration an
23、d Standardization9.1 Preparation of Calibration StandardsPrepare stan-dards using fresh compounds listed in Table 2 according toPractice D4307. Weigh the components directly into thevolumetric flasks specified and record the mass to the nearest0.1 mg. Dilute the volumetric flasks to mark with pyridi
24、ne.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 means ofmicrolitre syringes to 10 mL septa vials.Add to each of the fivestandard solutions 100 L of MSTFA. Close the vial
25、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 automatic sam-pler, transfer an aliquot of the solution into a glass GC vial andseal with a TFE-fluorocarbonlined cap.9.4 Standardizat
26、ionAnalyze 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 the analysis. Obtain a chromatogramand peak integration report. For each reference substance,determine the response rat
27、io (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.amti5 Wi/Ws! (2)where:Wi= mass of reference substance, andWs= mass of internal standard.9.4.1 Prepare a calibration curve for each reference co
28、mpo-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 should be at least 0.99 or greater. If the above criteria for3Rea
29、gent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the American Chemical Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole Dorset, U.K., and the United States Pharmacopeia an
30、dNational Formulary, 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 minDefect
31、orType Flame ionizationTemperature 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-octadecenoylg
32、lycerol(diolein)2465-32-9 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 102r2are not met, rerun the calibration or check instrumentpara
33、meters 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 = average values for all rspidata points.9.6 Calibration FunctionsFor each reference calibrationfunctions are calculated i
34、n the form:AxAis5FaxSWxWisDG1 bx(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 standard,ax= slope of the calibration function, andbx= intercept of the calibration function.10. Procedure10.1 Set the instr
35、ument 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 exactly 100 L of each internalstandard and MSTFA. Shake the vials, and allow to set for 15to 20 min at room temperatu
36、re. 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 and peak integration report.10.3 Peak IdentificationIdentify peaks by comparison ofretention times to the standards.
37、 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 according to carbon numbers (CN).10.4 Monoglyceride consists of the four overlapping peakswith relative retention times
38、 (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 should not be included in thecalculation of monoglyceride.10.5 Diglyceride is also primarily separated according tocarbon
39、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 be attributed to diglyceride. Carbon number alsoseparates triglyceride. Peaks with RRT of 1.16 to 1.31 (CN 52,54, 56
40、, 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 triglyceride. Usingthe slope and y-intercept of the calibration functions, calculatethe mass of each as follows:11.1.1 Gly
41、cerin:G 5FWis1agGSFAgAisG 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 Standard 1, mg,W = weight of sample, mg,ag= slope of the calibration function,bg= intercept of the calibration function.11.1
42、.2 Individual Glyceride:Glj5FWis2aolGSFAgljAis2G bo1DF100WG(8)where:Glj= mass percentage of individual glyceride in sample,Aglj= peak area of individual glyceride,Ais2= peak area of Internal Standard 2,Wis2= weight of Internal Standard 2, mg,W = weight of sample, mg,aol= slope of the calibration fun
43、ction for mono, di-, ortriolein, andbol= intercept of the calibration function for mono, di, ortriolein.11.1.3 Calculation of Total Glycerin:total glycerin 5 free glycerin 1 bound glycerin (9)where:free glycerin = glycerin determined in Eq 7,bound glycerin = ( (GlM,GlD,GlT)TABLE 3 Standard Solutions
44、Standard 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 100L of butanetriol stock solution 100 100 100 100 100L of tricaprin stock solution 100 100
45、 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 0.83-0.86monolinolenin, and monostearinTricaprin 1.00Internal Standard 2Diglyceride 2 1.05-
46、1.09Triglyceride 2 1.16-1.31D6584 103where:GlM= 0.2591 3(monoglyceride, mass % determined inEq 8,GlD= 0.1488 3(diglyceride, mass % determined in Eq8, andGlT= 0.1044 3(triglyceride, mass % determined in Eq8.11.2 Report the total monoglyceride, total diglyceride, totaltriglyceride, and free and total
47、glycerin to the nearest 0.001mass %.12. Precision and Bias12.1 The precision of this procedure, as determined bystatistical examination of the 2006 interlaboratory test results,4obtained from 12 laboratories on 12 B-100 biodiesel samplesfrom a variety of sources, is as follows:4Supporting data have
48、been filed at ASTM International Headquarters and maybe obtained by requesting Research Report RR:D02-1603.FIG. 1 Reference ChromatogramsD6584 10412.1.1 RepeatabilityThe difference between successiveresults obtained by the same operator with the same apparatusunder constant operating conditions on i
49、dentical test material,would in the long run, in the normal and correct operation ofthe test method, exceed the following values in on case intwenty.12.1.1.1 Total Glycerin Repeatability:r 5 5.405E202* TG 1 0.5164! (10)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 2.339E202* FG 11.000E204!0.4888(11)FG = the calculated result for free glycerin expressed as amass % of the glycerin content in the sample, an
