1、Designation: D6584 10aStandard 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.1. Scope*1.1 This test method cover
3、s the quantitative determinationof total monoglycerides, total diglycerides, total triglycerides,and free and total glycerin in B-100 methyl esters by gaschromatography. The range of detection for free glycerin is0.005 to 0.05 mass %, and total glycerin from 0.05 to 0.5 mass%. This procedure is not
4、applicable to vegetable oil methylesters obtained from lauric oils, such as coconut oil and palmkernel 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 concer
5、ns, 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 applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D4307 Practice for Preparation of Liquid
6、Blends for Use asAnalytical StandardsE355 Practice for Gas Chromatography Terms and Rela-tionshipsE594 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 ch
7、ain fatty acids derived from vegetable oils oranimal fats.3.1.2 bonded glycerin, nglycerin portion of the mono-,di-, and triglyceride molecules.3.1.3 total glycerin, nsum of free and bonded glycerin.3.2 This test method makes reference to many common gaschromatographic procedures, terms, and relatio
8、nships. 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 four reference materia
9、ls. Mono-, di-, and trig-lycerides are determined by comparing to monoolein, diolein,and triolein standards respectively. Average conversion factorsare applied to the mono-, di-, and triglycerides to calculate thebonded glycerin content of the sample.5. Significance and Use5.1 Free and bonded glycer
10、in 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 atinjection nozzles,
11、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 % phenylpoly-dimethylsiloxane bond
12、ed and cross linked phase internal coat-ing. The column should have an upper temperature limit of 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 efficie
13、ncy and selectivity can be used. It isrecommended 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.1This test method is under the jurisdiction of ASTM CommitteeD02 onPetroleu
14、m Products and Lubricants and is the direct responsibility of D02.04.0L onGas Chromatography Methods.Current edition approved Nov. 1, 2010. Published January 2011. Originallyapproved in 2000. Last previous edition approved in 2010 as D658410a. DOI:10.1520/D6584-10A.2For referenced ASTM standards, vi
15、sit 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 International
16、, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.6.2 Electronic 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 timess
17、hall be measured 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 Materials7.1 Purity of Rea
18、gentsReagent 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 be usedprovided it is fir
19、st 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 or helium of high purit
20、y. 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 Screw Cap Vials, with poly
21、tetrafluoroethylene (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 throughout the system. If lea
22、ks 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 thevolumetric flasks s
23、pecified 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 means ofmicrolitre syri
24、nges to 10 mL septa vials.Add to each of the fivestandard solutions 100 L of 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 automatic sam-pler, transfer an ali
25、quot of the solution into a glass GC vial andseal 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 the analysis. Obtain
26、 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.amti5 Wi/Ws! (2)where:Wi= mass of reference
27、substance, andWs= mass of internal standard.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 calibr
28、ation set using Eq 3. The r2value should 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:3Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington,
29、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, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.TABLE 1 Operating
30、 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 ionizationTemperature 380CCarrier GasType Hydrogen or helium measured at 50CFlow ra
31、te 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)2465-32-9 50 101,2,3-Tri cis-octadecenoylglycerol(triolein)122-32-7 50 10(S) - (
32、-) -1,2,4-Butanetriol - (InternalStandard 1)42890-76-6 25 251,2,3-Tridecanolylglycerol (tricaprin) -(Internal Standard 2)621-71-6 80 10D6584 10a2x 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 a
33、ll rspidata points.9.6 Calibration FunctionsFor each reference calibrationfunctions are calculated in 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 th
34、e 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 exactly 100 L of
35、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 and peak integra
36、tion 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. The mono-, di, andtriglycerides are separated according to car
37、bon numbers (CN).10.4 Monoglycerides consist 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 should not be inclu
38、ded in thecalculation of monoglycerides.10.5 Diglycerides are 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 be attribu
39、ted to diglycerides. Carbon number alsoseparates triglycerides. 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 triglycerides.
40、Usingthe slope and y-intercept of the calibration functions, calculatethe mass of each as follows:11.1.1 Glycerin: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
41、 = weight of sample, mg,ag= slope of the calibration function,bg= intercept of the calibration function.11.1.2 Individual Glycerides:Glj5FWis2aolGSFAgljAis2G bo1DF100WG(8)where:Glj= mass percentage of individual glycerides in sample,Aglj= peak area of individual glyceride,Ais2= peak area of Internal
42、 Standard 2,Wis2= weight of Internal Standard 2, mg,W = 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 Calculation of Total Glycerin:total glycerin 5 free glycerin 1 bound glycerin (
43、9)where:free glycerin = glycerin determined in Eq 7,bound glycerin = ( (GlM,GlD,GlT)where: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 monoglycerides, total dig
44、lycerides,total triglycerides, and free and total glycerin to the nearest0.001 mass %.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
45、solution 10 20 40 70 100L 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 0.76Monoo
46、lein, monolinolein 2 0.83-0.86monolinolenin, and monostearinTricaprin 1.00Internal Standard 2Diglycerides 2 1.05-1.09Triglycerides 2 1.16-1.31D6584 10a312. Precision and Bias12.1 The precision of this procedure, as determined bystatistical examination of the 2006 interlaboratory test results,4is as
47、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 normal and correct operation ofthe test method, exceed the following values in on cas
48、e intwenty.12.1.1.1 Total Glycerin Repeatability:r 5 5.405E202* X 1 0.5164! (10)X = calculated result in mass %, andr = repeatability.12.1.1.2 Free Glycerin Repeatability:r 5 2.339E202* X 11.000E204!0.4888(11)X = calculated result in mass %, andr = repeatability.12.1.1.3 Total Monoglyceride Repeatab
49、ility:r 5 0.1452 * X0.6995(12)12.1.1.4 Total Diglyceride Repeatability:r 5 0.1304 * X 1 1.310E02!0.3784(13)12.1.1.5 Total Triglyceride Repeatability:r 5 0.3528 * X 1 9.100E03!0.5780(14)12.1.2 ReproducibilityThe difference between two singleand independent results, obtained by different operators work-ing in different laboratories on identical material, would in thelong run, in the normal and correct operation of the testmethod, exceed the following values only in one case intwenty.12.1.2.1 Total Glycerin Reproducibility:R
