1、Designation: D 6584 08An American National StandardStandard Test Method forDetermination of Free and Total Glycerin in B-100 BiodieselMethyl Esters by Gas Chromatography1This standard is issued under the fixed designation D 6584; the number immediately following the designation indicates the year of
2、original 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 the quantitative determinationof f
3、ree 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 applicable to vegetable oil methylesters obtained from lauric oils, such as coconut oil and palmkernel
4、 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 use. It is theresponsibility of the user of this standard to establish
5、 appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 4307 Practice for Preparation of Liquid Blends for Use asAnalytical StandardsE 355 Practice for Gas Chromatography Terms and Rela-tionshipsE
6、594 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 vegetable oils oranimal fats.3.1.2 bonded glycerin, nglycerin portion
7、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 relationships. Detaileddefinitions can be found in Practices E 355 and E 594.4. Summary of Test Method4.1
8、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-lycerides are determined by comparing to monoolein, diolein,and triolein
9、 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 glycerin content reflects the quality ofbiodiesel. A high content of free glycerin may cause problemsdu
10、ring 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 E 355 for spe-cific design
11、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
12、f at1This test method is under the jurisdiction of ASTM CommitteeD02 onPetroleum Products and Lubricants and is the direct responsibility of D02.04.0L onGas Chromatography Methods.Current edition approved Oct. 15, 2008. Published November 2008. Originallyapproved in 2000. Last previous edition appro
13、ved in 2007 as D 658407.2For 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.TABLE 1 Operating ConditionsInjectorC
14、ool 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 rate 3 mL/min1*A Summa
15、ry of Changes section appears at the end of this standard.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.least 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 s
16、atisfactory. Any column with better or equivalentchromatographic efficiency 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.6.2
17、 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 timesshall be measured by computer or electronic integration.6.2.2 This device must be capable
18、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 ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is
19、 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 first ascertained that the reagent is of sufficientpurity to permit its use without lessenin
20、g 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 purity. Addi-tional purification is recommended by the use of molecularsieves or other suitabl
21、e 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 polytetrafluoroethylene (PTFE)-faced septa, 10 mL capacity.8. Preparation of Apparatus8.1 Ins
22、tall 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 leaks are found,tighten or replace fittings and recheck for leaks before proceed-ing.9. Cali
23、bration and Standardization9.1 Preparation of Calibration StandardsPrepare stan-dards using fresh compounds listed in Table 2 according toPractice D 4307. Weigh the components directly into thevolumetric flasks specified and record the mass to the nearest0.1 mg. Dilute the volumetric flasks to mark
24、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 syringes to 10 mL septa vials.Add to each of the fivestandard solutions 100 L of MSTFA. Clos
25、e 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 aliquot of the solution into a glass GC vial andseal with a TFE-fluorocarbonlined cap.9.4 S
26、tandardizationAnalyze 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 r
27、esponse 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 substance, andWs= mass of internal standard.9.4.1 Prepare a calibration curve for each r
28、eference 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 should be at least 0.99 or greater. If the above crite
29、ria 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)3Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by th
30、e American Chemical Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole Dorset, U.K., and the United States Pharmacopeia andNational Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.TABLE 2 Stock SolutionsCompound CAS No.ApproximateMass (mg)VolumetricFlask Size
31、(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) - (-) -1,2,4-Butanetriol - (InternalStandard 1)42890-76-6 25 251,2,3-Tridecanolylglycerol (tr
32、icaprin) -(Internal Standard 2)621-71-6 80 10D6584082and: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 in the form:AxAis5F
33、axSWxWisDG1 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 instrument operating va
34、riables 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 temperature. Add approximat
35、ely 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. For identificatio
36、n 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 carbon numbers (CN).10.4 Monoglycerides consist of the four overlapping peakswith relative retention times (RRT) of 0.76
37、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 monoglycerides.10.5 Diglycerides are also primarily separated according tocarbon number, but
38、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 diglycerides. Carbon number alsoseparates triglycerides. Peaks with RRT of 1.16 to 1.31 (CN 52,54, 56, and 58)
39、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. Usingthe slope and y-intercept of the calibration functions, calculatethe mass of each as follows:11.1.1 Glycerin:G 5F
40、Wis1agGSFAgAisG 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.11.1.2 Individ
41、ual Glycerides:Glj5FWis2aolGSFAgljAis2G bo1DF100WG(8)where:Glj= mass percentage of individual glycerides 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 function fo
42、r 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)where:GlM= 0.2591 3(monoglyceride,
43、 mass % determined inEq 8,GlD= 0.1488 3(diglyceride, mass % determined in Eq8, andGlT= 0.1044 3(triglyceride, mass % determined in Eq8.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 s
44、tock 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 100 100 100 100 100TABLE 4 Approximate Relative Retention TimesComponent Use InternalStandardRelative RetentionTimeGlycerin 1 0.851,2,4 Butanetrio
45、l 1.00Internal Standard 1Monopalmitin 2 0.76Monoolein, monolinolein 2 0.83-0.86monolinolenin, and monostearinTricaprin 1.00Internal Standard 2Diglycerides 2 1.05-1.09Triglycerides 2 1.16-1.31D658408311.2 Report the free and total glycerin to the nearest 0.001mass %.12. Precision and Bias12.1 The pre
46、cision of this procedure, as determined bystatistical examination of the 2006 interlaboratory test results,4is 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,w
47、ould 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* X 1 0.5164! (10)X = calculated result in mass %, and4Supporting data have been filed at ASTM International Headquarters an
48、d maybe obtained by requesting Research Report RR: D021603.FIG. 1 Reference ChromatogramsD6584084r = 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.12.1.2 ReproducibilityThe difference between two singlea
49、nd 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 5 0.4928 * X 1 2.510E202! (12)X = calculated result in mass %, andR = reproducibility.NOTE 1The total data (with no common transform) precision in theD2PP output includes a caveat that the precision is only applicable above0.13. The other sa
