1、Designation: E 1758 01Standard Test Method forDetermination of Carbohydrates in Biomass by HighPerformance Liquid Chromatography1This standard is issued under the fixed designation E 1758; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revi
2、sion, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.INTRODUCTIONThe carbohydrates making up a major portion of biomass samples are polysaccharides constructedprima
3、rily of glucose, xylose, arabinose, galactose, and mannose subunits. The polysaccharides presentin a biomass sample can be hydrolyzed to their component sugar monomers by sulfuric acid in atwo-stage hydrolysis process. These monosaccharides can then be quantified by ion-moderatedpartition HPLC.1. Sc
4、ope1.1 This test method covers the determination of carbohy-drates present in a biomass sample, expressed as the percent,by mass, of each sugar on a 105C dried mass basis.NOTE 1The percent sugar must be corrected for the water ofhydrolysis before calculating the actual mass percent of the polysaccha
5、-ride in the original biomass sample.1.2 Sample materials suitable for this procedure includehard and soft woods, herbaceous materials (such as switchgrassand sericea), agricultural residues (such as corn stover, wheatstraw, and bagasse), wastepaper (such as office waste, box-board, and newsprint),
6、acid or alkaline-pretreated biomass(washed free of any residual acid or alkali), and the solidfraction of fermentation residues. All results are reportedrelative to the 105C oven-dried mass of the sample.1.3 The options for the types of samples to be analyzed inthis test method are as follows:1.3.1
7、Prepared Biomass Samples:1.3.1.1 Air Dried (%Tad)The percent, by mass, of totalsolids of the air-dried sample.1.3.1.2 45C Dried (%T45)The percent, by mass, of totalsolids of the 45C dried sample.1.3.1.3 Freeze Dried (%Tfd)The percent, by mass, of totalsolids of the freeze dried sample.1.3.2 Extracti
8、ves-Free Sample (%Text)The percent, bymass, of total solids of the extracted sample determined at105C.1.4 The values stated in SI units are to be regarded as thestandard.1.5 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility
9、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. Specific precau-tionary statements are given in Note 3 and Note 4.2. Referenced Documents2.1 ASTM Standards:2D 1193 Specification for Reagent Wa
10、terE 1690 Test Method for the Determination of EthanolExtractives in BiomassE 1721 Test Method for the Determination of Acid-Insoluble Residue in BiomassE 1756 Test Method for the Determination ofTotal Solids inBiomassE 1757 Practice for Preparation of Biomass for Composi-tional Analysis3. Terminolo
11、gy3.1 Definitions 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 edition approved November 10, 2001. Published February 2002.Originally published
12、 as E 1758-95. Last previous edition E 175895e1.2For 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.1Copyright AS
13、TM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.1 as received biomassthe biomass material as it isreceived in its field or process collected state.3.1.2 oven-dried massthe moisture-free mass of a biom-ass sample dried at 105C as described in
14、Test Method E 1756.3.1.3 prepared biomassmaterial that has been treatedaccording to Practice E 1757 in order to raise the total solidscontent above 85 %, by mass, based on an oven-dried solidsmass.3.2 Abbreviations:AbbreviationsAbbreviations of stan-dards used in the procedure, and definitions of te
15、rms used inthe calculations are as follows:3.2.1 C1known concentration of sugar recovery standardbefore hydrolysis, in mg/mL.3.2.2 C2concentration of sugar recovery standard de-tected by HPLC after hydrolysis, in mg/mL.3.2.3 Ccorrconcentration of sugar in hydrolyzed samplecorrected for hydrolysis, i
16、n mg/mL.3.2.4 Csplconcentration of sugar in hydrolyzed sampledetected by HPLC, in mg/mL.3.2.5 CVS (calibration verification standard)standardsused in determining the quality of the calibration curve as wellas the quality of the standard reagents used in preparing thecalibration standards.3.2.6 m1ini
17、tial mass of sample, in mg.3.2.7 % extractivesthe percentage, by mass, of extractivesin the prepared biomass sample as described in Test MethodE 1690.3.2.8 %Rsrspercent recovery of sugar recovery standard,as determined in 13.2.3.2.9 %sugarextractives-freethe percentage, by mass, ofsugar on an extrac
18、tives-free 105C dry weight basis, asdetermined in 13.6.1.3.2.10 % sugarwhole samplethe corrected mass percentsugar value on an extractives-free basis corrected to an asreceived (whole sample) 105C dry mass basis.3.2.11 %T45percentage, by mass, of total solids of thesample prepared by drying at 45C,
19、as described by PracticeE 1757.3.2.12 %T105percentage, by mass, of total solids in thesample, dried at 105C, as determined by Test Method E 1756.3.2.13 %Tadpercentage, by mass, of total solids of theair-dried sample determined at 105C as described by TestMethod E 1756.3.2.14 %Textpercentage, by mass
20、, of total solids of theextracted sample determined at 105C as described by TestMethod E 1756.3.2.15 %Tfdpercentage, by mass, of total solids of thesample prepared by freeze drying, as described by Test MethodE 1756.3.2.16 %Tpreppercentage, by mass, of total solids of thesample prepared by freeze dr
21、ying, %Tfd, or by drying at 45C,%T45, as determined by Practice E 1757.3.2.17 SRS (sugar recovery standards)standards used todetermine sugar recovery after hydrolysis.3.2.18 VFvolume of filtrate, 87.0 mL.4. Significance and Use4.1 The percentage, by mass, of sugar content is used inconjunction with
22、other assays to determine the total composi-tion of biomass samples.5. Interferences5.1 Samples with high protein content may result in thepercentage, by mass, of sugar values being biased low, as aconsequence of protein binding with some monosaccharides.5.2 Test specimens not suitable for analysis
23、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.6. Apparatus6.1 Analytical Balance, readable to 0.1 mg.6.2 Autoclave, capable of maintaining 121 6 3C.6.3
24、Convection Ovens, temperature control to 45 6 3 and105 6 3C.6.4 Desiccator, using anhydrous calcium sulfate.6.5 Guard Columns, cartridges appropriate for the columnused.NOTE 2Deashing guard column cartridges from BioRad,3of the ionicform H+/CO3, are an option when using an HPX-87P3column, orequivale
25、nt. These cartridges are effective in eliminating baseline ramping.6.6 Hewlett Packard4Model 1090 HPLC, or equivalent,with refractive index detector.6.7 HPLC Columns, BioRad HPX-87C3or HPX-87P,3orboth, or equivalent.6.8 Water Bath, set at 30 6 1C.7. Reagents and Materials7.1 Chemicals:7.1.1 Purity o
26、f ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.5Other grades may be used,provided it
27、is first ascertained that the reagent is of sufficientlyhigh purity to permit its use without lessening the accuracy ofthe determination.7.1.2 Purity of WaterUnless otherwise indicated, refer-ences to water shall be understood to mean reagent water asdefined by Type 1 of Specification D 1193.7.1.3 C
28、alcium Carbonate.7.1.4 High-Purity Sugars (98 % +, By Mass)Two sets ofglucose, xylose, galactose, arabinose, and mannose, meeting3BioRad Aminext, HPX-87C and Aminext HPX-87P, available from BioRad,Main Office, 3300 Regatta Boulevard, Richmond, CA94804 has been found suitablefor this purpose.4Availab
29、le from Hewlett-Packard, HPAnalytical Direct, 2850 Centerville Road,Wilmington, DE 19808.5Reagent 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
30、 for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville,MD.E1758012the requirements described above, dried at 45C. The sugarsare used in preparing calibration standards, calibration veri
31、fi-cation standards (CVS), and sugar recovery standards (SRS).The sugars used in preparing the calibration standards shouldbe from a source (manufacturer or lot) other than that used inpreparing the CVS. Either set of sugars may be used forpreparing the SRS solutions used in determining sugar recov-
32、eries after hydrolysis.7.1.5 Sulfuric Acid Solution (72 % w/w or 12.00 6 0.02M)Slowly add 665 mLof concentrated sulfuric acid (H2SO4)to 300 mL of water while cooling in an ice bath and stirring.Allow to come to room temperature.Adjust the relative densityto 1.6389 6 0.0012 at 15.6C/15.6C.7.2 Materia
33、ls:7.2.1 Autosampler Vials, with crimp top seals to fit.7.2.2 Disposable Syringes, 3 mL.7.2.3 Disposable Syringe Filters, nylon, 0.2 m.7.2.4 Glass Serum Bottles, crimp top style, 125 mL, withrubber stoppers and aluminum seals to fit.8. Hazards8.1 Handle the sulfuric acid carefully to avoid contact w
34、ithskin or clothing, as it is corrosive.8.2 The glass bottles are hot and may be pressurized afterthe autoclave step. Use caution when handling.9. Sampling, Test Specimens, and Test Units9.1 Test specimens suitable for analysis by this procedureare:9.1.1 Prepared biomass prepared according to Practi
35、ceE 1757, and9.1.2 Extractives-free material prepared according to TestMethod E 1690.10. Calibration and Standardization10.1 Prepare a series of three to six sugar standards indeionized water at concentrations appropriate for preparingcalibration curves to quantitfy each sugar of interest. AnHPX-87C
36、3column, or equivalent, is used to analyze glucose,xylose, and arabinose. If mannose and galactose are also to bequantified, an HPX-87P3column, or equivalent, must be usedinstead. Typically, the concentrations of these sugar standardscover the range starting at the detection limit of the instrumenta
37、nd extending up to 4.0 mg/mL.10.2 Prepare an independent CVS, as described in 8.1.2, foreach set of calibration standards, using sugars obtained from asource other than that used in preparing the calibration stan-dards. The CVS will contain precisely known amounts of eachsugar contained in the calib
38、ration standards, at a concentrationin the middle of the validated range of the calibration curve.The CVS will be analyzed after each calibration curve and atregular intervals in the HPLC sequence, as dictated by goodlaboratory practice. The CVS is used in confirming the qualityof the calibration cu
39、rve(s) and the standard reagents used inpreparing the calibration standards. An additional benefit isobtained by bracketing groups of samples in the sequence withthe CVS, assuring the analyst of the quality of the calibrationcurve throughout the run.11. Procedure11.1 An overview of the overall analy
40、tical sequence is asfollows:11.1.1 Hydrolysis of sample with 72 % sulfuric acid,11.1.2 Hydrolyzate dilution and autoclaving,11.1.3 Filtration of insolubles if separate analysis is desired,11.1.4 Neutralization of hydrolyzate,11.1.5 Filtration of sample prior to HPLC analysis,11.1.6 HPLC analysis of
41、sugar standards, CVS, SRS, andhydrolyzate samples, and11.1.7 Calculation of sugar contents.11.2 For prepared biomass samples, determine the totalsolids by Test Method E 1756 and record the total solids valueas %T105. This prepared sample should be stored in a mannerto ensure its moisture content doe
42、s not change before theanalysis begins.11.2.1 If Test Method A of this practice is used (air drying),determine the total solids content of this prepared sample byTest Method E 1756 and record the total solids value as %Tad.11.2.2 If Test Method B of this practice is used (drying at45C), record the t
43、otal solids calculated in this practice, %T45,as %Tprep.11.2.3 If Test Method C of this practice is used (freezedrying), record the total solids calculated in this practice, %Tfd,as %Tprep.11.3 If extractives-free material is used, determine the totalsolids content of the extractive-free material by
44、 Test MethodE 1756 and record this value as %Text.11.4 Weigh 300 6 10 mg of the prepared or extractives-freesample to the nearest 0.1 mg and place in 16x 100 mm glasstest tube. Record as m1, the initial mass of sample in grams.NOTE 3Warning: 72 % w/w sulfuric acid is very corrosive andshould be hand
45、led by trained personnel only.11.5 Add 3.00 6 0.01 mL (4.92 6 0.01 g) of 72 % w/wH2SO4to the test tube containing the sample and stir for 1 minor until thoroughly mixed.11.6 Place the test tube containing the sample into the waterbath controlled to 30 6 1C and hydrolyze for 1h. Stirapproximately eve
46、ry 15 min to ensure the sample is completelymixed and wet.11.7 Weigh out 300 6 10 mg of each high purity sugarstandard (dried at 45C), described in 8.1.4, to the nearest 0.1mg and place each in its own individual 16x 100 mm glass testtube. Add acid and hydrolyze these sugars as described in theprevi
47、ous two steps. These SRSs will be taken through theremaining steps in the procedure in parallel with the samples.The calculated recovery of the SRS will be used to correct forlosses caused by the destruction of sugars during the hydrolysisprocess.11.8 Transfer each hydrolyzate to a glass bottle and
48、dilute to4 % w/w acid concentration by adding 84.00 6 0.04 mLwater.Be careful to transfer all the residual solids along with thehydrolysis liquor. The total mass added to the tared bottle is89.22 g (0.3 g sample, 4.92 g 72 % w/w H2SO4, and 84.00 gdeionized water). Because the relative density of the
49、 4 % w/wacid solution is 1.0250, the total volume of solution, VF, is 87.0mL.E175801311.9 Stopper the bottles and crimp the aluminum seals intoplace in preparation for the next step.11.10 Set the autoclave to a liquid vent cycle to prevent lossof sample from the bottle in the event of a loose crimp seal.Autoclave the sample in the sealed bottle for1hat1216 3C.NOTE 4Warning: Handle the sealed bottle with caution after theautoclave step, as it may have become pressurized.11.11 After completing the autoclave cycle, allow thebott
