ASTM E1758-01(2020)
(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, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific precautionary statements are given in Note 3 and Note 4.
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
General Information
Relations
Standards Content (Sample)
This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E1758 − 01 (Reapproved 2020)
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-
1.5 This standard does not purport to address all of the
drates present in a biomass sample, expressed as the percent,
safety concerns, if any, associated with its use. It is the
by mass, of each sugar on a 105°C dried mass basis.
responsibility of the user of this standard to establish appro-
NOTE 1—The percent sugar must be corrected for the water of
priate safety, health, and environmental practices and deter-
hydrolysis before calculating the actual mass percent of the polysaccha-
mine the applicability of regulatory limitations prior to use.
ride in the original biomass sample.
SpecificprecautionarystatementsaregiveninNote3 and Note
1.2 Sample materials suitable for this procedure include
4.
hardandsoftwoods,herbaceousmaterials(suchasswitchgrass
1.6 This international standard was developed in accor-
and sericea), agricultural residues (such as corn stover, wheat
dance with internationally recognized principles on standard-
straw, and bagasse), wastepaper (such as office waste,
ization established in the Decision on Principles for the
boxboard, and newsprint), acid or alkaline-pretreated biomass
Development of International Standards, Guides and Recom-
(washed free of any residual acid or alkali), and the solid
mendations issued by the World Trade Organization Technical
fraction of fermentation residues. All results are reported
Barriers to Trade (TBT) Committee.
relative to the 105°C oven-dried mass of the sample.
2. Referenced Documents
1.3 The options for the types of samples to be analyzed in
this test method are as follows:
2.1 ASTM Standards:
1.3.1 Prepared Biomass Samples:
D1193Specification for Reagent Water
1.3.1.1 Air Dried (%T )—The percent, by mass, of total
ad E1690Test Method for Determination of Ethanol Extrac-
solids of the air-dried sample.
tives in Biomass
1.3.1.2 45 °C Dried (%T )—The percent, by mass, of total
45 E1721Test Method for Determination of Acid-Insoluble
solids of the 45°C dried sample.
Residue in Biomass
1.3.1.3 Freeze Dried (%T )—The percent, by mass, of total
fd E1756Test Method for Determination of Total Solids in
solids of the freeze dried sample.
Biomass
1.3.2 Extractives-Free Sample (%T )—The percent, by
ext E1757Practice for Preparation of Biomass for Composi-
mass, of total solids of the extracted sample determined at
tional Analysis
105°C.
3. Terminology
3.1 Definitions of Terms Specific to This Standard:
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
Current edition approved July 1, 2020. Published August 2020. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved in 1995. Last previous edition approved in 2015 as E1758–01(2015). Standards volume information, refer to the standard’s Document Summary page on
DOI: 10.1520/E1758-01R20. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1758 − 01 (2020)
3.1.1 as received biomass—the biomass material as it is 3.2.17 SRS (sugar recovery standards)—standards used to
received in its field or process collected state. determine sugar recovery after hydrolysis.
3.2.18 V —volume of filtrate, 87.0 mL.
3.1.2 oven-dried mass—themoisture-freemassofabiomass
F
sample dried at 105°C as described in Test Method E1756.
4. Significance and Use
3.1.3 prepared biomass—material that has been treated
4.1 The percentage, by mass, of sugar content is used in
according to Practice E1757 in order to raise the total solids
conjunction with other assays to determine the total composi-
content above 85%, by mass, based on an oven-dried solids
tion of biomass samples.
mass.
3.2 Abbreviations—Abbreviations of standards used in the
5. Interferences
procedure, and definitions of terms used in the calculations are
5.1 Samples with high protein content may result in the
as follows:
percentage, by mass, of sugar values being biased low, as a
3.2.1 C —known concentration of sugar recovery standard
consequence of protein binding with some monosaccharides.
before hydrolysis, in mg/mL.
5.2 Test specimens not suitable for analysis by this proce-
3.2.2 C —concentrationofsugarrecoverystandarddetected
dure include alkaline and acid-pretreated biomass samples that
by HPLC after hydrolysis, in mg/mL.
have not been washed. Unwashed pretreated biomass samples
3.2.3 C — concentration of sugar in hydrolyzed sample containing free acid or alkali may change visibly on heating.
corr
corrected for hydrolysis, in mg/mL.
6. Apparatus
3.2.4 C — concentration of sugar in hydrolyzed sample
spl
6.1 Analytical Balance, readable to 0.1 mg.
detected by HPLC, in mg/mL.
6.2 Autoclave, capable of maintaining 121 6 3°C.
3.2.5 CVS (calibration verification standard)—standards
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
105 6 3°C.
calibration standards.
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
NOTE2—DeashingguardcolumncartridgesfromBioRad, oftheionic
E1690.
+ − 3
form H /CO , are an option when using an HPX-87P column, or
equivalent.Thesecartridgesareeffectiveineliminatingbaselineramping.
3.2.8 %R — percent recovery of sugar recovery standard,
srs
as determined in 13.2.
6.6 Hewlett Packard Model 1090 HPLC, or equivalent,
with refractive index detector.
3.2.9 %sugar —the percentage, by mass, of
extractives-free
3 3
sugar on an extractives-free 105°C dry weight basis, as
6.7 HPLC Columns, BioRad HPX-87C or HPX-87P, or
determined in 13.6.1.
both, or equivalent.
3.2.10 % sugar —thecorrectedmasspercentsugar
6.8 Water Bath, set at 30 6 1°C.
whole sample
value on an extractives-free basis corrected to an as received
7. Reagents and Materials
(whole sample) 105°C dry mass basis.
7.1 Chemicals:
3.2.11 %T —percentage, by mass, of total solids of the
7.1.1 Purity of Reagents—Reagent grade chemicals shall be
sample prepared by drying at 45°C, as described by Practice
used in all tests. Unless otherwise indicated, it is intended that
E1757.
all reagents conform to the specifications of the Committee on
3.2.12 %T —percentage, by mass, of total solids in the
Analytical Reagents of theAmerican Chemical Society where
sample,driedat105°C,asdeterminedbyTestMethodE1756.
such specifications are available. Other grades may be used,
3.2.13 %T — percentage, by mass, of total solids of the
ad
air-dried sample determined at 105°C as described by Test
The sole source of supply of the apparatus known to the committee at this time
Method E1756.
is BioRad Aminex®, HPX-87C and Aminex® HPX-87P, available from BioRad,
3.2.14 %T — percentage, by mass, of total solids of the
Main Office, 3300 Regatta Boulevard, Richmond, CA 94804. If you are aware of
ext
alternative suppliers, please provide this information to ASTM International
extracted sample determined at 105°C as described by Test
Headquarters.Your comments will receive careful consideration at a meeting of the
Method E1756.
responsible technical committee, which you may attend.
Available from Hewlett-Packard, HPAnalytical Direct, 2850 Centerville Road,
3.2.15 %T — percentage, by mass, of total solids of the
fd
Wilmington, DE 19808.
samplepreparedbyfreezedrying,asdescribedbyTestMethod
Reagent Chemicals, American Chemical Society Specifications , American
E1756.
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
listed by the American Chemical Society, see Analar Standards for Laboratory
3.2.16 %T — percentage, by mass, of total solids of the
prep
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
samplepreparedbyfreezedrying,%T ,orbydryingat45°C,
fd and National Formulary,U.S.PharmaceuticalConvention,Inc.(USPC),Rockville,
%T , as determined by Practice E1757. MD.
E1758 − 01 (2020)
provided it is first ascertained that the reagent is of sufficiently sugar contained in the calibration standards, at a concentration
high purity to permit its use without lessening the accuracy of in the middle of the validated range of the calibration curve.
the determination. The CVS will be analyzed after each calibration curve and at
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
11.1.4 Neutralization of hydrolyzate,
0.02 M)—Slowly add 665 mL of concentrated sulfuric acid
11.1.5 Filtration of sample prior to HPLC analysis,
(H SO ) to 300 mL of water while cooling in an ice bath and
2 4
11.1.6 HPLC analysis of sugar standards, CVS, SRS, and
stirring. Allow to come to room temperature. Adjust the
hydrolyzate samples, and
relative density to 1.6389 6 0.0012 at 15.6°C⁄15.6°C.
11.1.7 Calculation of sugar contents.
7.2 Materials:
11.2 For prepared biomass samples, determine the total
7.2.1 Autosampler Vials, with crimp top seals to fit.
solids by Test Method E1756 and record the total solids value
7.2.2 Disposable Syringes, 3 mL.
as %T . This prepared sample should be stored in a manner
7.2.3 Disposable Syringe Filters, nylon, 0.2 µm.
to ensure its moisture content does not change before the
7.2.4 Glass Serum Bottles, crimp top style, 125 mL, with
analysis begins.
rubber stoppers and aluminum seals to fit.
11.2.1 IfTest MethodAof this practice is used (air drying),
determine the total solids content of this prepared sample by
8. Hazards
Test Method E1756 and record the total solids value as %T .
ad
8.1 Handle the sulfuric acid carefully to avoid contact with
11.2.2 If Test Method B of this practice is used (drying at
skin or clothing, as it is corrosive.
45°C), record the total solids calculated in this practice, %T ,
as %T .
8.2 The glass bottles are hot and may be pressurized after
prep
11.2.3 If Test Method C of this practice is used (freeze
the autoclave step. Use caution when handling.
drying),recordthetotalsolidscalculatedinthispractice, %T ,
fd
9. Sampling, Test Specimens, and Test Units as %T .
prep
9.1 Test specimens suitable for analysis by this procedure 11.3 If extractives-free material is used, determine the total
are: solids content of the extractive-free material by Test Method
9.1.1 Prepared biomass prepared according to Practice E1756 and record this value as %T .
ext
E1757, and
11.4 Weigh300 610mgofthepreparedorextractives-free
9.1.2 Extractives-free material prepared according to Test
sample to the nearest 0.1 mg and place in 16x 100 mm glass
Method E1690.
test tube. Record as m , the initial mass of sample in grams.
NOTE 3—Warning: 72% w/w sulfuric acid is very corrosive and
10. Calibration and Standardization
should be handled by trained personnel only.
10.1 Prepare a series of three to six sugar standards in
11.5 Add 3.00 6 0.01 mL (4.92 6 0.01 g) of 72% w/w
deionized water at concentrations appropriate for preparing
H SO tothetesttubecontainingthesampleandstirfor1min
2 4
calibration curves to quantitfy each sugar of interest. An
or until thoroughly mixed.
HPX-87C column, or equivalent, is used to analyze glucose,
xylose, and arabinose. If mannose and galactose are also to be 11.6 Placethetesttubecontainingthesampleintothewater
quantified, an HPX-87P column, or equivalent, must be used bath controlled to 30 6 1°C and hydrolyze for 1h. Stir
instead. Typically, the concentrations of these sugar standards approximatelyevery15mintoensurethesampleiscompletely
mixed and wet.
cover the range starting at the detection limit of the instrument
and extending up to 4.0 mg/mL.
11.7 Weigh out 300 6 10 mg of each high purity sugar
10.2 PrepareanindependentCVS,asdescribedin8.1.2,for standard (dried at 45°C), described in 8.1.4, to the nearest
each set of calibration standards, using sugars obtained from a 0.1mg and place each in its own individual 16x 100 mm glass
source other than that used in preparing
...
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