1、Designation: E 1821 08Standard Test Method forDetermination of Carbohydrates in Biomass by GasChromatography1This standard is issued under the fixed designation E 1821; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of la
2、st revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.INTRODUCTIONThis test method gives a reproducible way to quantitatively determine in lignocellulosic materialsthe kind and amount o
3、f 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 using capillary ga
4、s 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, herbaceous ma
5、terials, 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 residues.1.3 T
6、he 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 by mass, based
7、 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 extractedsample.
8、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 This standard doe
9、s 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 forspecific hazards s
10、tatements.2. Referenced Documents2.1 ASTM Standards:2D 1193 Specification for Reagent WaterE 1690 Test Method for Determination of Ethanol Extrac-tives in BiomassE 1721 Test Method for Determination of Acid-InsolubleResidue in BiomassE 1756 Test Method for Determination of Total Solids inBiomassE 17
11、57 Practice for Preparation of Biomass for Composi-tional Analysis3. Terminology3.1 Descriptions of Terms Specific to This Standard:1This test method is under the jurisdiction of ASTM Committee E48 onBiotechnology and is the direct responsibility of Subcommittee E48.05 on BiomassConversion.Current e
12、dition approved May 1, 2008. Published May 2008. Originallyapproved in 1996. Last previous edition approved in 2007 as E 1821-01(2007).2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume inf
13、ormation, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.1 anhydrosugars, nthe nominal repeating unit of apolysaccharide. When polysaccharides undergo acid hydroly-
14、sis, each repeating unit adds a single molecule of water to formthe free monosaccharide that is analyzed. The extra weightfrom this water of hydrolysis must be taken in to accountwhencalculating the actual mass percent of the polysaccharidein the original biomass sample.3.1.2 as received biomass, nm
15、aterial 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 E 1690.3.1.4 oven-dried mass, nthe moisture-free mass of anybiomass sample (as received, prepared, extractives-free, etc.)dried
16、at 105C as described in Test Method E 1756.3.1.5 prepared biomass, nas received biomass materialthat has been treated according to Practice E 1757 in order toraise the total solids content above 85 %, based on an oven-dried solids weight.3.1.6 structural carbohydrates, npolysaccharides thatcannot be
17、 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 glucose.3.2 Abbreviations:3.2.1 %Anhydroextthe percent by mass
18、 of the anhydro-sugar on an extractives-free, oven-dried mass basis.3.2.2 %Anhydrowholethe percent by mass of the anhydro-sugar, on an oven-dried mass basis.3.2.3 ARc(Amount Ratio)ratio of the concentration(amount) of monosaccharide c to the concentration (amount) ofinternal standard in the specimen
19、.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 electronicintegrator.3.2.6 Cavgaverage concentration of monosaccharide
20、 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 and specimen, in mg/mL.3.2.9 Csconcentration of monosaccha
21、ride 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 value measured.3.2.12 %extractivesthe percentage by mass of e
22、xtractivesin the extracted specimen as described in Test Method E 1690.3.2.13 kconstant used to convert the mass of monosac-charide to the mass of anhydrosugar from which it is derived.For arabinose and xylose, k = 0.88 (m/z 132/150); for man-nose, galactose and glucose, k = 0.90 (m/z 162/180).3.2.1
23、4 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 solution, cor-rected for hydrolysis losses, in mg.3.2.17 RRav
24、gaveraged 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 (inositol) in the specimen.3.2.19 RRSTDresponse ratio of m
25、onosaccharide 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 standard(inositol) for a given injection of the specim
26、en.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 PracticeE 1757.3.2.23 %T105percentage by mass, of total solids of thespecimen, dried at 105C, as determined by Test MethodE 1756.3.2.24 %Tadpercen
27、tage by mass, of total solids of theair-dried specimen determined at 105C as described by TestMethod E 1756.3.2.25 %Textpercentage by mass, of total solids of theextracted specimen determined at 105C as described by TestMethod E 1756.3.2.26 %Tfdpercentage by mass, of total solids of thespecimen prep
28、ared by freeze drying, as described by PracticeE 1757.3.2.27 %Tpreppercentage, by mass, of total solids of thespecimen prepared by freeze drying,% Tfd, or by drying at45C, %T45, as determined by Practice E 1757.4. Significance and Use4.1 The structural carbohydrate content is used in conjunc-tion wi
29、th 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 hydrolysis will biasthe results low because dimeric and o
30、ligomeric 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 of acetic acidfrom the methylene chloride extract
31、 prior to GC analysis canresult 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 Test specimens not suitable for analysis by this proce-dure incl
32、ude alkaline and acid-pretreated biomass samples thatE1821082have not been washed. Unwashed pretreated biomass samplescontaining free acid or alkali may change visibly on heating.5.4 Materials containing nonstructural carbohydrates alsoare unsuitable for this procedure since nonstructural carbohy-dr
33、ates 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 105 6 3C.6.4 Desiccator, containing anhydrous calcium su
34、lfate.6.5 Gas Chromatograph, equipped with electronic integra-tor, capillary split injection port, flame ionization detector withmake-up gas, 250 m 3 15 m fused-silica capillary columncoated with 50 % cyanopropylphenyl methylpolysiloxane, 0.25m film thickness (DB-2253or equivalent).6.6 Ice Bath.6.7
35、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. Unless otherwise indicated, it is intended that allreag
36、ents 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 shall be 98+ mass % purity. Other chemicalgrades may
37、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 water as defined by Type 1 ofSpecification D 1193.7.
38、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 (;3M)Dilute 5.06 0.1 mL of concentrated ammonium hydroxide (NH4OH)with 20.06 0.1 mL of water. Prepare fresh before each use.7.1.7 Mono
39、saccharide 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 weeks.Arabinose (C5H10O5) 90110 mgXylose (C5H10O5) 65
40、0750 mgMannose (C6H12O6) 90110 mgGalactose (C6H12O6) 90110 mgGlucose (C6H12O6) 19002100 mg7.1.8 Monosaccharide Stock B SolutionPrepare in man-ner identical to monosaccharide stock A solution.7.1.9 Dichloromethane, (CH2Cl2).7.1.10 Inositol Solution (20 mg/mL)Dissolve 5.000 60.0025 g of inositol (C6H1
41、2O6, 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 (recordeach actual weight to the nearest 0.1 mg). Dissolve
42、 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)(CH3).7.1.13 Potassium Borohydride Solution, (0.15 g
43、/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) solution. Prepare immediatelybefore use. Discard afte
44、r 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 mL with water.7.1.15 Sulfuric Acid Solution (12 M)Sl
45、owly 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 (24.0 60.2 N).7.2 Materials:7.2.1 Glass Filtering Cruci
46、bles, 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.7.2.4 Vials,133 32 mm crimp-top style withpolytetrafluoroethylene-faced rubber septum and aluminumcrimp
47、 seals or equivalent.8. Hazards8.1 Do not permit 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
48、 Solutions of potassium borohydride will spontaneouslyevolve hydrogen gas on standing. To prevent pressurization, donot seal bottles. Ensure adequate ventilation around such3DB-225 is a trademark of Agilent Technologies, Inc., 5301 Stevens CreekBoulevard, Santa Clara CA 95051.4Reagent Chemicals, Ame
49、rican 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., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.E1821083solutions to avert the accumulation of flammable hydrogen gas.Wet potassium borohydride is highly flammable.8.4 Methylene chloride is a very
