Designation: E1758 − 01 (Reapproved 2015)
Standard Test Method for
Determination of Carbohydrates in Biomass by High Performance Liquid Chromatography 1 This standard is issued under the fixed designation E1758; the number immediately following the designation indicates the year of original origin al adoption or, in the case of revis revision, ion, the year of last revision. revision. A number in paren parenthese thesess indicates the year of last reappr reapproval. oval. A superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
The carbohydrates carbohydrates making up a major portion of biomas biomasss samples are polys polysacchar accharides ides constructed constructed primarily of glucose, xylose, arabinose, galactose, and mannose subunits. The polysaccharides present in a biomass sample can be hydrolyzed to their component sugar monomers by sulfuric acid in a two-st two -stage age hyd hydrol rolysi ysiss pro process cess.. The These se mon monosa osacch cchari arides des can the then n be qua quanti ntified fied by ion ion-mo -moder derated ated partition HPLC. 1.4 The values values stated in SI uni units ts are to be reg regard arded ed as the standard.
1. Sco Scope pe 1.1 This test method covers the determination determination of carbohydrates present in a biomass sample, expressed as the percent, by mass, of each sugar on a 105°C dried mass basis.
standard d doe doess not purport purport to add addre ress ss all of the 1.5 This standar safet sa fetyy co conc ncern erns, s, if an anyy, as asso soci ciat ated ed wi with th its us use. e. It is th thee responsibility of the user of this standard to establish appro priate safety and health practices and determine the applicability of regulatory limitations prior to use. Specific precau and Note Note 4. 4. tionary statements are given in Note in Note 3 and
NOTE 1—Th 1—Thee pe perc rcen entt su suga garr mu must st be co corr rrec ecte ted d fo forr th thee wa water ter of hydrolysis before calculating the actual mass percent of the polysaccharide in the original biomass sample.
1.2 Sam Sample ple mat materia erials ls sui suitab table le for this pro proced cedure ure inc includ ludee hard and soft woods, herbaceous materials (such as switchgrass and sericea), agricultural residues (such as corn stover, wheat straw str aw,, an and d ba bagas gasse) se),, wa waste stepa paper per (s (suc uch h as of offfice wa waste ste,, boxboard, boxbo ard, and newsp newsprint), rint), acid or alkalin alkaline-pre e-pretreated treated biomass (washe (wa shed d fre freee of any res residu idual al acid or alk alkali) ali),, and the sol solid id fractio fra ction n of fer fermen mentati tation on res residu idues. es. All res result ultss are rep report orted ed relative to the 105°C oven-dried mass of the sample.
2. Referenc Referenced ed Documents 2.1 ASTM Standards: 2 D1193 Specification D1193 Specification for Reagent Water E1690 Test Metho Method d for Determ Determinatio ination n of Ethano Ethanoll Extrac Extrac-tives in Biomass E1721 Test Met Method hod for Dete Determi rminat nation ion of Acid Acid-In -Insol solubl ublee Residue in Biomass E1756 Test Met Method hod for Det Determ erminat ination ion of Tota otall Sol Solids ids in Biomass E1757 Pra Practic cticee for Pre Prepar paratio ation n of Biom Biomass ass for Com Compos posiitional Analysis
1.3 The options options for the types of samples samples to be ana analyz lyzed ed in this test method are as follows: 1.3.1 Prepared Biomass Samples: 1.3.1.1 Air Dried (%T ad )— The The percent, by mass, of total solids of the air-dried sample. 1.3.1.2 45°C Dried (%T 45)—The percent, by mass, of total solids of the 45°C dried sample. 1.3.1.3 Freeze Dried (%T fd )— The The percent, by mass, of total solids of the freeze dried sample. 1.3.2 Extractives-Fr The per percen cent, t, by Extractives-Free ee Samp Sample le (%T ext )— The mass,, of tot mass total al sol solids ids of the ext extrac racted ted sam sample ple determined determined at 105°C.
3. Terminology 3.1 Definitions of Terms Specific to This Standard: received ived bioma biomass— ss— the 3.1.1 as rece t he bi biom omas asss ma mater terial ial as it is received in its field or process collected state. 3.1.2 oven-dried mass— the the moisture-free mass of a biomass sample dried at 105°C as described in Test Method E1756 E1756..
1
Thiss tes Thi testt met method hod is und under er the jur jurisd isdict iction ion of AS ASTM TM Com Committ mittee ee E48 on Bioenergy and Industrial Chemicals from Biomass and is the direct responsibility of Subcommittee E48.05 Subcommittee E48.05 on Biomass Conversion. Current edition approved June 1, 2015. Published July 2015. Originally approved in 199 1995. 5. Las Lastt pre previo vious us edi editio tion n app approv roved ed in 200 2007 7 as E17 E1758– 58–01( 01(200 2007). 7). DOI DOI:: 10.1520/E1758-01R15.
2 For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at
[email protected]. For Annual Book of ASTM Standards volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
E1758 − 01 (2015) 3.1.3 prepar m ateria iall th that at ha hass be been en tr trea eated ted prepared ed bioma biomass— ss— mater according to Practice E1757 in order to raise the total solids content above 85 %, by mass, based on an oven-dried solids mass.
4. Signi Significanc ficancee and Use 4.1 The percenta percentage, ge, by mass mass,, of sugar content content is use used d in conjunction with other assays to determine the total composition of biomass samples.
3.2 Abbreviations—Abbreviations of standards used in the procedure, and definitions of terms used in the calculations procedure, calculations are as follows: 3.2.1 C 1 — known known concentration of sugar recovery standard before hydrolysis, in mg/mL.
5. Interfere Interferences nces 5.1 Samples Samples with high protein protein con conten tentt may result result in the percentage, by mass, of sugar values being biased low, as a consequence of protein binding with some monosaccharides.
3.2.2 C 2 — concentration concentration of sugar recovery standard detected by HPLC after hydrolysis, in mg/mL.
5.2 Test specimens not suitable suitable for analysis by this proce proce-dure include alkaline and acid-pretreated biomass samples that have not been washed. Unwashed pretreated biomass samples containing free acid or alkali may change visibly on heating.
3.2.3 C corr — concentration of sugar in hydrolyzed sample corrected for hydrolysis, in mg/mL. 3.2.4 C spl — concentrat concentration ion of sugar in hydro hydrolyzed lyzed sample detected by HPLC, in mg/mL.
6. Appar Apparatus atus 6.1 Analytical Balance, readable to 0.1 mg.
3.2.5 CVS (calib (calibration ration verific verification ation stand standard ard)— )— standards used in determining the quality of the calibration curve as well as the quality of the standard reagents used in preparing the calibration standards.
6.2 Autoclave, capable of maintaining 121
6
3°C.
6.3 Convection Ovens, temperature control to 45 105 6 3°C.
6 3
and
6.4 Desiccator, using anhydrous calcium sulfate.
3.2.6 m1 — initial initial mass of sample, in mg.
6.5 Guard Columns, cartridges appropriate for the column used.
3.2.7 % extractives— the the percentage, by mass, of extractives in the prepared biomass sample as described in Test Method E1690.. E1690
NOTE 2—Deashing guard column cartridges from BioRad,3 of the ionic form fo rm H+ /CO3−, ar aree an op opti tion on wh when en us usin ing g an HP HPXX-87 87P P3 colum column, n, or equivalent. These cartridges are effective in eliminating baseline ramping.
3.2.8 %Rsrs — percent percent recovery of sugar recovery standard, as determined in 13.2.
Packard d 4 Mod 6.6 Hewlett Packar Model el 109 1090 0 HPL HPLC, C, or equ equiva ivalen lent, t, with refractive index detector.
3.2.9 %sugar extractives-free — the t he pe perc rcen enta tage ge,, by mass mass,, of suga su garr on an ex extra tract ctiv iveses-fr free ee 10 105° 5°C C dr dry y we weig ight ht ba basi sis, s, as determined in 13.6.1.
6.7 HPLC Colum HPX-87P, 3 or Columns, ns, BioRad HPX-87C3 or HPX-87P, both, or equivalent.
3.2.10 % sugar whole sample — the the corrected mass percent sugar value on an extractives-free basis corrected to an as received (whole sample) 105°C dry mass basis.
6.8 Water Bath, set at 30
6
1°C.
7. Reag Reagents ents and Materials Materials
3.2.11 %T 45 — percen percentag tage, e, by mass mass,, of tot total al sol solids ids of the sample prepared by drying at 45°C, as described by Practice E1757.. E1757
7.1 Chemicals: 7.1.1 Purity of Reagents— Reagent Reagent grade chemicals chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all reagents conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society where such specifications are available.5 Other grades may be used, provided prov ided it is first ascerta ascertained ined that the reagent is of sufficientl sufficiently y high purity to permit its use without lessening the accuracy of the determination.
3.2.12 %T 105 — percentage, percentage, by mass, of total solids in the sample, dried at 105°C, as determined by Test Method E1756 E1756.. 3.2.13 %T ad — percentage, by mass, of total solids of the air-dr air -dried ied sam sample ple det determ ermine ined d at 105 105°C °C as des describ cribed ed by Test Method E1756 Method E1756.. 3.2.14 %T ext — percentage, by mass, of total solids of the extracted extracte d sam sample ple det determ ermine ined d at 105 105°C °C as des descri cribed bed by Test Method E1756 Method E1756..
3
The sole source of supply of the apparatus known to the committee at this time is BioRa BioRad d Aminex Aminex®, ®, HPX-8 HPX-87C 7C and Aminex® HPX-87P, HPX-87P, avail available able from BioRa BioRad, d, Main Office, 3300 Regatta Boulevard, Richmond, CA 94804. If you are aware of alternative altern ative suppliers, suppliers, pleas pleasee provid providee this informa information tion to ASTM Intern Internationa ationall Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee,1 which you may attend. 4 Available from Hewlett-Packard, HPAnalytical Direct, 2850 Centerville Road, Wilmington, DE 19808. 5 Reagent Chemicals, American Chemical Society Specifications , Americ American an Chemical Society, Washington, DC. For suggestions on the testing of reagents not Standard ardss for Labor Laboratory atory listed liste d by the American Chemical Chemical Soci Society ety,, see Analar Stand Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville, MD.
3.2.15 %T fd — percentage, by mass, of total solids of the sample prepared by freeze drying, as described by Test Method E1756.. E1756 3.2.16 %T prep — percentage, percentage, by mass, of total solids of the sample prepared by freeze drying, % T fd , or by drying at 45°C, % T 45, as determined by Practice E1757 Practice E1757.. 3.2.17 SRS (sugar recovery standards)— standards standards used to determine sugar recovery after hydro determine hydrolysis. lysis. 3.2.18 V F — volume volume of filtrate, 87.0 mL. 2
E1758 − 01 (2015) 7.1.2 Purity Unless otherwise indicat indicated, ed, refer refer-Purity of Wate ater— r— Unless ences to water shall be understood to mean reagent water as defined by Type 1 of Specification D1193 D1193.. 7.1.3 Calcium Carbonate. 7.1.4 High-Purity Sugars (98 % + , By Mass)— Two Two sets of glucose, xylose, galactose, arabinose, and mannose, meeting the requirements described above, dried at 45°C. The sugars are used in preparing calibration standards, calibration verification standards (CVS), and sugar recovery standards (SRS). The sugars used in preparing the calibration standards should be from a source (manufacturer or lot) other than that used in prep pr epar arin ing g th thee CVS CVS.. Ei Eith ther er set of su suga gars rs ma may y be us used ed fo forr preparing the SRS solutions used in determining sugar recoveries after hydr hydrolysis olysis.. 7.1.5 Sulfuric Sulfuric Aci Acid d Sol Soluti ution on (72 % w/w or 12. 12.00 00 6 0.02 Slowly add 665 mL of concentrated sulfuric acid (H 2SO4) M)— Slowly to 300 mL of water while cooling in an ice bath and stirring. Allow to come to room temperature. temperature. Adjus Adjustt the relativ relativee density to 1.6389 6 0.0012 at 15.6°C/15.6°C.
regular intervals in the HPLC sequence, as dictated by good laboratory practice. The CVS is used in confirming the quality of the calibration curve(s) and the standard reagents used in preparing prepa ring the calibra calibration tion standards. standards. An additio additional nal benefi benefitt is obtained by bracketing groups of samples in the sequence with the CVS, assuring the analyst of the quality of the calibration curve throughout the run. 11. Proced Procedure ure 11.1 11 .1 An ove overvi rview ew of the overall overall ana analyti lytical cal seq sequen uence ce is as follows: 11.1.1 11 .1.1 Hydr Hydrolysis olysis of sample with 72 % sulfu sulfuric ric acid, 11.1.2 11 .1.2 Hydr Hydrolyzat olyzatee dilutio dilution n and autoclaving, autoclaving, 11.1.3 11 .1.3 Filtrat Filtration ion of insolubles if separate analysis is desired, 11.1.4 11 .1.4 Neutr Neutralizatio alization n of hydro hydrolyzate, lyzate, 11.1.5 11 .1.5 Filtrat Filtration ion of sample prior to HPLC analysis, 11.1.6 11 .1.6 HPLC analysis of sugar standards, standards, CVS, SRS, and hydrolyzate hydr olyzate samples, and 11.1.7 11 .1.7 Calcula Calculation tion of sugar contents. contents.
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 µm. 7.2.4 Glass Serum Bottles, crimp top style, 125 mL, with rubber stoppers and aluminum seals to fit.
11.2 For pre 11.2 prepar pared ed bio biomas masss sam sample ples, s, det determ ermine ine the tot total al solids by Test Method E1756 E1756 and and record the total solids value as %T 105. This prepared sample should be stored in a manner to ens ensure ure its moi moistu sture re con content tent does not change change bef before ore the analysis begins. 11.2.1 11 .2.1 If Test Test Method A of this practice practice is used (air drying), drying), determine the total solids content of this prepared sample by Test Method E1756 Method E1756 and and record the total solids value as %T ad . 11.2.2 11 .2.2 If Test Test Method B of this practice is used (drying at 45°C), record the total solids calculated in this practice, %T 45, as %T prep. 11. 1.2. 2.3 3 If Tes estt Me Meth thod od C of th this is pr prac actic ticee is us used ed (f (fre reeze eze drying), record the total solids calculated in this practice, %T fd , as %T prep.
8. Haza Hazards rds 8.1 Handl Handlee the sulfu sulfuric ric acid carefully to avoid contact contact with skin or clothing, as it is corrosive. 8.2 The glass bottles bottles are hot and may be pressuri pressurized zed after the autoclave step. Use caution when handling. 9. Sampling, Test Test Specimens, and Test Test Units 9.1 Test specimens suitable for analysis by this proce procedure dure are: 9.1.1 9.1 .1 Prepar Prepared ed bio biomass mass pre prepar pared ed acco accordi rding ng to Prac Practice tice E1757,, and E1757 9.1.2 Extract Extractives-f ives-free ree materia materiall prepar prepared ed accord according ing to Test Method E1690 Method E1690..
11.3 If extractives-fre 11.3 extractives-freee material is used, determine determine the total solids content of the extractive-free material by Test Method E1756 and E1756 and record this value as %T ext . 11.4 Weigh 300 6 10 mg of the prepared or extractives-free sample to the nearest 0.1 mg and place in 16x 100 mm glass test tube. Record as m1, the initial mass of sample in grams.
10. Calibration and Standardizatio Standardization n 10.1 Pr 10.1 Prep epar aree a se seri ries es of th thre reee to si six x su suga garr st stan anda dard rdss in deionized deioni zed water at concen concentration trationss appro appropriate priate for prepa preparing ring calibra cali bration tion cur curves ves to qua quanti ntitfy tfy each sug sugar ar of int intere erest. st. An HPX-87C3 column, or equivalent, is used to analyze glucose, xylose, and arabinose. If mannose and galactose are also to be quantified, an HPX-87P 3 column, or equivalent, must be used instead.. Typical instead ypically ly,, the concen concentration trationss of these sugar standards cover the range starting at the detection limit of the instrument and extending up to 4.0 mg/mL.
NOTE 3—Warning: 72 % w/w sulfuric sulfuric aci acid d is ver very y cor corros rosive ive and should be handled by trained personnel only.
11.5 Add 3.00 6 0.01 mL (4.92 6 0.01 g) of 72 % w/w 11.5 H2SO4 to the test tube containing the sample and stir for 1 min or until thoroughly mixed. 11.6 11 .6 Place the test tube containing containing the sample sample into the water bath co bath cont ntro roll lled ed to 30 6 1°C 1°C an and d hy hydr drol olyz yzee fo forr 1h 1h.. Sti Stirr approximately every 15 min to ensure the sample is completely mixed and wet.
10.2 Prepar Preparee an independent independent CVS, as descri described bed in 8.1.2, for each set of calibration standards, using sugars obtained from a source other than that used in preparing the calibration standards.. The CVS will contai dards contain n precis precisely ely known amounts of each sugar contained contained in the calibration standards, standards, at a conce concentratio ntration n in the middle of the validated range of the calibration curve. The CVS will be analyzed after each calibration curve and at
11.7 Weigh out 300 6 10 mg of each high purity sugar 11.7 standard (dried at 45°C), described in 8.1.4, to the nearest 0.1 mg and place each in its own individual 16x 100 mm glass test tube. Add acid and hydrolyze these sugars as described in the previo pre vious us two steps. These SRS’s will be tak taken en thr throug ough h the remaining steps in the procedure in parallel with the samples. 3
E1758 − 01 (2015) The calculated recovery of the SRS will be used to correct for losses caused by the destruction of sugars during the hydrolysis process.
11.17 Analyz 11.17 Analyzee the calibration sugar standards, standards, the CVS’s, the hydrolyzed SRS’s, and the hydrolyzed samples by HPLC using usi ng eith either er the HPX HPX-87 -87C C3 or HPX-8 HPX-87P 7P3 col column umn,, or thei theirr equiva equ ivalent lents, s, as des descri cribed bed in 11.1. 11.1. For ma many ny an anal alys yses es,, it is useful to run the same samples on both columns and compare the results.
11.8 Tran 11.8 Transfer sfer each hydrolyzate hydrolyzate to a glass bottle and dilute to 4 % w/w acid conce concentratio ntration n by adding 84.00 84.00 6 0.04 mL water. Be careful to transfer all the residual solids along with the hydrolysis liquor. The total mass added to the tared bottle is 89.22 g (0.3 g sample, 4.92 g 72 % w/w H 2SO4, and 84.00 g deionized water). Because the relative density of the 4 % w/w acid solution is 1.0250, the total volume of solution, V F , is 87.0 mL.
11.18 11 .18 The following instrumental instrumental conditions are used for both the HPX both HPX-87 -87C C3 and the HPX HPX-87 -87P P3 colu columns mns,, or the their ir equivalents: 11.18.1 Sample Volume— 50 50 µL, 11.18.2 Eluant— 0.2 0 .2 µm fil filter tered ed,, 18 meg megoh ohm m de deio ioni nize zed d water (de-gassed with helium or vacuum), 11.18.3 Flow Rate— 0.6 0.6 mL/min, 11.18.4 Column Temperature— 85°C, 85°C, 11.18.5 Detector— refractive refractive index, and 11.18.6 Run Time— 20 20 min data collection, 15 min post-run.
11.9 Stopp 11.9 Stopper er the bottles and crimp the alumin aluminum um seals into place in preparation for the next step. 11.10 11 .10 Set the autoclave autoclave to a liquid vent vent cycle to prevent loss of sample from the bottle in the event of a loose crimp seal. Autoclave the sample in the sealed bottle for 1 h at 121 6 3°C. NOTE 4—Warning: Handle Handle the sea sealed led bot bottle tle wit with h cau cautio tion n aft after er the autoclave step, as it may have become pressurized.
12. Calc Calculat ulation ion
11. 1.1 11 Af Afte terr co comp mplet leting ing th thee au auto tocla clave ve cy cycle cle,, all allow ow th thee bottles to cool for about 20 min at room temperature before removing the seals and stoppers.
12.1 Create a calibration calibration curve by linear regression regression analysis analysis for each sugar to be quantified. From these curves, determine thee co th conc ncen entr trati ation on in mg mg/mL /mL of th thee su suga gars rs pr pres esen entt in ea each ch solution analyzed by HPLC.
11. 1.12 12 Th These ese au auto tocla clave ved d so solu lutio tions ns ma may y als also o be us used ed fo forr determining acid-insoluble determining acid-insoluble residu residuee or acid-s acid-soluble oluble lignin, or both, in parallel with this carbohydrate determination.
12.2 Calcula Calculate te the amoun amountt of sugar recovered, recovered, in percent, from each sugar recovery standard taken through the two-stage hydrolysis. This will estimate the amount of each individual sugar destroyed during the hydr hydrolysis olysis procedure: procedure:
NOTE 5—If aci acid-i d-inso nsolub luble le res residu iduee or aci acid-s d-solu oluble ble lig lignin nin,, or bot both, h, determinations are to be conducted on a sample, the residual solids must be collected by filtering the hydrolyzate through an ashed and weighed filtering crucible before proceeding with the carbohydrate determination. Refe Re ferr to Tes estt Me Meth thod od E1721 for for det details ails.. If an aci acid-s d-solu oluble ble lign lignin in determination is to be conducted, a portion of the filtrate must be reserved for analysis. Acid-soluble lignin should be analyzed within 24 h, preferably within 6 h of hydrolysis.
% recoverysrs 5
C 2 C 1
3 100 %
(1)
where: known concentrat concentration ion of sug sugar ar recovery recovery standard standard beC 1 = known fore hydrolysis, in mg/mL, and C 2 = conce concentratio ntration n of sugar recovery recovery standard standard detected by HPLC after hydrolysis, in mg/mL.
11.1 1.13 3 Tr Trans ansfer fer a 20 mL aliquot aliquot of eac each h hyd hydrol rolyza yzate, te, or filtrate, to 50 mL Erlenmeyer flasks. 11.14 Neutr 11.14 Neutralize alize with calcium carbonate carbonate to a pH between 5 and 6. Do not ove overr-neu neutra tralize lize.. Add the calc calcium ium car carbon bonate ate slowly to avoid problems with foaming.
12.3 Use the per 12.3 percen centag tagee rec recove overy ry of the sug sugar ar rec recove overy ry standar stan dard d to cor correc rectt sug sugar ar con concen centra tratio tion n val values ues (in mg/ mg/mL) mL) obtained from HPLC for each hydrolyzed sample:
11.1 1.15 5 Filt Filter er eac each h neu neutra tralize lized d hyd hydrol rolyza yzate te dir directl ectly y into a capped test tube using a 3 mL syringe with a 0.2 µm filter attac att ache hed d an and d pl place ace in ic icee ba bath th.. If th thee hy hydr drol olyz yzat atee is to be analyzed without dilution, filter an additional portion directly into an auto autosam sampler pler vial. If the sol solutio ution n req requir uires es dilu dilution tion,, withdraw the necessary amount, dilute as required, then filter the diluted sample into an autosampler vial.
C spl C corr 5
% R srs
(2 )
100%
where: %Rsrs = percen percentt rec recove overy ry of sug sugar ar reco recover very y stan standar dard, d, as determined determ ined in in 12.2 12.2,, C corr = con concen centra tration tion of sugar sugar in hyd hydrol rolyze yzed d sample corcorrected for hydrolysis, in mg/mL, and C spl = con concen centra tration tion of sugar sugar in hydrolyz hydrolyzed ed sample sample detected by HPLC, in mg/mL.
NOTE 6—The initial glucose concentrations of the samples could be determined using an alternative technique, such as a glucose analyzer,6 to predict whether or not the sugars in the samples will fall within the linear range of the analysis.
11.16 Place the remainder 11.16 remainder of each filtered sample sample into the refrigerator as soon as possible, and reserve in case a repeat analysis is required. The samples should be stored for no longer than two weeks.
12.4 If the bio 12.4 biomass mass was pre prepar pared ed acco accordi rding ng to Par Partt A of Practice E1757 Practice E1757,, calculate the percent, by mass, of each sugar presentt in the as-rec presen as-received eived sample, on a 105° 105°C C dried mass basis as follows: % sugar 5
6
A YSI, Model 2700 Select, available from Yellow Yellow Springs Instrument Co., Inc., Yellow Springs, OH 45387, has been found to be satisfactory for this purpose.
4
C corr 3 V F
% T ad m1 3 100%
3 100 %
(3)
E1758 − 01 (2015) where: = ini initial tial mas masss of samp sample, le, in mg, m 1 = volume volume of filtr filtrate, ate, 87. 87.0 0 mL, V F C corr = con concen centrat tration ion of sugar sugar in hyd hydrol rolyze yzed d sample sample corrected rec ted for hydrolys hydrolysis, is, as dete determi rmined ned in 13. 13.3, 3, in mg/mL, and percen cent, t, by mas mass, s, of total solids solids of the air-dr air-dried ied %T ad = per sample determined at 105°C as described by Test Method E1756 Method E1756..
m1 = ini initial tial mas masss of of extra extracted cted sam sample ple,, in mg, and %T ext = percen percent, t, by mass, of total solids solids of the extracted extracted sample determined at 105°C as described by Test Method E1756 Method E1756..
12.6.2 Cor 12.6.2 Correc rectt the percent, percent, by mas mass, s, of sugar value value on an extrac ext ractive tives-f s-free ree bas basis, is, calc calcula ulated ted abo above, ve, to an as rec receiv eived ed (whole sample) 105°C dried mass basis as follows: % sugar whole sample 5 % sugar extractives2 free 3
12.5 If the biomass was prepared prepared according according to Part B or C of Practice E1757 Practice E1757 calculate calculate the percent, by mass, of each sugar present in the as-received hydrolyzed sample, on a 105°C dried mass basis as follows: % sugar 5
where: C corr V F m1 %T 105
%T prep
C corr 3 V F
% T 105 m1 3 % T prep
3 100 %
where: % sugar extractives-free
(4)
% extractives
m1 3
% T ext
3 100 %
= the perc percent, ent, by mass, mass, of of sugar sugar on an extrac ext ractive tives-f s-free ree 105 105°C °C dri dried ed mas masss basis, as determined in 12.6.1 in 12.6.1,, and = the per percen cent, t, by mas mass, s, of ext extrac ractive tivess in the extracted sample as described in Test Method E1690 Method E1690..
13. Repo Report rt 13.1 13. 1 Rep Report ort the per percen cent, t, by mass, of sug sugar ar present present in the sample, to two decimal places, on a 105°C dried mass basis. 14. Pre Precisi cision on and Bias 14.1 Da 14.1 Data ta ob obtai taine ned d by re repl plica icate te te testi sting ng of gl gluc ucos osee in a 3 hybrid poplar in one laboratory, using a HPX-87P column, gave ga ve a st stan anda dard rd de devi viati ation on of 1. 1.90 90 %, an and d a co coef efffic icien ientt of 7,8 variation percent (CV) of 3.95 %.
12.6 If the biomass was prepa prepared red according according to Test Method E1690,, fir E1690 first st cal calcu cula late te th thee pe perc rcen ent, t, by ma mass ss,, of ea each ch su suga garr present on an extractives-free 105°C dried mass basis and then correct this value to an as received (whole sample) 105°C dried mass basis. 12.6.1 Calculate the percent, by mass, of sugar sugar present on an extractives-free basis as follows: % sugar extractives2 free 5
100% (6 )
= correc corrected ted sugar sugar concent concentrat ration ion of the hydroly hydrolyzed zed sample, in mg/mL, = vol volume ume of filtr filtrate, ate, 87. 87.0 0 mL, = ini initial tial mas masss of pre prepar pared ed sam sample, ple, in mg, = pe perc rcen ent, t, by ma mass ss,, of total total so solid lidss in the sa samp mple, le, dried dri ed at 105 105°C, °C, as det determ ermine ined d by Test Test Meth Method od E1756,, and E1756 = pe perc rcen ent, t, by mass, mass, of total total so solid lidss of the sa samp mple le prepared by freeze drying, %T fd , or by drying at 45°C, %T45, as determined by Practice E1757 Practice E1757..
C corr 3 V F
~ 100% 2 % extractives!
14.2 Dat 14.2 Dataa obt obtain ained ed by rep replica licate te tes testing ting of glu glucos cosee in an extrac ext ractive tives-f s-free ree hyb hybrid rid pop poplar lar in five lab labora orator tories ies,, usi using ng a HPX-87P3 column, gave a standard deviation of 1.90 %, by mass, and a CV, by mass, of 4.0 %. 7,8 15. Keyw Keywords ords 15.1 agricu agricultural ltural residue; bioma biomass; ss; carbo carbohydr hydrates; ates; fermen fermen-tation residue; herba herbaceous; ceous; wastepaper; wood
(5)
100% 7
Ehrman Ehr man,, C. I., and Him Himmel mel,, M. E. E.,, “S “Simu imulta ltaneo neous us Sac Saccha charifi rificat cation ion and Fermentation of Pretreated Biomass: Improving Mass Balance Closure,” Biotechnology Techniques, 8(2), 1994, pp. 99–104. 8 Vinzant, T. B., Ponfick, L., Nagle, N. J., Ehrman, C. I., Reynolds, J. B., and Himmel, M. E., “SSF Comparison of Selected Woods from Southern Sawmills” Applied Biochemical Biotechnology, 45/46, 1994, pp. 611–626.
where: corrected ted sugar concentrat concentration ion of the hydrolyze hydrolyzed d C corr = correc sample, in mg/mL, V F = vol volume ume of filtr filtrate, ate, 87. 87.0 0 mL,
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