ASTM E1758-01(2015)
(Test Method)Standard Test Method for Determination of Carbohydrates in Biomass by High Performance Liquid Chromatography
Standard Test Method for Determination of Carbohydrates in Biomass by High Performance Liquid Chromatography
SIGNIFICANCE AND USE
4.1 The percentage, by mass, of sugar content is used in conjunction with other assays to determine the total composition of biomass samples.
SCOPE
1.1 This test method covers the determination of carbohydrates present in a biomass sample, expressed as the percent, by mass, of each sugar on a 105°C dried mass basis.
Note 1: The percent sugar must be corrected for the water of hydrolysis before calculating the actual mass percent of the polysaccharide in the original biomass sample.
1.2 Sample materials suitable for this procedure include hard and soft woods, herbaceous materials (such as switchgrass and sericea), agricultural residues (such as corn stover, wheat straw, and bagasse), wastepaper (such as office waste, boxboard, and newsprint), acid or alkaline-pretreated biomass (washed free of any residual acid or alkali), and the solid fraction of fermentation residues. All results are reported relative to the 105°C oven-dried mass of the sample.
1.3 The options for the types of samples to be analyzed in this test method are as follows:
1.3.1 Prepared Biomass Samples:
1.3.1.1 Air Dried (%Tad)—The percent, by mass, of total solids of the air-dried sample.
1.3.1.2 45°C Dried (%T45)—The percent, by mass, of total solids of the 45°C dried sample.
1.3.1.3 Freeze Dried (%Tfd)—The percent, by mass, of total solids of the freeze dried sample.
1.3.2 Extractives-Free Sample (%T ext)—The percent, by mass, of total solids of the extracted sample determined at 105°C.
1.4 The values stated in SI units are to be regarded as the standard.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Specific precautionary statements are given in Note 3 and Note 4.
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Designation: E1758 − 01 (Reapproved 2015)
Standard Test Method for
Determination of Carbohydrates in Biomass by High
Performance Liquid Chromatography
This standard is issued under the fixed designation E1758; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
The carbohydrates making up a major portion of biomass samples are polysaccharides constructed
primarilyofglucose,xylose,arabinose,galactose,andmannosesubunits.Thepolysaccharidespresent
in a biomass sample can be hydrolyzed to their component sugar monomers by sulfuric acid in a
two-stage hydrolysis process. These monosaccharides can then be quantified by ion-moderated
partition HPLC.
1. Scope 1.4 The values stated in SI units are to be regarded as the
standard.
1.1 This test method covers the determination of carbohy-
drates present in a biomass sample, expressed as the percent,
1.5 This standard does not purport to address all of the
by mass, of each sugar on a 105°C dried mass basis.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
NOTE 1—The percent sugar must be corrected for the water of
priate safety and health practices and determine the applica-
hydrolysis before calculating the actual mass percent of the polysaccha-
ride in the original biomass sample.
bility of regulatory limitations prior to use. Specific precau-
tionary statements are given in Note 3 and Note 4.
1.2 Sample materials suitable for this procedure include
hardandsoftwoods,herbaceousmaterials(suchasswitchgrass
2. Referenced Documents
and sericea), agricultural residues (such as corn stover, wheat
straw, and bagasse), wastepaper (such as office waste,
2.1 ASTM Standards:
boxboard, and newsprint), acid or alkaline-pretreated biomass
D1193Specification for Reagent Water
(washed free of any residual acid or alkali), and the solid
E1690Test Method for Determination of Ethanol Extrac-
fraction of fermentation residues. All results are reported
tives in Biomass
relative to the 105°C oven-dried mass of the sample.
E1721Test Method for Determination of Acid-Insoluble
1.3 The options for the types of samples to be analyzed in
Residue in Biomass
this test method are as follows:
E1756Test Method for Determination of Total Solids in
1.3.1 Prepared Biomass Samples:
Biomass
1.3.1.1 Air Dried (%T )—The percent, by mass, of total
E1757Practice for Preparation of Biomass for Composi-
ad
solids of the air-dried sample.
tional Analysis
1.3.1.2 45°C Dried (%T )—The percent, by mass, of total
solids of the 45°C dried sample.
3. Terminology
1.3.1.3 Freeze Dried (%T )—The percent, by mass, of total
fd
3.1 Definitions of Terms Specific to This Standard:
solids of the freeze dried sample.
3.1.1 as received biomass—the biomass material as it is
1.3.2 Extractives-Free Sample (%T )—The percent, by
ext
received in its field or process collected state.
mass, of total solids of the extracted sample determined at
105°C.
3.1.2 oven-dried mass—themoisture-freemassofabiomass
sample dried at 105°C as described in Test Method E1756.
This test method is under the jurisdiction of ASTM Committee E48 on
BioenergyandIndustrialChemicalsfromBiomassandisthedirectresponsibilityof
Subcommittee E48.05 on Biomass Conversion. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
CurrenteditionapprovedJune1,2015.PublishedJuly2015.Originallyapproved contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
in 1995. Last previous edition approved in 2007 as E1758–01(2007). DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/E1758-01R15. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1758 − 01 (2015)
3.1.3 prepared biomass—material that has been treated 4. Significance and Use
according to Practice E1757 in order to raise the total solids
4.1 The percentage, by mass, of sugar content is used in
content above 85%, by mass, based on an oven-dried solids
conjunction with other assays to determine the total composi-
mass.
tion of biomass samples.
3.2 Abbreviations—Abbreviations of standards used in the
procedure, and definitions of terms used in the calculations are 5. Interferences
as follows:
5.1 Samples with high protein content may result in the
3.2.1 C —known concentration of sugar recovery standard
1 percentage, by mass, of sugar values being biased low, as a
before hydrolysis, in mg/mL.
consequence of protein binding with some monosaccharides.
3.2.2 C —concentrationofsugarrecoverystandarddetected
5.2 Test specimens not suitable for analysis by this proce-
by HPLC after hydrolysis, in mg/mL.
dure include alkaline and acid-pretreated biomass samples that
have not been washed. Unwashed pretreated biomass samples
3.2.3 C — concentration of sugar in hydrolyzed sample
corr
containing free acid or alkali may change visibly on heating.
corrected for hydrolysis, in mg/mL.
3.2.4 C — concentration of sugar in hydrolyzed sample
spl
6. Apparatus
detected by HPLC, in mg/mL.
6.1 Analytical Balance, readable to 0.1 mg.
3.2.5 CVS (calibration verification standard)— standards
6.2 Autoclave, capable of maintaining 121 6 3°C.
used in determining the quality of the calibration curve as well
6.3 Convection Ovens, temperature control to 45 6 3 and
as the quality of the standard reagents used in preparing the
calibration standards. 105 6 3°C.
6.4 Desiccator, using anhydrous calcium sulfate.
3.2.6 m —initial mass of sample, in mg.
6.5 Guard Columns, cartridges appropriate for the column
3.2.7 % extractives—thepercentage,bymass,ofextractives
used.
in the prepared biomass sample as described in Test Method
E1690.
NOTE2—DeashingguardcolumncartridgesfromBioRad, oftheionic
+ − 3
form H /CO , are an option when using an HPX-87P column, or
3.2.8 %R — percent recovery of sugar recovery standard,
srs
equivalent.Thesecartridgesareeffectiveineliminatingbaselineramping.
as determined in 13.2.
6.6 Hewlett Packard Model 1090 HPLC, or equivalent,
3.2.9 %sugar —the percentage, by mass, of
extractives-free with refractive index detector.
sugar on an extractives-free 105°C dry weight basis, as
3 3
6.7 HPLC Columns, BioRad HPX-87C or HPX-87P, or
determined in 13.6.1.
both, or equivalent.
3.2.10 % sugar —thecorrectedmasspercentsugar
whole sample
6.8 Water Bath, set at 30 6 1°C.
value on an extractives-free basis corrected to an as received
(whole sample) 105°C dry mass basis.
7. Reagents and Materials
3.2.11 %T —percentage, by mass, of total solids of the
7.1 Chemicals:
sample prepared by drying at 45°C, as described by Practice
7.1.1 Purity of Reagents—Reagent grade chemicals shall be
E1757.
used in all tests. Unless otherwise indicated, it is intended that
3.2.12 %T —percentage, by mass, of total solids in the all reagents conform to the specifications of the Committee on
sample, dried at 105°C, as determined by Test Method E1756.
Analytical Reagents of theAmerican Chemical Society where
such specifications are available. Other grades may be used,
3.2.13 %T — percentage, by mass, of total solids of the
ad
provided it is first ascertained that the reagent is of sufficiently
air-dried sample determined at 105°C as described by Test
high purity to permit its use without lessening the accuracy of
Method E1756.
the determination.
3.2.14 %T — percentage, by mass, of total solids of the
ext
extracted sample determined at 105°C as described by Test
Method E1756.
Thesolesourceofsupplyoftheapparatusknowntothecommitteeatthistime
is BioRad Aminex®, HPX-87C and Aminex® HPX-87P, available from BioRad,
3.2.15 %T — percentage, by mass, of total solids of the
fd
Main Office, 3300 Regatta Boulevard, Richmond, CA 94804. If you are aware of
samplepreparedbyfreezedrying,asdescribedbyTestMethod
alternative suppliers, please provide this information to ASTM International
E1756.
Headquarters.Your comments will receive careful consideration at a meeting of the
responsible technical committee, which you may attend.
3.2.16 %T — percentage, by mass, of total solids of the
prep 4
AvailablefromHewlett-Packard,HPAnalyticalDirect,2850CentervilleRoad,
samplepreparedbyfreezedrying, %T ,orbydryingat45°C,
fd Wilmington, DE 19808.
Reagent Chemicals, American Chemical Society Specifications , American
%T , as determined by Practice E1757.
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
3.2.17 SRS (sugar recovery standards)—standards used to
listed by the American Chemical Society, see Analar Standards for Laboratory
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
determine sugar recovery after hydrolysis.
and National Formulary,U.S.PharmaceuticalConvention,Inc.(USPC),Rockville,
3.2.18 V —volume of filtrate, 87.0 mL. MD.
F
E1758 − 01 (2015)
7.1.2 Purity of Water—Unless otherwise indicated, refer- regular intervals in the HPLC sequence, as dictated by good
ences to water shall be understood to mean reagent water as laboratory practice.The CVS is used in confirming the quality
defined by Type 1 of Specification D1193. of the calibration curve(s) and the standard reagents used in
7.1.3 Calcium Carbonate. preparing the calibration standards. An additional benefit is
7.1.4 High-Purity Sugars (98 %+, By Mass)—Two sets of obtainedbybracketinggroupsofsamplesinthesequencewith
glucose, xylose, galactose, arabinose, and mannose, meeting the CVS, assuring the analyst of the quality of the calibration
the requirements described above, dried at 45°C. The sugars curve throughout the run.
are used in preparing calibration standards, calibration verifi-
cation standards (CVS), and sugar recovery standards (SRS).
11. Procedure
The sugars used in preparing the calibration standards should
11.1 An overview of the overall analytical sequence is as
be from a source (manufacturer or lot) other than that used in
follows:
preparing the CVS. Either set of sugars may be used for
11.1.1 Hydrolysis of sample with 72% sulfuric acid,
preparing the SRS solutions used in determining sugar recov-
11.1.2 Hydrolyzate dilution and autoclaving,
eries after hydrolysis.
11.1.3 Filtrationofinsolublesifseparateanalysisisdesired,
7.1.5 Sulfuric Acid Solution (72 % w/w or 12.00 6 0.02
M)—Slowlyadd665mLofconcentratedsulfuricacid(H SO ) 11.1.4 Neutralization of hydrolyzate,
2 4
to 300 mL of water while cooling in an ice bath and stirring.
11.1.5 Filtration of sample prior to HPLC analysis,
Allowtocometoroomtemperature.Adjusttherelativedensity
11.1.6 HPLC analysis of sugar standards, CVS, SRS, and
to 1.6389 6 0.0012 at 15.6°C/15.6°C.
hydrolyzate samples, and
7.2 Materials: 11.1.7 Calculation of sugar contents.
7.2.1 Autosampler Vials, with crimp top seals to fit.
11.2 For prepared biomass samples, determine the total
7.2.2 Disposable Syringes, 3 mL.
solids by Test Method E1756 and record the total solids value
7.2.3 Disposable Syringe Filters, nylon, 0.2 µm.
as %T . This prepared sample should be stored in a manner
7.2.4 Glass Serum Bottles, crimp top style, 125 mL, with
to ensure its moisture content does not change before the
rubber stoppers and aluminum seals to fit.
analysis begins.
11.2.1 IfTest MethodAof this practice is used (air drying),
8. Hazards
determine the total solids content of this prepared sample by
8.1 Handle the sulfuric acid carefully to avoid contact with
Test Method E1756 and record the total solids value as %T .
ad
skin or clothing, as it is corrosive.
11.2.2 If Test Method B of this practice is used (drying at
8.2 The glass bottles are hot and may be pressurized after
45°C), record the total solids calculated in this practice, %T ,
the autoclave step. Use caution when handling.
as %T .
prep
11.2.3 If Test Method C of this practice is used (freeze
9. Sampling, Test Specimens, and Test Units
drying),recordthetotalsolidscalculatedinthispractice, %T ,
fd
9.1 Test specimens suitable for analysis by this procedure as %T .
prep
are:
11.3 If extractives-free material is used, determine the total
9.1.1 Prepared biomass prepared according to Practice
solids content of the extractive-free material by Test Method
E1757, and
E1756 and record this value as %T .
ext
9.1.2 Extractives-free material prepared according to Test
11.4 Weigh300 610mgofthepreparedorextractives-free
Method E1690.
sample to the nearest 0.1 mg and place in 16x 100 mm glass
10. Calibration and Standardization test tube. Record as m , the initial mass of sample in grams.
10.1 Prepare a series of three to six sugar standards in
NOTE 3—Warning: 72% w/w sulfuric acid is very corrosive and
deionized water at concentrations appropriate for preparing
should be handled by trained personnel only.
calibration curves to quantitfy each sugar of interest. An
11.5 Add 3.00 6 0.01 mL (4.92 6 0.01 g) of 72% w/w
HPX-87C column, or equivalent, is used to analyze glucose,
H SO tothetesttubecontainingthesampleandstirfor1min
2 4
xylose, and arabinose. If mannose and galactose are also to be
or until thoroughly mixed.
quantified, an HPX-87P column, or equivalent, must be used
11.6 Placethetesttubecontainingthesampleintothewater
instead. Typically, the concentrations of these sugar standards
bath controlled to 30 6 1°C and hydrolyze for 1h. Stir
cover the range starting at the detection limit of the instrument
approximatelyevery15mintoensurethesampleiscompletely
and extending up to 4.0 mg/mL.
mixed and wet.
10.2 PrepareanindependentCVS,asdescribedin8.1.2,for
each set of calibration standards, using sugars obtained from a 11.7 Weigh out 300 6 10 mg of each high purity sugar
source other than that used in preparing the calibration stan- standard (dried at 45°C), described in 8.1.4, to the nearest 0.1
dards.The CVS will contain precisely known amounts of each mgandplaceeachinitsownindividual16x100mmglasstest
sugar contained in the calibration standards, at a concentration tube.Add acid and hydrolyze these sugars as described in the
in the middle of the validated range of the calibration curve. previous two steps. These SRS’s will be taken through the
The CVS will be analyzed after each calibration curve and at remaining steps in the procedure in parallel with the samples.
E1758 − 01 (2015)
The calculated recovery of the SRS will be used to correct for 11.17 Analyze the calibration sugar standards, the CVS’s,
lossescausedbythedestructionofsugarsduringthehydrolysis the hydrolyzed SRS’s,
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E1758 − 01 (Reapproved 2007) E1758 − 01 (Reapproved 2015)
Standard Test Method for
Determination of Carbohydrates in Biomass by High
Performance Liquid Chromatography
This standard is issued under the fixed designation E1758; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
The carbohydrates making up a major portion of biomass samples are polysaccharides 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-stage hydrolysis process. These monosaccharides can then be quantified by ion-moderated
partition HPLC.
1. Scope
1.1 This test method covers the determination of carbohydrates present in a biomass sample, expressed as the percent, by mass,
of each sugar on a 105°C dried mass basis.
NOTE 1—The percent sugar must be corrected for the water of hydrolysis before calculating the actual mass percent of the polysaccharide in the original
biomass sample.
1.2 Sample materials suitable for this procedure include hard and soft woods, herbaceous materials (such as switchgrass and
sericea), agricultural residues (such as corn stover, wheat straw, and bagasse), wastepaper (such as office waste, boxboard, and
newsprint), acid or alkaline-pretreated biomass (washed free of any residual acid or alkali), and the solid fraction of fermentation
residues. All results are reported relative to the 105°C oven-dried mass of the sample.
1.3 The options for the types of samples to be analyzed in this test method are as follows:
1.3.1 Prepared Biomass Samples:
1.3.1.1 Air Dried (%T )—The percent, by mass, of total solids of the air-dried sample.
ad
1.3.1.2 45°C Dried (%T )—The percent, by mass, of total solids of the 45°C dried sample.
1.3.1.3 Freeze Dried (%T )—The percent, by mass, of total solids of the freeze dried sample.
fd
1.3.2 Extractives-Free Sample (%T )—The percent, by mass, of total solids of the extracted sample determined at 105°C.
ext
1.4 The values stated in SI units are to be regarded as the standard.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory
limitations prior to use. Specific precautionary statements are given in Note 3 and Note 4.
2. Referenced Documents
2.1 ASTM Standards:
D1193 Specification for Reagent Water
E1690 Test Method for Determination of Ethanol Extractives in Biomass
E1721 Test Method for Determination of Acid-Insoluble Residue in Biomass
E1756 Test Method for Determination of Total Solids in Biomass
E1757 Practice for Preparation of Biomass for Compositional Analysis
This test method is under the jurisdiction of ASTM Committee E48 on Bioenergy and Industrial Chemicals from Biomass and is the direct responsibility of Subcommittee
E48.05 on Biomass Conversion.
Current edition approved Nov. 15, 2007June 1, 2015. Published January 2008July 2015. Originally approved in 1995. Last previous edition approved in 20012007 as
1758–01.E1758–01(2007). DOI: 10.1520/E1758-01R07.10.1520/E1758-01R15.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. 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
E1758 − 01 (2015)
3. Terminology
3.1 Definitions of Terms Specific to This Standard:
3.1.1 as received biomass—the biomass material as it is received in its field or process collected state.
3.1.2 oven-dried mass—the moisture-free mass of a biomass sample dried at 105°C as described in Test Method E1756.
3.1.3 prepared biomass—material that has been treated according to Practice E1757 in order to raise the total solids content
above 85 %, by mass, based on an oven-dried solids mass.
3.2 Abbreviations—Abbreviations of standards used in the procedure, and definitions of terms used in the calculations are as
follows:
3.2.1 C —known concentration of sugar recovery standard before hydrolysis, in mg/mL.
3.2.2 C —concentration of sugar recovery standard detected by HPLC after hydrolysis, in mg/mL.
3.2.3 C — concentration of sugar in hydrolyzed sample corrected for hydrolysis, in mg/mL.
corr
3.2.4 C — concentration of sugar in hydrolyzed sample detected by HPLC, in mg/mL.
spl
3.2.5 CVS (calibration verification standard)— 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.
3.2.6 m —initial mass of sample, in mg.
3.2.7 % extractives—the percentage, by mass, of extractives in the prepared biomass sample as described in Test Method E1690.
3.2.8 %R — percent recovery of sugar recovery standard, as determined in 13.2.
srs
3.2.9 %sugar —the percentage, by mass, of sugar on an extractives-free 105°C dry weight basis, as determined in
extractives-free
13.6.1.
3.2.10 % sugar —the corrected mass percent sugar value on an extractives-free basis corrected to an as received
whole sample
(whole sample) 105°C dry mass basis.
3.2.11 %T —percentage, by mass, of total solids of the sample prepared by drying at 45°C, as described by Practice E1757.
3.2.12 %T —percentage, by mass, of total solids in the sample, dried at 105°C, as determined by Test Method E1756.
3.2.13 %T — percentage, by mass, of total solids of the air-dried sample determined at 105°C as described by Test Method
ad
E1756.
3.2.14 %T — percentage, by mass, of total solids of the extracted sample determined at 105°C as described by Test Method
ext
E1756.
3.2.15 %T — percentage, by mass, of total solids of the sample prepared by freeze drying, as described by Test Method E1756.
fd
3.2.16 %T — percentage, by mass, of total solids of the sample prepared by freeze drying, % T , or by drying at 45°C, %
prep fd
T , as determined by Practice E1757.
3.2.17 SRS (sugar recovery standards)—standards used to determine sugar recovery after hydrolysis.
3.2.18 V —volume of filtrate, 87.0 mL.
F
4. Significance and Use
4.1 The percentage, by mass, of sugar content is used in conjunction with other assays to determine the total composition of
biomass samples.
5. Interferences
5.1 Samples with high protein content may result in the percentage, by mass, of sugar values being biased low, as a consequence
of protein binding with some monosaccharides.
5.2 Test specimens not suitable for analysis by this procedure 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.
6. Apparatus
6.1 Analytical Balance, readable to 0.1 mg.
6.2 Autoclave, capable of maintaining 121 6 3°C.
6.3 Convection Ovens, temperature control to 45 6 3 and 105 6 3°C.
6.4 Desiccator, using anhydrous calcium sulfate.
6.5 Guard Columns, cartridges appropriate for the column used.
E1758 − 01 (2015)
3 + − 3
NOTE 2—Deashing guard column cartridges from BioRad, of the ionic form H /CO , are an option when using an HPX-87P column, or equivalent.
These cartridges are effective in eliminating baseline ramping.
6.6 Hewlett Packard Model 1090 HPLC, or equivalent, with refractive index detector.
3 3
6.7 HPLC Columns, BioRad HPX-87C or HPX-87P, or both, or equivalent.
6.8 Water Bath, set at 30 6 1°C.
7. Reagents and Materials
7.1 Chemicals:
7.1.1 Purity of Reagents—Reagent grade 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. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
7.1.2 Purity of Water—Unless otherwise indicated, references to water shall be understood to mean reagent water as defined by
Type 1 of Specification D1193.
7.1.3 Calcium Carbonate.
7.1.4 High-Purity Sugars (98 % +, By Mass)—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 preparing the CVS. Either set of sugars may be used for preparing the SRS
solutions used in determining sugar recoveries after hydrolysis.
7.1.5 Sulfuric Acid Solution (72 % w/w or 12.00 6 0.02 M)—Slowly add 665 mL of concentrated sulfuric acid (H SO ) to 300
2 4
mL of water while cooling in an ice bath and stirring. Allow to come to room temperature. Adjust the relative density to 1.6389
6 0.0012 at 15.6°C/15.6°C.
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.
8. Hazards
8.1 Handle the sulfuric acid carefully to avoid contact with skin or clothing, as it is corrosive.
8.2 The glass bottles are hot and may be pressurized after the autoclave step. Use caution when handling.
9. Sampling, Test Specimens, and Test Units
9.1 Test specimens suitable for analysis by this procedure are:
9.1.1 Prepared biomass prepared according to Practice E1757, and
9.1.2 Extractives-free material prepared according to Test Method E1690.
10. Calibration and Standardization
10.1 Prepare a series of three to six sugar standards in deionized water at concentrations appropriate for preparing calibration
curves to quantitfy each sugar of interest. An HPX-87C column, or equivalent, is used to analyze glucose, xylose, and arabinose.
If mannose and galactose are also to be quantified, an HPX-87P column, or equivalent, must be used instead. Typically, the
concentrations of these sugar standards cover the range starting at the detection limit of the instrument and extending up to 4.0
mg/mL.
10.2 Prepare an independent CVS, as described 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 contain precisely known amounts of each sugar
contained in the calibration standards, at a concentration in the middle of the validated range of the calibration curve. The CVS
will be analyzed after each calibration curve and at 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 the calibration
The sole source of supply of the apparatus known to the committee at this time is BioRad Aminex®, HPX-87C and Aminex® HPX-87P, available from BioRad, Main
Office, 3300 Regatta Boulevard, Richmond, CA 94804. If you are aware of alternative suppliers, please provide this information to ASTM International Headquarters. Your
comments will receive careful consideration at a meeting of the responsible technical committee, which you may attend.
Available from Hewlett-Packard, HP Analytical Direct, 2850 Centerville Road, Wilmington, DE 19808.
Reagent Chemicals, American Chemical Society Specifications , American Chemical Society, Washington, DC. For suggestions on the testing of reagents not listed by
the American Chemical Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National
Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville, MD.
E1758 − 01 (2015)
standards. An additional benefit 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. Procedure
11.1 An overview of the overall analytical sequence is as follows:
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 sugar standards, CVS, SRS, and hydrolyzate samples, and
11.1.7 Calculation of sugar contents.
11.2 For prepared biomass samples, determine the total solids by Test Method E1756 and record the total solids value as %T .
This prepared sample should be stored in a manner to ensure its moisture content does not change before the analysis begins.
11.2.1 If Test Method A of this practice is used (air drying), determine the total solids content of this prepared sample by Test
Method E1756 and record the total solids value as %T .
ad
11.2.2 If Test Method B of this practice is used (drying at 45°C), record the total solids calculated in this practice, %T , as
%T .
prep
11.2.3 If Test Method C of this practice is used (freeze drying), record the total solids calculated in this practice, %T , as %T .
fd prep
11.3 If extractives-free material is used, determine the total solids content of the extractive-free material by Test Method 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 m , the initial mass of sample in grams.
NOTE 3—Warning: 72 % w/w sulfuric acid is very corrosive 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 H SO to the test tube containing the sample and stir for 1 min or until
2 4
thoroughly mixed.
11.6 Place the test tube containing the sample into the water bath controlled to 30 6 1°C and hydrolyze for 1h. Stir
approximately every 15 min to ensure the sample is complet
...
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