1、Designation: D 6584 07An 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 (e) indicates an editorial change since the last revision or reapproval.1. Scope*1.1 This test method covers the quantitative determinationof
3、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 applicable to vegetable oil methylesters obtained from lauric oils, such as coconut oil and palmkerne
4、l 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 establis
5、h 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, nis the glycerin
7、portion of themono-, di-, and triglyceride molecules.3.1.3 total glycerin, nis the sum of free and bondedglycerin.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 T
8、est 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 materials. Mono-, di-, and trig-lycerides are determined by comparing to monoolein, diolei
9、n,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 glycerin content reflects the quality ofbiodiesel. A high content of free glycerin may ca
10、use 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, pistons, and valves.6. Apparatus6.1 Chromatographic SystemSee Practice E 355 for sp
11、e-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 bonded and cross linked phase internal coat-ing. The column should have an upper tempe
12、rature 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 efficiency and selectivity can be used. It isrecommended thata2to5metre 0.53 mm high temp
13、erature1This 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 Jan. 1, 2007. Published February 2007. Originallyapproved in 2000. Last previous edition app
14、roved in 2000 as D 658400e1.2For 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.1*A Summary of Changes section ap
15、pears at the end of this standard.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.guard column be installed from the injector to the analyticalcolumn. This allows the use of autoinjectors and also increasescolumn life.6.2 Electronic D
16、ata 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 performing
17、 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 tha
18、tall 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 accurac
19、y 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 to r
20、emove 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 cond
21、ition 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 and S
22、tandardization9.1 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 with pyridine
23、.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 an
24、d 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 Standardizatio
25、nAnalyze 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 ratio
26、 (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 comp
27、o-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 forr2are
28、not met, rerun the calibration or check instrumentparameters and hardware.3Reagent 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 Laboratory
29、Chemicals, BDH Ltd., Poole Dorset, U.K., and the United States Pharmacopeia andNational 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 1
30、5C / 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/minTABLE 2 Stock SolutionsCompound CAS No.ApproximateMass (mg)VolumetricFlask Size(mL)Glycerin 56-81-5 25 501-Mono cis
31、-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 (tricaprin) -(Internal Standard 2)621-7
32、1-6 80 10D6584072r25( xy2#( 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
33、in the form:Wx/Wis5 ax3 Ax/Ais! 1 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 i
34、nstrument 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 tempe
35、rature. 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 standa
36、rds. 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 carbon numbers (CN).10.4 Monoglycerides consist of the four overlapping peakswith relative retentio
37、n 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 included in thecalculation of monoglycerides.10.5 Diglycerides are also primarily separated according
38、 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 attributed to diglycerides. Carbon number alsoseparates triglycerides. Peaks with RRT of 1.16 to 1.31 (
39、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. Usingthe slope and y-intercept of the calibration functions, calculatethe mass of each as follow
40、s:11.1.1 Glycerin:G 5 ag3 Ag/Ais11 bg! 3 Wis13 100/W (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 calibra
41、tion function.11.1.2 Individual Glycerides:Gli5 ao3 Agli/Ais21 bo1! 3 Wis23 100/W (8)where:Gli= mass percentage of individual glycerides in sample,Agli= peak area of individual glyceride,Ais2= peak area of Internal Standard 2,Wis2= weight of Internal Standard2,mg,W = weight of sample, mg,aol= slope
42、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 (9)where:free glycerin = glycerin determined in Eq 7,bound glycerin = ( (GlM,GlD,GlT)wher
43、e: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.TABLE 3 Standard SolutionsStandard Solution Number 1 2 3 4 5L of glycerin stock solution 10 30 50 70 100L of monoolein stock solution 2
44、0 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 100 100 100 100 100TABLE 4 Approximate Relative Retention TimesComponent Use InternalStandardRelative RetentionTimeG
45、lycerin 1 0.851,2,4 Butanetriol 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 1.16-1.31D658407311.2 Report the free and total glycerin to the nearest 0.001mass %.12.
46、Precision and Bias12.1 The precision 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 condition
47、s on identical 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* X 1 0.5164! (10)X = calculated result in mass %, and4Supporting data have been filed at ASTM
48、 International Headquarters and maybe obtained by requesting Research Report RR: D021603.FIG. 1 Reference ChromatogramsD6584074r = 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
49、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 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 ap
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