1、Designation: D6584 131D6584 17Standard 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 fol
2、lowing the 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 NOTEThe formats of Eq 11
3、and Eq 16 were editorially corrected in April 2014.1. Scope*1.1 This test method covers the quantitative determination of total monoglyceride, total diglyceride, total triglyceride, and freeand total glycerin in B-100 methyl esters by gas chromatography. The range of quantitation for monoglyceride i
4、s 0.009 to 0.77860mass %,0.009 % to 0.77860 % by mass, for diglyceride is 0.092353 to 0.54475 mass %,% to 0.54475 % by mass, and fortriglyceride is 0.00092857 to 1.3881 mass %.0.00092857 % to 1.3881 % by mass. The range of quantitation for free glycerin is0.00057140.0005714 % to 0.019533 0.019533 %
5、by mass % and for total glycerin from 0.0090714 to 0.42767 mass%.0.0090714 % to 0.42767 % by mass. This procedure is not applicable to vegetable oil methyl esters obtained from lauric oils,such as coconut oil and palm kernel oil.1.2 The values stated in SI units are to be regarded as standard. No ot
6、her units of measurement are included in this standard.1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and determine the applicability
7、of regulatorylimitations prior to use.1.4 This international standard was developed in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Development of International Standards, Guides and Recommendations issuedby the World Trade
8、 Organization Technical Barriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2D4307 Practice for Preparation of Liquid Blends for Use as Analytical StandardsE355 Practice for Gas Chromatography Terms and RelationshipsE594 Practice for Testing Flame Ionization Detectors Used in
9、 Gas or Supercritical Fluid Chromatography3. Terminology3.1 Definitions:3.1.1 biodiesel (B-100), nfuel comprised of mono-alkyl esters of long chain fatty acids derived from vegetable oils or animalfats.3.1.2 bonded glycerin, nglycerin portion of the mono-, di-, and triglyceride molecules.3.2 Definit
10、ions 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 gas chromatographic procedures, terms, and relationships. Detaileddefinitions can be found in Practices E355 and E594.1 This test method is under the jur
11、isdiction of ASTM Committee D02 on Petroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility of D02.04.0Lon Gas Chromatography Methods.Current edition approved May 1, 2013June 1, 2017. Published May 2013July 2017. Originally approved in 2000. Last previous edition approved in
12、 20102013 asD6584D6584 1310a1. DOI: 10.1520/D6584-13E01.10.1520/D6584-17.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the A
13、STM website.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consu
14、lt prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.*A Summary of Changes section appears at the end of this standardCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken,
15、PA 19428-2959. United States14. Summary of Test Method4.1 The sample is analyzed by gas chromatography, after silyating with N-methyl-N-trimethylsilyltrifluoracetamide (MSTFA).Calibration is achieved by the use of two internal standards and four reference materials. Mono-, di-, and triglyceride ared
16、etermined by comparing to monoolein, diolein, and triolein standards respectively. Average conversion factors are applied tomono-, di-, and triglycerides to calculate the bonded glycerin content of the sample.5. Significance and Use5.1 Free and bonded glycerin content reflects the quality of biodies
17、el.Ahigh content of free glycerin may cause problems duringstorage, or in the fuel system, due to separation of the glycerin. A high total glycerin content can lead to injector fouling and mayalso contribute to the formation of deposits at injection nozzles, pistons, and valves.6. Apparatus6.1 Chrom
18、atographic SystemSee Practice E355 for specific designations and definitions.6.1.1 Gas Chromatograph (GC)The system must be capable of operating at the conditions given in Table 1.6.1.2 Column, open tubular column with a 5 % phenylpolydimethylsiloxane bonded and cross linked phase internal coating.T
19、hecolumn should have an upper temperature limit of at least 400C.400 C. Columns, either 10 m or 15 m 10 m or 15 m in length,with a 0.32 mm 0.32 mm internal diameter, and a 0.1 m 0.1 m film thickness have been found satisfactory. Any column withbetter or equivalent chromatographic efficiency and sele
20、ctivity can be used. It is recommended that a 2 to 5 metre 0.53 mm 2 mto 5 m-0.53 mm high temperature guard column be installed from the injector to the analytical column. This allows the use ofautoinjectors and also increases column life.6.2 Electronic Data Acquisition System:6.2.1 Integrator or Co
21、mputer, capable of providing real time graphic and digital presentation of the chromatographic data isrecommended for use. Peak areas and retention times shall be measured by computer or electronic integration.6.2.2 This device must be capable of performing multilevel internal-standard-type calibrat
22、ions and be able to calculate thecorrelation coefficient (r2) and internal standard calculations for each data set.7. Reagents and Materials7.1 Purity of ReagentsReagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that allreagents conform to the specificati
23、ons of the Committee on Analytical Reagents of the American Chemical Society where suchspecifications are available.3 Other grades may be used provided it is first ascertained that the reagent is of sufficient purity topermit its use without lessening the accuracy of the determination.7.2 n-Heptane,
24、 reagent grade.7.3 N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), reagent grade.7.4 Pyridine, reagent grade.3 Reagent Chemicals, American Chemical Society Specifications, American Chemical Society, Washington, DC. For Suggestions on the testing of reagents not listed bythe American Chemical So
25、ciety, see Annual Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and NationalFormulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.TABLE 1 Operating ConditionsInjectorCool on column injectionSample size 1 LColumn Temperature Progr
26、amInitial temperature 50C hold 1 minInitial temperature 50 C hold 1 minRate 1 15C / min to 180CRate 1 15 C / min to 180 CRate 2 7C / min to 230CRate 2 7 C / min to 230 CRate 3 30C / min 380C hold 10 minRate 3 30 C / min 380 C hold 10 minDefectorType Flame ionizationTemperature 380CTemperature 380 CC
27、arrier GasType Hydrogen or helium measured at 50CType Hydrogen or helium measured at50 CFlow rate 3 mL/minD6584 1727.5 Carrier Gas, hydrogen or helium of high purity. Additional purification is recommended by the use of molecular sieves orother suitable agents to remove water, oxygen, and hydrocarbo
28、ns.Available pressure must be sufficient to ensure a constant carriergas flow rate.7.6 Microlitre Syringes, 100 L and 250 L 100 L and 250 L capacity.7.7 Screw Cap Vials, with polytetrafluoroethylene (PTFE)-faced septa, 10 mL 10 mL capacity.8. Preparation of Apparatus8.1 Install and condition the col
29、umn in accordance with manufacturer or suppliers instructions. After conditioning, attachcolumn outlet to flame ionization detector inlet and check for leaks throughout the system. If leaks are found, tighten or replacefittings and recheck for leaks before proceeding.9. Calibration and Standardizati
30、on9.1 Preparation of Calibration StandardsPrepare standards using fresh compounds listed in Table 2 according to PracticeD4307. Weigh the components directly into the volumetric flasks specified and record the mass to the nearest 0.1 mg. Dilute thevolumetric flasks to mark with pyridine. Store the c
31、alibration standards in a refrigerator when not in use.9.2 Standard SolutionsPrepare the five standard solutions in Table 3 by transferring the specified volumes by means ofmicrolitre syringes to 10 mL 10 mL septa vials. Add to each of the five standard solutions 100 L 100 L of MSTFA. Close thevial
32、and shake. Allow the vial to stand for 1515 min to 20 min 20 min at room temperature. Add approximately 8 mL 8 mLn-Heptane to the vial and shake.9.3 Chromatographic AnalysisIf using an automatic sampler, transfer an aliquot of the solution into a glass GC vial and sealwith a TFE-fluorocarbonlined ca
33、p.9.4 StandardizationAnalyze the calibration standards under the same operating conditions as the sample solutions. Inject 1L 1 L of the reaction mixture into the cool on-column injection port and start the analysis. Obtain a chromatogram and peakintegration report. For each reference substance, det
34、ermine the response ratio (rspi) and amount ratio (amti) for each componentusing Eq 1 and 2.rspi 5Ai/As! (1)where:Ai = area of reference substance, andAs = area of internal standard.TABLE 2 Stock SolutionsCompound CAS No.ApproximateMass (mg)VolumetricFlask Size(mL)Glycerin 56-81-5 25 501-Mono cis-9-
35、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)621-71-6
36、 80 10D6584 173amti 5Wi/Ws! (2)where:Wi = mass of reference substance, andWs = mass of internal standard.9.4.1 Prepare a calibration curve for each reference component by plotting the response ratios (rspi), as the y-axis, versus theamount ratios (amti), as the x-axis.9.5 Calculate the correlation c
37、oefficient r2 value for each reference component in the calibration set using Eq 3. The r2 valueshould be at least 0.99 or greater. If the above criteria for r2 are not met, rerun the calibration or check instrument parameters andhardware.r25 (xy!2(x2!(y2! (3)where:x 5Xi 2x (4)y 5Yi 2y (5)and:Xi = a
38、mti ratio data point,x = average values for all amti data pointsYi = corresponding rspi data points,y = average values for all rspi data points.9.6 Calibration FunctionsFor each reference calibration functions are calculated in the form:AxAis 5FaxSWxWisDG1bx (6)where:Wx = mass of reference substance
39、, 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 variables to the values specified in Table 1. Weigh
40、 to the nearest 0.1 mg approximately 100mg 0.1 mg approximately 100 mg of sample directly into a 10 mL 10 mL septa vial. Using microlitre syringes, add exactly 100L 100 L of each internal standard and MSTFA. Shake the vials, and allow to set for 1515 min to 20 min 20 min at roomtemperature. Add appr
41、oximately 8 mL 8 mL of n-Heptane to the vial and shake.10.2 Inject 1 L 1 L of the reaction mixture into the cool on-column injection port and start the analysis. Obtain achromatogram and peak integration report.10.3 Peak IdentificationIdentify peaks by comparison of retention times to the standards.
42、 For identification of additionalpeaks, use the relative retention times given in Table 4 and the reference chromatograms given in Fig. 1. Mono-, di-, andtriglycerides are separated according to carbon numbers (CN).10.4 Monoglyceride consists of the four overlapping peaks with relative retention tim
43、es (RRT) of 0.76 and 0.83 to 0.86 withrespect to the internal standard tricaprin.Apair of peaks, methyl esters with a carbon number of 24, may appear with RRT of 0.80to 0.82, and should not be included in the calculation of monoglyceride.TABLE 3 Standard SolutionsStandard Solution Number 1 2 3 4 5L
44、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 100 100 100 100 100D6584 17410.5 Diglycerid
45、e is also primarily separated according to carbon number, but due to varying double bonds in the molecules,baseline resolution of the peaks does not occur. The grouping of 3 to 4 peaks with RRT of 1.05 to 1.09 (CN 34, 36, and 38) shallbe attributed to diglyceride. Carbon number also separates trigly
46、ceride. 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 the peaks identified as glycerin, mono-, di-, and triglyceride. Using theslope and y-intercept of the calibration funct
47、ions, calculate the mass of each as follows:11.1.1 Glycerin:G 5FWis1agGSF AgAisG 2bgDF 100W G (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 cali
48、bration function,bg = intercept of the calibration function.11.1.2 Individual Glyceride:Glj 5FWis2aolGSF AgljAis2G2bo1DF100W G (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 S
49、tandard 2, mg,W = weight of sample, mg,aol = slope of the calibration function for mono, di-, or triolein, andbol = intercept of the calibration function for mono, di, or triolein.11.1.3 Calculation of Total Glycerin:total glycerin5free glycerin1bound glycerin (9)total glycerin5free glycerin1bonded glycerin (9)where:free glycerin = glycerin determined in Eq 7,bound glycerin = (GlM, GlD, GlT)bonded glycerin = (GlM, GlD, GlT)where:GlM = 0.2591 monoglyceride, mass % determined in Eq 8,GlD = 0.1488
