ASTM E1821-08(2020)
(Test Method)Standard Test Method for Determination of Carbohydrates in Biomass by Gas Chromatography
Standard Test Method for Determination of Carbohydrates in Biomass by Gas Chromatography
SIGNIFICANCE AND USE
4.1 The structural carbohydrate content is used in conjunction with other assays to determine the total composition of biomass samples.
SCOPE
1.1 This test method describes the determination of structural carbohydrates present in a biomass sample, expressed as the percent mass of an oven-dried sample basis of each anhydrosugar.
1.2 Sample materials suitable for this procedure include hard and softwoods, herbaceous materials, such as sericea and switchgrass, agricultural residues, such as corn stover, wheat straw, and bagasse, wastepaper, such as boxboard, office waste, and newsprint, acid or alkaline-pretreated biomass, washed free of any residual acid or alkali, and the solid fraction of fermentation residues.
1.3 The options for the types of samples to be analyzed in this procedure are:
1.3.1 Prepared Biomass Samples:
1.3.1.1 Air Dried Material—Results are reported as the percent by mass, based on the oven-dried mass of the air-dried sample.
1.3.1.2 45 °C Dried Material—Results are reported as the percent by mass, based on the oven-dried mass of the 45 °C dried sample.
1.3.1.3 Freeze Dried Material—Results are reported as the percent by mass, based on the oven-dried mass of the freeze dried sample.
1.3.2 Extractives-Free Sample—Results are reported as the percent by mass, based on the oven-dried mass of the extracted sample.
1.4 This standard method is generally not suitable for samples that contain soluble, nonstructural carbohydrates unless they are removed prior to analysis.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 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. See Section 8 for specific hazards statements.
1.7 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: E1821 − 08 (Reapproved 2020)
Standard Test Method for
Determination of Carbohydrates in Biomass by Gas
Chromatography
This standard is issued under the fixed designation E1821; 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
This test method gives a reproducible way to quantitatively determine in lignocellulosic materials
thekindandamountofthestructuralcarbohydratesmadefromarabinose,xylose,mannose,galactose,
and glucose. This way is accomplished by first hydrolyzing the carbohydrates to their constituent
monosaccharides. Subsequent derivatization produces the corresponding alditol acetates that are
quantified using capillary gas chromatography.
1. Scope 1.4 This standard method is generally not suitable for
samples that contain soluble, nonstructural carbohydrates un-
1.1 This test method describes the determination of struc-
less they are removed prior to analysis.
tural carbohydrates present in a biomass sample, expressed as
the percent mass of an oven-dried sample basis of each 1.5 The values stated in SI units are to be regarded as
anhydrosugar. standard. No other units of measurement are included in this
standard.
1.2 Sample materials suitable for this procedure include
1.6 This standard does not purport to address all of the
hard and softwoods, herbaceous materials, such as sericea and
safety concerns, if any, associated with its use. It is the
switchgrass, agricultural residues, such as corn stover, wheat
responsibility of the user of this standard to establish appro-
straw,andbagasse,wastepaper,suchasboxboard,officewaste,
priate safety, health, and environmental practices and deter-
and newsprint, acid or alkaline-pretreated biomass, washed
mine the applicability of regulatory limitations prior to use.
free of any residual acid or alkali, and the solid fraction of
See Section 8 for specific hazards statements.
fermentation residues.
1.7 This international standard was developed in accor-
1.3 The options for the types of samples to be analyzed in
dance with internationally recognized principles on standard-
this procedure are:
ization established in the Decision on Principles for the
1.3.1 Prepared Biomass Samples:
Development of International Standards, Guides and Recom-
1.3.1.1 Air Dried Material—Results are reported as the
mendations issued by the World Trade Organization Technical
percent by mass, based on the oven-dried mass of the air-dried
Barriers to Trade (TBT) Committee.
sample.
1.3.1.2 45 °C Dried Material—Results are reported as the
2. Referenced Documents
percent by mass, based on the oven-dried mass of the 45°C
2.1 ASTM Standards:
dried sample.
D1193Specification for Reagent Water
1.3.1.3 Freeze Dried Material—Results are reported as the
E1690Test Method for Determination of Ethanol Extrac-
percent by mass, based on the oven-dried mass of the freeze
tives in Biomass
dried sample.
E1721Test Method for Determination of Acid-Insoluble
1.3.2 Extractives-Free Sample—Results are reported as the
Residue in Biomass
percentbymass,basedontheoven-driedmassoftheextracted
E1756Test Method for Determination of Total Solids in
sample.
Biomass
E1757Practice for Preparation of Biomass for Composi-
tional Analysis
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 1996. Last previous edition approved in 2015 as E1821–08(2015). Standards volume information, refer to the standard’s Document Summary page on
DOI: 10.1520/E1821-08R20. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1821 − 08 (2020)
3. Terminology 3.2.12 %extractives—the percentage by mass of extractives
in the extracted specimen as described in Test Method E1690.
3.1 Definitions of Terms Specific to This Standard:
3.2.13 k—constantusedtoconvertthemassofmonosaccha-
3.1.1 anhydrosugars, n—the nominal repeating unit of a
ride to the mass of anhydrosugar from which it is derived. For
polysaccharide. When polysaccharides undergo acid
arabinose and xylose, k =0.88 (m⁄z 132/150); for mannose,
hydrolysis,eachrepeatingunitaddsasinglemoleculeofwater
galactose and glucose, k=0.90 (m/z 162/180).
to form the free monosaccharide that is analyzed. The extra
weight from this water of hydrolysis must be taken in to
3.2.14 LF—loss factor for monosaccharide c. Used to cor-
account when calculating the actual mass percent of the
rect for the amount of monosaccharide lost through degrada-
polysaccharide in the original biomass sample.
tion during acid hydrolysis of biomass.
3.1.2 as received biomass, n—materialasitisreceivedinits
3.2.15 m —initial mass of the biomass specimen, in mg.
I
field or process collected state.
3.2.16 m —mass of monosaccharide in solution, cor-
corr
3.1.3 extractives-free biomass—air-dried solids left after rected for hydrolysis losses, in mg.
biomass has been treated according to Test Method E1690.
3.2.17 RR —averaged response ratio of monosaccharide c
avg
to the internal standard (inositol) in the calibration standard.
3.1.4 oven-dried mass, n—the moisture-free mass of any
Derived from multiple injections of the same calibration
biomass sample (as received, prepared, extractives-free, etc.)
standard.
dried at 105°C as described in Test Method E1756.
3.2.18 RR —response ratio of monosaccharide c to the
3.1.5 prepared biomass, n—as received biomass material s
internal standard (inositol) in the specimen.
that has been treated according to Practice E1757 in order to
raise the total solids content above 85%, based on an oven-
3.2.19 RR —response ratio of monosaccharide c to the
STD
dried solids weight.
internal standard (inositol) in the calibration standard.
3.1.6 structural carbohydrates, n—polysaccharides that 3.2.20 RRF (Relative Response Factor of monosaccharide
cannot be removed by extraction with solvents and are liber- c)—this is the ratio of the detector response for monosaccha-
ated from the biomass solids with dilute acid hydrolysis. For ride c versus the detector response for the internal standard
the purpose of this test method, the monosaccharides that are (inositol) for a given injection of the specimen.
considered present are arabinose, xylose, mannose, galactose,
3.2.21 V —87 mL, volume of hydrolysis solution.
f
and glucose.
3.2.22 %T —percentage by mass, of total solids of the
3.2 Abbreviations:
specimenpreparedbydryingat45°C,asdescribedbyPractice
3.2.1 %Anhydro —the percent by mass of the anhydro-
E1757.
ext
sugar on an extractives-free, oven-dried mass basis.
3.2.23 %T —percentage by mass, of total solids of the
3.2.2 %Anhydro —the percent by mass of the
specimen, dried at 105°C, as determined by Test Method
whole
anhydrosugar, on an oven-dried mass basis.
E1756.
3.2.3 AR (Amount Ratio)—ratio of the concentration 3.2.24 %T —percentage by mass, of total solids of the
c ad
air-dried specimen determined at 105°C as described by Test
(amount)ofmonosaccharide ctotheconcentration(amount)of
internal standard in the specimen. Method E1756.
3.2.25 %T —percentage by mass, of total solids of the
3.2.4 area —reported area counts for the monosaccharide c
ext
c
extracted specimen determined at 105°C as described by Test
peak in the chromatogram, as integrated by the electronic
Method E1756.
integrator.
3.2.26 %T —percentage by mass, of total solids of the
3.2.5 area —reported area counts for the internal standard fd
IS
specimen prepared by freeze drying, as described by Practice
peak in the chromatogram, as integrated by the electronic
E1757.
integrator.
3.2.27 %T —percentage, by mass, of total solids of the
prep
3.2.6 C —average concentration of monosaccharide c in
avg
specimen prepared by freeze drying,% T , or by drying at
specimen s, in mg/mL, averaged across multiple injections of fd
45°C, %T , as determined by Practice E1757.
specimen s.
3.2.7 C —original concentration of monosaccharide c in
4. Significance and Use
LF
loss factor sample, in mg/mL.
4.1 The structural carbohydrate content is used in conjunc-
3.2.8 C —concentration of internal standard (inositol) in
tion with other assays to determine the total composition of
IS
the calibration standards and specimen, in mg/mL. biomass samples.
3.2.9 C —concentrationofmonosaccharide cinspecimen s,
s
5. Interferences
measured by gas chromatography (GC), in mg/mL.
5.1 The results of structural carbohydrate analysis are af-
3.2.10 C —concentration of monosaccharide c in the
STD
fected by incomplete hydrolysis of biomass or hydrolysis
calibration standard, in mg/mL.
conditions that are too severe. Incomplete hydrolysis will bias
3.2.11 CV (coeffıcient of variation)—the estimated standard the results low because dimeric and oligomeric carbohydrates
deviation divided by the average value measured. are not quantified. Hydrolysis conditions that are too severe
E1821 − 08 (2020)
degrade the liberated monosaccharides into materials that are 7.1.2 Purity of Water—Unless otherwise indicated, refer-
not quantified by this procedure, again biasing the results low. ences to water mean reagent water as defined by Type 1 of
Specification D1193.
5.2 Incomplete neutralization and removal of acetic acid
7.1.3 Acetic Acid (CH COOH), glacial.
from the methylene chloride extract prior to GC analysis can
7.1.4 Acetic Anhydride ((CH CO) O).
result in ghost peaks appearing in the chromatogram or 3 2
7.1.5 Ammonium Hydroxide, (NH OH), concentrated
carryover of monosaccharides from one injection to the next 4
(28–30 wt% NH ).
(owing to buildup of monosaccharides in the injection port), 3
7.1.6 Ammonium Hydroxide Solution (;3 M)—Dilute5.0 6
leading to erroneous quantitation.
0.1 mL of concentrated ammonium hydroxide (NH OH) with
5.3 Test specimens not suitable for analysis by this proce-
20.06 0.1 mL of water. Prepare fresh before each use.
dure include alkaline and acid-pretreated biomass samples that
7.1.7 Monosaccharide Stock A Solution—Combine the fol-
have not been washed. Unwashed pretreated biomass samples
lowing monosaccharides. Weigh each monosaccharide in the
containing free acid or alkali may change visibly on heating.
following nominal amounts (record each actual mass to the
5.4 Materials containing nonstructural carbohydrates also
nearest 0.1 mg). Dissolve in water and dilute to 100 mL. Store
are unsuitable for this procedure since nonstructural carbohy-
at 4°C and discard after four weeks.
drates may undergo degradation to materials that are not
Arabinose (C H O ) 90–110 mg
5 10 5
quantified in this procedure.
Xylose (C H O ) 650–750 mg
5 10 5
Mannose (C H O ) 90–110 mg
6 12 6
Galactose (C H O ) 90–110 mg
6. Apparatus
6 12 6
Glucose (C H O ) 1900–2100 mg
6 12 6
6.1 Analytical Balance, readable to 0.1 mg.
7.1.8 Monosaccharide Stock B Solution—Prepareinmanner
6.2 Autoclave, capable of maintaining 121 6 3°C.
identical to monosaccharide stock A solution.
6.3 Convection Ovens, temperature controlled to 45 63°C 7.1.9 Dichloromethane, (CH Cl ).
2 2
and 105 6 3°C. 7.1.10 Inositol Solution (20 mg/mL)—Dissolve 5.000 6
0.0025 g of inositol (C H O , 98+wt%) in water and dilute
6 12 6
6.4 Desiccator, containing anhydrous calcium sulfate.
to 250 mL. Store at 4°C and discard after one week.
6.5 Gas Chromatograph, equipped with electronic
7.1.11 Loss Factor Standard Stock Solution—Combine to-
integrator, capillary split injection port, flame ionization detec-
gether each of the following monosaccharides. Weigh each
tor with make-up gas, 250 µm×15 m fused-silica capillary
monosaccharide in the following nominal amounts (record
column coated with 50 % cyanopropylphenyl
each actual weight to the nearest 0.1 mg). Dissolve in water
methylpolysiloxane, 0.25 µm film thickness (DB-225 or
and dilute to 100 mL. Store at 4°C and discard after four
equivalent).
weeks.
6.6 Ice Bath.
Arabinose (C H O ) 900–1100 mg
5 10 5
Mannose (C H O ) 900–1100 mg
6 12 6
6.7 Ultrasonic Bath.
Galactose (C H O ) 900–1100 mg
6 12 6
Xylose (C H O ) 900–1100 mg
5 10 5
6.8 Vortex Mixer, or equivalent method to rapidly mix
Glucose (C H O ) 900–1100 mg
6 12 6
solutions in a test tube.
7.1.12 1-Methylimidazole, ((C H N −)(CH )).
3 3 2 3
6.9 Water Bath, setable to 30 6 1°C and 40 6 1°C.
7.1.13 Potassium Borohydride Solution, (0.15 g/mL)—
Dissolve 7.50 6 0.05 g potassium borohydride (KBH)in40
7. Reagents and Materials
mLof ;3Mammoniumhydroxide(NH OH)solution.Usean
7.1 Chemicals:
ultrasonic bath to get the salt to dissolve in a reasonable
7.1.1 Purity of Reagents—Use reagent grade chemicals in
amount of time. Dilute to 50.0 6 0.1 mL with ;3M
all tests. Unless otherwise indicated, it is intended that all
ammoniumhydroxide(NH OH)solution.Prepareimmediately
reagents conform to the specifications of the Committee on
before use. Discard after 6 h. This quantity is sufficient for 50
Analytical Reagents of theAmerican Chemical Society where
specimens and calibration standards.
such specifications are available. Monosaccharides used to
7.1.14 Potassium Hydroxide Solution (3.5 M)—Dissolve
prepare the monosaccharide stock solutions and loss factor
58.06 0.5 g of potassium hydroxide (KOH, 85 wt%) in
standard solutions shall be 98+mass% purity. Other chemical
200mL water. Allow to cool to room temperature before
grades may be substituted, provided it is first ascertained that
diluting to 250 mL with water.
the reagent is of sufficiently high purity to permit its use
7.1.15 Sulfuric Acid Solution (12 M)—Slowly add 665 mL
without lessening the accuracy of the determination.
of96wt%sulfuricacid(H SO )to300mLofwatercooledin
2 4
an ice bath with stirring. Allow solution to come to room
DB-225 is a trademark of Agilent Technologies, Inc., 5301 Stevens Creek temperature and dilute to 1 L. Check the concentration by
Boulevard, Santa Clara CA 95051.
titration and adjust the concentration to 12.0 6 0.1 M (24.0 6
Reagent Chemicals, American Chemical Society Specifications, American
0.2 N).
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
listed by the American Chemical Society, see An
...
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