1、Designation: D6584 10a1D6584 13Standard 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 fo
2、llowing 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 NOTECorrected formatting
3、 and spacing in Eqs 12-19 and added previous Summary of Changes editorially in March2011.1. 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 ran
4、ge of quantitation for monoglyceride is 0.1000.009 to1.0000.77860 mass %, for diglyceride is 0.0500.092353 to 0.5000.54475 mass %, and for triglyceride is 0.0500.00092857 to0.5001.3881 mass %. The range of quantitation for free glycerin is 0.0050.0005714 to 0.050.019533 mass % and for total glycerin
5、from 0.050.0090714 to 0.50.42767 mass %. This procedure is not applicable to vegetable oil methyl esters obtained from lauricoils, such as coconut oil and palm kernel oil.1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.3
6、 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 of regulatorylimitations prior to use.2. Referenced Docume
7、nts2.1 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 Gas or Supercritical Fluid Chromatography3. Terminology3.1 Definitions:3.1.1
8、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 Definitions of Terms Specific to This Standard:3.2.1 total glycerin, nsum of free and
9、 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 E594.4. Summary of Test Method4.1 The sample is analyzed by gas chromatography, after silyating with N-methyl-N-trimet
10、hylsilyltrifluoracetamide (MSTFA).Calibration is achieved by the use of two internal standards and four reference materials. Mono-, di-, and triglyceride are1 This test method is under the jurisdiction of ASTM Committee D02 on Petroleum Products and Lubricants and is the direct responsibility of D02
11、.04.0L on GasChromatography Methods.Current edition approved Nov. 1, 2010May 1, 2013. Published January 2011May 2013. Originally approved in 2000. Last previous edition approved in 2010 asD658410.10a1. DOI: 10.1520/D6584-10A.10.1520/D6584-13.2 For referencedASTM standards, visit theASTM website, www
12、.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 ASTM website.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of wha
13、t changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the
14、 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, PA 19428-2959. United States1determined by comparing to monoolein, diolein, and triolein standards respectively. Average conversion
15、 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 biodiesel.Ahigh content of free glycerin may cause problems duringstorage, or in the fuel system, due to sep
16、aration 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 Chromatographic SystemSee Practice E355 for specific designations and definitions.6.1.1 Gas Chromatograph
17、(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.Thecolumn should have an upper temperature limit of at least 400C. Columns, either 10 m or 15 m in len
18、gth, with a 0.32 mm internaldiameter, and a 0.1 m film thickness have been found satisfactory. Any column with better or equivalent chromatographicefficiency and selectivity can be used. It is recommended that a 2 to 5 metre 0.53 mm high temperature guard column be installedfrom the injector to the
19、analytical column. This allows the use of autoinjectors and also increases column life.6.2 Electronic Data Acquisition System:6.2.1 Integrator or Computer, capable of providing real time graphic and digital presentation of the chromatographic data isrecommended for use. Peak areas and retention time
20、s shall be measured by computer or electronic integration.6.2.2 This device must be capable of performing multilevel internal-standard-type calibrations and be able to calculate thecorrelation coefficient (r2) and internal standard calculations for each data set.7. Reagents and Materials7.1 Purity o
21、f ReagentsReagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that allreagents conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society where suchspecifications are available.3 Other grades may be used provided
22、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, reagent grade.3 Reagent Chemicals, American Chemical Society Specifications, American Chemical Society, Washington, DC. For Suggestions on the testing
23、of reagents not listed bythe American Chemical Society, 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
24、 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/minTABLE 1 Operating Cond
25、itionsInjectorCool 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
26、mL/minD6584 1327.3 N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), reagent grade.7.4 Pyridine, reagent grade.7.5 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 hydrocarbon
27、s.Available pressure must be sufficient to ensure a constant carriergas 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 condition the column in accordance with
28、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 Standardization9.1 Preparation of Ca
29、libration 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 calibration standards in
30、 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 septa vials. Add to each of the five standard solutions 100 L of MSTFA. Close the vial and shake.Allow the vial to stand
31、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 sampler, transfer an aliquot of the solution into a glass GC vial and sealwith a TFE-fluorocarbonlined cap.9.4 StandardizationAnalyze the calibration standar
32、ds under the same operating conditions as the sample solutions. Inject 1L 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, determine the response ratio (rspi) and amount ratio (amti)
33、 for each componentusing Eq 1 and 2.rspi 5Ai/As! (1)where:Ai = area of reference substance, andTABLE 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(diol
34、ein)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 10TABLE 2 Stock SolutionsCompound CAS No.ApproximateMass (mg)VolumetricFlask Size(m
35、L)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 (tric
36、aprin) -(Internal Standard 2)621-71-6 80 10D6584 133As = area of internal standard.amti 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 th
37、eamount ratios (amti), as the x-axis.9.5 Calculate the correlation coefficient 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 andha
38、rdware.r25 (xy!2(x2!(y2! (3)where:x 5Xi 2x (4)x 5Xi 2x (4)y 5Yi 2y (5)y 5Yi 2y (5)and:Xi = amti 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 function
39、s are calculated in the form:AxAis 5FaxSWxWisDG1bx (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. Pr
40、ocedure10.1 Set the instrument operating variables to the values specified in Table 1. Weigh to the nearest 0.1 mg approximately 100mg of sample directly into a 10 mLsepta vial. Using microlitre syringes, add exactly 100 Lof each internal standard and MSTFA.Shake the vials, and allow to set for 15 t
41、o 20 min at room temperature.Add approximately 8 mLof n-Heptane to the vial and shake.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
42、solution 10 20 40 70 100L of butanetriol stock solution 100 100 100 100 100L of tricaprin stock solution 100 100 100 100 100TABLE 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 soluti
43、on 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 13410.2 Inject 1 L of the reaction mixture into the cool on-column injection port and start the analysis. Obtain a chromatogramand p
44、eak integration report.10.3 Peak IdentificationIdentify peaks by comparison of retention times to the standards. 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 accord
45、ing to carbon numbers (CN).10.4 Monoglyceride consists of the four overlapping peaks with relative retention times (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 no
46、t be included in the calculation of monoglyceride.10.5 Diglyceride 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) shall
47、be attributed to diglyceride. Carbon number also separates triglyceride. 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 trig
48、lyceride. Using theslope and y-intercept of the calibration functions, 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 Intern
49、al Standard 1, mg,W = weight of sample, mg,ag = slope of the calibration 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,TABLE 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 1.00
