1、Designation: E 1821 01Standard 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 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of
9、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 statements.2. Referenced Documents2.1 ASTM Standards:2D 1193 Specification for Reagent WaterE 1690 Test Method for Determinat
10、ion 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 1757 Practice for Preparation of Biomass for Composi-tional Analysis3. Terminology3.1 Descriptions of Terms Specific to This S
11、tandard:3.1.1 anhydrosugars, nthe nominal repeating unit of apolysaccharide. When polysaccharides undergo acid hydroly-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 account1T
12、his test method is under the jurisdiction of ASTM Committee E48 onBiotechnology and is the direct responsibility of Subcommittee E48.05 on BiomassConversion.Current edition approved Nov. 10, 2001. Published February 2002. Originallypublished as E 1821-96. Last previous edition E1821-96.2For referenc
13、ed 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.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Co
14、nshohocken, PA 19428-2959, United States.whencalculating the actual mass percent of the polysaccharidein 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
15、 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 at 105C as described in Test Method E 1756.3.1.5 prepared biomass, nas received biomass materialthat has been treated according to Practic
16、e 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 removed by extraction with solvents and are liber-ated from the biomass solids with dilute acid hydrolysis. Forthe purpose of this test m
17、ethod, the monosaccharides that areconsidered present are arabinose, xylose, mannose, galactose,and glucose.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 the anhydro-sugar, on an ove
18、n-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.3.2.4 areacreported area counts for the monosaccharide cpeak in the chromatogram, as integrated by the electronicintegrator.3.2.5 areaISr
19、eported area counts for the internal standardpeak in the chromatogram, as integrated by the electronicintegrator.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 fac
20、tor 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 monosaccharide c in specimen s,measured by gas chromatography (GC), in mg/mL.3.2.10 CSTDconcentration of monosaccharide c in thecalibration standard
21、, 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 extractivesin the extracted specimen as described in Test Method E 1690.3.2.13 kconstant used to convert the mass of monosac-charide to the
22、 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).3.2.14 LFloss factor for monosaccharide c. Used to cor-rect for the amount of monosaccharide lost through degrada-tion during acid hydrolysis o
23、f 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 RRavgaveraged response ratio of monosaccharide cto the internal standard (inositol) in the calibration standard.Derived from multiple injectio
24、ns 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 monosaccharide c to theinternal standard (inositol) in the calibration standard.3.2.20 RRF (Relative Response Factor of monosaccharidec)thi
25、s 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 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,
26、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 %Tadpercentage by mass, of total solids of theair-dried specimen determined at 105C as described by TestMethod E 1756.3.2.25 %Textpercentage by mass
27、, 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 prepared by freeze drying, as described by PracticeE 1757.3.2.27 %Tpreppercentage, by mass, of total solids of thespecimen prepared by freeze
28、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 with other assays to determine the total composition ofbiomass samples.5. Interferences5.1 The results of structural carbohydrate analysis a
29、re af-fected by incomplete hydrolysis of biomass or hydrolysisconditions that are too severe. Incomplete hydrolysis will biasthe results low because dimeric and oligomeric carbohydratesare not quantified. Hydrolysis conditions that are too severedegrade the liberated monosaccharides into materials t
30、hat arenot quantified by this procedure, again biasing the results low.5.2 Incomplete neutralization and removal of acetic acidfrom the methylene chloride extract prior to GC analysis canresult in ghost peaks appearing in the chromatogram orcarryover of monosaccharides from one injection to the next
31、(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 include alkaline and acid-pretreated biomass samples thathave not been washed. Unwashed pretreated biomass samplescontaining free acid or alka
32、li may change visibly on heating.5.4 Materials containing nonstructural carbohydrates alsoare unsuitable for this procedure since nonstructural carbohy-drates may undergo degradation to materials that are notquantified in this procedure.E18210126. Apparatus6.1 Analytical Balance, readable to 0.1 mg.
33、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 sulfate.6.5 Gas Chromatograph, equipped with electronic integra-tor, capillary split injection port, flame ionization detector withmake-up g
34、as, 250 m 3 15 m fused-silica capillary columncoated with 50 % cyanopropylphenyl methylpolysiloxane, 0.25m film thickness (DBy-2253or equivalent).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
35、 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 allreagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are a
36、vailable.4Monosaccharides used toprepare the monosaccharide stock solutions and loss factorstandard solutions 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 ac
37、curacy 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 1193.7.1.3 Acetic Acid (CH3COOH), glacial.7.1.4 Acetic Anhydride (CH3CO)2O).7.1.5 Ammonium Hydroxide, (NH4OH), concentrated(2830 wt % NH3).7.1.6
38、 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 Monosaccharide Stock A SolutionCombine the fol-lowing monosaccharides. Weigh each monosaccharide in thefollowing nominal amounts (record each
39、 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) 650750 mgMannose (C6H12O6) 90110 mgGalactose (C6H12O6) 90110 mgGlucose (C6H12O6) 19002100 mg7.1.8 Monosaccharide Stock B SolutionPrepare in
40、 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 (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
41、-gether each of the following monosaccharides. Weigh eachmonosaccharide in the following nominal amounts (recordeach 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 mgGalactos
42、e (C6H12O6) 9001100 mgXylose (C5H10O5) 9001100 mgGlucose (C6H12O6) 9001100 mg7.1.12 1-Methylimidazole, (C3H3N2)(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 t
43、he 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 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
44、 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)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 roomtemp
45、erature and dilute to 1 L. Check the concentration bytitration and adjust the concentration to 12.06 0.1 M (24.0 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
46、 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 seals or equivalent.8. Hazards8.1 Do not permit sulfuric acid, glacial acetic acid, aceticanhydride, or potassium hydroxide to contact sk
47、in 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. Ensu
48、re adequate ventilation around suchsolutions to avert the accumulation of flammable hydrogen gas.Wet potassium borohydride is highly flammable.8.4 Methylene chloride is a very volatile solvent and is bothtoxic and a suspected carcinogen. Handle only with adequateventilation.3DBy-225 is a trademark o
49、f J for mannose, galac-tose and glucose, k = 0.90 (162/180),mi= initial mass of specimen, in mg, and%Tad= percent by mass, of total solids of theair-dried specimen determined by TestMethod E 1756.12.5.2 If the biomass was prepared in accordance withPractice E 1757, calculate the percent by mass of each anhy-drosugar in the as received sample, on an oven-dried massbasis, as follows:%Anhydrowhole5mcorr3 km13%T105%Tprep3 100 % (11)where:%Anhydrowhole= the percent by mass, of the anhydro-sugar in the sample, o
