1、Designation: E1821 08 (Reapproved 2015)Standard Test Method forDetermination of Carbohydrates in Biomass by GasChromatography1This standard is issued under the fixed designation E1821; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision
2、, 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.INTRODUCTIONThis test method gives a reproducible way to quantitatively determine in lignocellulosic materialsthe ki
3、nd and amount of the structural carbohydrates made from arabinose, xylose, mannose, galactose,and glucose. This way is accomplished by first hydrolyzing the carbohydrates to their constituentmonosaccharides. Subsequent derivatization produces the corresponding alditol acetates that arequantified usi
4、ng capillary gas chromatography.1. Scope1.1 This test method describes the determination of struc-tural carbohydrates present in a biomass sample, expressed asthe percent mass of an oven-dried sample basis of eachanhydrosugar.1.2 Sample materials suitable for this procedure includehard and softwoods
5、, herbaceous materials, such as sericea andswitchgrass, agricultural residues, such as corn stover, wheatstraw, and bagasse, wastepaper, such as boxboard, office waste,and newsprint, acid or alkaline-pretreated biomass, washedfree of any residual acid or alkali, and the solid fraction offermentation
6、 residues.1.3 The options for the types of samples to be analyzed inthis procedure are:1.3.1 Prepared Biomass Samples:1.3.1.1 Air Dried MaterialResults are reported as thepercent by mass, based on the oven-dried mass of the air-driedsample.1.3.1.2 45C Dried MaterialResults are reported as thepercent
7、 by mass, based on the oven-dried mass of the 45Cdried sample.1.3.1.3 Freeze Dried MaterialResults are reported as thepercent by mass, based on the oven-dried mass of the freezedried sample.1.3.2 Extractives-Free SampleResults are reported as thepercent by mass, based on the oven-dried mass of the e
8、xtractedsample.1.4 This standard method is generally not suitable forsamples that contain soluble, nonstructural carbohydrates un-less they are removed prior to analysis.1.5 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.6 Th
9、is 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 appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. See Section 8 forspe
10、cific hazards statements.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterE1690 Test Method for Determination of Ethanol Extrac-tives in BiomassE1721 Test Method for Determination of Acid-InsolubleResidue in BiomassE1756 Test Method for Determination of Total Solids in
11、BiomassE1757 Practice for Preparation of Biomass for Composi-tional Analysis3. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 anhydrosugars, nthe nominal repeating unit of apolysaccharide. When polysaccharides undergo acid1This test method is under the jurisdiction of ASTM Commi
12、ttee E48 onBioenergy and Industrial Chemicals from Biomass and is the direct responsibility ofSubcommittee E48.05 on Biomass Conversion.Current edition approved June 1, 2015. Published July 2015. Originally approvedin 1996. Last previous edition approved in 2008 as E1821-08. DOI: 10.1520/E1821-08R15
13、.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.Copyright ASTM International, 100 Barr Harbor Drive, PO Box
14、C700, West Conshohocken, PA 19428-2959. United States1hydrolysis, each repeating unit adds a single molecule of waterto form the free monosaccharide that is analyzed. The extraweight from this water of hydrolysis must be taken in toaccount whencalculating the actual mass percent of the poly-sacchari
15、de in the original biomass sample.3.1.2 as received biomass, nmaterial as it is received in itsfield or process collected state.3.1.3 extractives-free biomassair-dried solids left afterbiomass has been treated according to Test Method E1690.3.1.4 oven-dried mass, nthe moisture-free mass of anybiomas
16、s sample (as received, prepared, extractives-free, etc.)dried at 105C as described in Test Method E1756.3.1.5 prepared biomass, nas received biomass materialthat has been treated according to Practice E1757 in order toraise the total solids content above 85 %, based on an oven-dried solids weight.3.
17、1.6 structural carbohydrates, npolysaccharides thatcannot be removed by extraction with solvents and are liber-ated from the biomass solids with dilute acid hydrolysis. Forthe purpose of this test method, the monosaccharides that areconsidered present are arabinose, xylose, mannose, galactose,and gl
18、ucose.3.2 Abbreviations:3.2.1 %Anhydroextthe percent by mass of the anhydro-sugar on an extractives-free, oven-dried mass basis.3.2.2 %Anhydrowholethe percent by mass of theanhydrosugar, on an oven-dried mass basis.3.2.3 ARc(Amount Ratio)ratio of the concentration(amount) of monosaccharide c to the
19、concentration (amount) ofinternal standard in the specimen.3.2.4 areacreported area counts for the monosaccharide cpeak in the chromatogram, as integrated by the electronicintegrator.3.2.5 areaISreported area counts for the internal standardpeak in the chromatogram, as integrated by the electronicin
20、tegrator.3.2.6 Cavgaverage concentration of monosaccharide c inspecimen s, in mg/mL, averaged across multiple injections ofspecimen s.3.2.7 CLForiginal concentration of monosaccharide c inloss factor sample, in mg/mL.3.2.8 CISconcentration of internal standard (inositol) inthe calibration standards
21、and specimen, in mg/mL.3.2.9 Csconcentration of monosaccharide c in specimen s,measured by gas chromatography (GC), in mg/mL.3.2.10 CSTDconcentration of monosaccharide c in thecalibration standard, in mg/mL.3.2.11 CV (coeffcient of variation)the estimated standarddeviation divided by the average val
22、ue measured.3.2.12 %extractivesthe percentage by mass of extractivesin the extracted specimen as described in Test Method E1690.3.2.13 kconstant used to convert the mass of monosaccha-ride to the mass of anhydrosugar from which it is derived. Forarabinose and xylose, k = 0.88 (mz 132/150); for manno
23、se,galactose and glucose, k = 0.90 (m/z 162/180).3.2.14 LFloss factor for monosaccharide c. Used to cor-rect for the amount of monosaccharide lost through degrada-tion during acid hydrolysis of biomass.3.2.15 mIinitial mass of the biomass specimen, in mg.3.2.16 mcorrmass of monosaccharide in solutio
24、n, cor-rected for hydrolysis losses, in mg.3.2.17 RRavgaveraged response ratio of monosaccharide cto the internal standard (inositol) in the calibration standard.Derived from multiple injections of the same calibrationstandard.3.2.18 RRsresponse ratio of monosaccharide c to theinternal standard (ino
25、sitol) in the specimen.3.2.19 RRSTDresponse ratio of monosaccharide c to theinternal standard (inositol) in the calibration standard.3.2.20 RRF (Relative Response Factor of monosaccharidec)this is the ratio of the detector response for monosaccha-ride c versus the detector response for the internal
26、standard(inositol) for a given injection of the specimen.3.2.21 Vf87 mL, volume of hydrolysis solution.3.2.22 %T45percentage by mass, of total solids of thespecimen prepared by drying at 45C, as described by PracticeE1757.3.2.23 %T105percentage by mass, of total solids of thespecimen, dried at 105C,
27、 as determined by Test MethodE1756.3.2.24 %Tadpercentage by mass, of total solids of theair-dried specimen determined at 105C as described by TestMethod E1756.3.2.25 %Textpercentage by mass, of total solids of theextracted specimen determined at 105C as described by TestMethod E1756.3.2.26 %Tfdperce
28、ntage by mass, of total solids of thespecimen prepared by freeze drying, as described by PracticeE1757.3.2.27 %Tpreppercentage, by mass, of total solids of thespecimen prepared by freeze drying,% Tfd, or by drying at45C, %T45, as determined by Practice E1757.4. Significance and Use4.1 The structural
29、 carbohydrate content is used in conjunc-tion with other assays to determine the total composition ofbiomass samples.5. Interferences5.1 The results of structural carbohydrate analysis are af-fected by incomplete hydrolysis of biomass or hydrolysisconditions that are too severe. Incomplete hydrolysi
30、s will biasthe results low because dimeric and oligomeric carbohydratesare not quantified. Hydrolysis conditions that are too severedegrade the liberated monosaccharides into materials that arenot quantified by this procedure, again biasing the results low.5.2 Incomplete neutralization and removal o
31、f acetic acidfrom the methylene chloride extract prior to GC analysis canE1821 08 (2015)2result in ghost peaks appearing in the chromatogram orcarryover of monosaccharides from one injection to the next(owing to buildup of monosaccharides in the injection port),leading to erroneous quantitation.5.3
32、Test specimens not suitable for analysis by this proce-dure include alkaline and acid-pretreated biomass samples thathave not been washed. Unwashed pretreated biomass samplescontaining free acid or alkali may change visibly on heating.5.4 Materials containing nonstructural carbohydrates alsoare unsu
33、itable for this procedure since nonstructural carbohy-drates may undergo degradation to materials that are notquantified in this procedure.6. Apparatus6.1 Analytical Balance, readable to 0.1 mg.6.2 Autoclave, capable of maintaining 121 6 3C.6.3 Convection Ovens, temperature controlled to 45 6 3Cand
34、105 6 3C.6.4 Desiccator, containing anhydrous calcium sulfate.6.5 Gas Chromatograph, equipped with electronicintegrator, capillary split injection port, flame ionization detec-tor with make-up gas, 250 m 15 m fused-silica capillarycolumn coated with 50 % cyanopropylphenylmethylpolysiloxane, 0.25 m f
35、ilm thickness (DB-2253orequivalent).6.6 Ice Bath.6.7 Ultrasonic Bath.6.8 Vortex Mixer, or equivalent method to rapidly mixsolutions in a test tube.6.9 Water Bath, setable to 30 6 1C and 40 6 1C.7. Reagents and Materials7.1 Chemicals:7.1.1 Purity of ReagentsUse reagent grade chemicals inall tests. Un
36、less otherwise indicated, it is intended that allreagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.4Monosaccharides used toprepare the monosaccharide stock solutions and loss factorstandard solutions
37、shall be 98+ mass % purity. Other chemicalgrades may be substituted, provided it is first ascertained thatthe reagent is of sufficiently high purity to permit its usewithout lessening the accuracy of the determination.7.1.2 Purity of WaterUnless otherwise indicated, refer-ences to water mean reagent
38、 water as defined by Type 1 ofSpecification D1193.7.1.3 Acetic Acid (CH3COOH), glacial.7.1.4 Acetic Anhydride (CH3CO)2O).7.1.5 Ammonium Hydroxide, (NH4OH), concentrated(2830 wt % NH3).7.1.6 Ammonium Hydroxide Solution (;3 M)Dilute 5.0 60.1 mL of concentrated ammonium hydroxide (NH4OH) with20.06 0.1
39、mL of water. Prepare fresh before each use.7.1.7 Monosaccharide Stock A SolutionCombine the fol-lowing monosaccharides. Weigh each monosaccharide in thefollowing nominal amounts (record each actual mass to thenearest 0.1 mg). Dissolve in water and dilute to 100 mL. Storeat 4C and discard after four
40、weeks.Arabinose (C5H10O5) 90110 mgXylose (C5H10O5) 650750 mgMannose (C6H12O6) 90110 mgGalactose (C6H12O6) 90110 mgGlucose (C6H12O6) 19002100 mg7.1.8 Monosaccharide Stock B SolutionPrepare in manneridentical to monosaccharide stock A solution.7.1.9 Dichloromethane, (CH2Cl2).7.1.10 Inositol Solution (
41、20 mg/mL)Dissolve 5.000 60.0025 g of inositol (C6H12O6, 98 + wt %) in water and diluteto 250 mL. Store at 4C and discard after one week.7.1.11 Loss Factor Standard Stock SolutionCombine to-gether each of the following monosaccharides. Weigh eachmonosaccharide in the following nominal amounts (record
42、each actual weight to the nearest 0.1 mg). Dissolve in waterand dilute to 100 mL. Store at 4C and discard after fourweeks.Arabinose (C5H10O5) 9001100 mgMannose (C6H12O6) 9001100 mgGalactose (C6H12O6) 9001100 mgXylose (C5H10O5) 9001100 mgGlucose (C6H12O6) 9001100 mg7.1.12 1-Methylimidazole, (C3H3N2)(
43、CH3).7.1.13 Potassium Borohydride Solution, (0.15 g/mL)Dissolve 7.50 6 0.05 g potassium borohydride (KBH4)in40mL of ;3 M ammonium hydroxide (NH4OH) solution. Use anultrasonic bath to get the salt to dissolve in a reasonableamount of time. Dilute to 50.0 6 0.1 mL with ;3Mammonium hydroxide (NH4OH) so
44、lution. Prepare immediatelybefore use. Discard after 6 h. This quantity is sufficient for 50specimens and calibration standards.7.1.14 Potassium Hydroxide Solution (3.5 M)Dissolve58.06 0.5 g of potassium hydroxide (KOH, 85 wt %) in 200mLwater.Allow to cool to room temperature before diluting to250 m
45、L with water.7.1.15 Sulfuric Acid Solution (12 M)Slowly add 665 mLof 96 wt % sulfuric acid (H2SO4) to 300 mL of water cooled inan ice bath with stirring. Allow solution to come to roomtemperature and dilute to 1 L. Check the concentration bytitration and adjust the concentration to 12.0 6 0.1 M (24.
46、0 60.2 N).7.2 Materials:7.2.1 Glass Filtering Crucibles, 50 mL, medium porosity,nominal pore size of 10 m.7.2.2 Glass Serum Bottles, 125 mL, crimp-top style withrubber stoppers and aluminum seals to fit.7.2.3 Vacuum Adaptor for Filtering Crucibles.3DB-225 is a trademark of Agilent Technologies, Inc.
47、, 5301 Stevens CreekBoulevard, Santa Clara CA 95051.4Reagent 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 LaboratoryChemicals, BDH Ltd.,
48、Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.E1821 08 (2015)37.2.4 Vials, 13 32 mm crimp-top style withpolytetrafluoroethylene-faced rubber septum and aluminumcrimp seals or equivalent.8. Hazards8.1 Do not per
49、mit sulfuric acid, glacial acetic acid, aceticanhydride, or potassium hydroxide to contact skin or clothing.They are corrosive. Wear protective clothing.8.2 After the autoclave step, the glass bottles are hot andmay be pressurized. Handle with caution.8.3 Solutions of potassium borohydride will spontaneouslyevolve hydrogen gas on standing. To prevent pressurization, donot seal bottles. Ensure adequate ventilation around suchsolutions to avert the accumulation of flammable hydrogen gas.Wet potassiu
