1、Designation: E1758 01 (Reapproved 2015)Standard Test Method forDetermination of Carbohydrates in Biomass by HighPerformance Liquid Chromatography1This standard is issued under the fixed designation E1758; the number immediately following the designation indicates the year oforiginal adoption or, in
2、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.INTRODUCTIONThe carbohydrates making up a major portion of biomass samples are polysaccharides c
3、onstructedprimarily 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-moderatedparti
4、tion HPLC.1. Scope1.1 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
5、 the polysaccha-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,boxboard, and
6、 newsprint), 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 f
7、ollows:1.3.1 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
8、.3.2 Extractives-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 there
9、sponsibility 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:2D1193 Specification fo
10、r 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 inBiomassE1757 Practice for Preparation of Biomass for Composi-tional Analysis3. Terminology
11、3.1 Definitions of Terms Specific to This Standard: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 biomasssample dried at 105C as described in Test Method E1756.1This test method is under the juri
12、sdiction of ASTM Committee 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 1995. Last previous edition approved in 2007 as E175801(2007
13、). DOI:10.1520/E1758-01R15.2For 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
14、Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.1.3 prepared biomassmaterial that has been treatedaccording to Practice E1757 in order to raise the total solidscontent above 85 %, by mass, based on an oven-dried solidsmass.3.2 AbbreviationsAbbreviations of standards
15、 used in theprocedure, and definitions of terms used in the calculations areas follows:3.2.1 C1known concentration of sugar recovery standardbefore hydrolysis, in mg/mL.3.2.2 C2concentration of sugar recovery standard detectedby HPLC after hydrolysis, in mg/mL.3.2.3 Ccorr concentration of sugar in h
16、ydrolyzed samplecorrected for hydrolysis, in mg/mL.3.2.4 Cspl concentration 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 p
17、reparing thecalibration standards.3.2.6 m1initial mass of sample, in mg.3.2.7 % extractivesthe percentage, by mass, of extractivesin the prepared biomass sample as described in Test MethodE1690.3.2.8 %Rsrs percent recovery of sugar recovery standard,as determined in 13.2.3.2.9 %sugarextractives-free
18、the percentage, by mass, ofsugar on an extractives-free 105C dry weight basis, asdetermined in 13.6.1.3.2.10 % sugarwhole samplethe corrected mass percent sugarvalue on an extractives-free basis corrected to an as received(whole sample) 105C dry mass basis.3.2.11 %T45percentage, by mass, of total so
19、lids of thesample prepared by drying at 45C, as described by PracticeE1757.3.2.12 %T105percentage, by mass, of total solids in thesample, dried at 105C, as determined by Test Method E1756.3.2.13 %Tad percentage, by mass, of total solids of theair-dried sample determined at 105C as described by TestM
20、ethod E1756.3.2.14 %Text percentage, by mass, of total solids of theextracted sample determined at 105C as described by TestMethod E1756.3.2.15 %Tfd percentage, by mass, of total solids of thesample prepared by freeze drying, as described by Test MethodE1756.3.2.16 %Tprep percentage, by mass, of tot
21、al solids of thesample prepared by freeze drying, %Tfd, or by drying at 45C,%T45, as determined by Practice E1757.3.2.17 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, o
22、f sugar content is used inconjunction with 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.
23、2 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.6. Apparatus6.1 Analytical Balance, readable to 0.1 mg.6.2 Auto
24、clave, capable of maintaining 121 6 3C.6.3 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 opti
25、on when using an HPX-87P3column, orequivalent. 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. Reagent
26、s and Materials7.1 Chemicals:7.1.1 Purity of 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 avail
27、able.5Other grades may be used,provided it is first ascertained that the reagent is of sufficientlyhigh purity to permit its use without lessening the accuracy ofthe determination.3The sole source of supply of the apparatus known to the committee at this timeis BioRad Aminex, HPX-87C and Aminex HPX-
28、87P, available from BioRad,Main Office, 3300 Regatta Boulevard, Richmond, CA 94804. If you are aware ofalternative suppliers, please provide this information to ASTM InternationalHeadquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee,1which
29、you may attend.4Available 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 Socie
30、ty, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville,MD.E1758 01 (2015)27.1.2 Purity of WaterUnless otherwise indicated, refer-ences to water shall be understo
31、od to mean reagent water asdefined by Type 1 of Specification D1193.7.1.3 Calcium Carbonate.7.1.4 High-Purity Sugars (98 % +, By Mass)Two sets ofglucose, xylose, galactose, arabinose, and mannose, meetingthe requirements described above, dried at 45C. The sugarsare used in preparing calibration stan
32、dards, calibration verifi-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 d
33、etermining sugar recov-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
34、15.6C/15.6C.7.2 Materials: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 caref
35、ully to avoid contact withskin 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 prepa
36、red according to PracticeE1757, and9.1.2 Extractives-free material prepared according to TestMethod E1690.10. 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 o
37、f interest. AnHPX-87C3column, 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 lim
38、it of the instrumentand 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 c
39、ontained in the calibration 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 quality
40、of the calibration curve(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
41、 of the overall analytical 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.
42、1.6 HPLC analysis of 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 E1756 and record the total solids valueas %T105. This prepared sample should be stored in a mannerto ensure its
43、moisture content does 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 E1756 and record the total solids value as %Tad.11.2.2 If Test Method B of this practice is used (drying a
44、t45C), record the total 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 extracti
45、ve-free material by Test MethodE1756 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
46、 andshould be handled 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. Sti
47、rapproximately every 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 des
48、cribed in theprevious two steps. These SRSs will be taken through theremaining steps in the procedure in parallel with the samples.E1758 01 (2015)3The calculated recovery of the SRS will be used to correct forlosses caused by the destruction of sugars during the hydrolysisprocess.11.8 Transfer each
49、hydrolyzate to a glass bottle and 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 4 % w/wacid solution is 1.0250, the total volume of solution, VF, is 87.0mL.11.9 Stopper the bottles and crimp the aluminum seals intoplace in preparation for the next step.11.10 Set the autocl
