ASTM E1821-08(2015)
(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 and health practices and determine the applicability of regulatory limitations prior to use. See Section 8 for specific hazards statements.
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Designation: E1821 − 08 (Reapproved 2015)
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 and health practices and determine the applica-
and newsprint, acid or alkaline-pretreated biomass, washed
bility of regulatory limitations prior to use. See Section 8 for
free of any residual acid or alkali, and the solid fraction of
specific hazards statements.
fermentation residues.
1.3 The options for the types of samples to be analyzed in
2. Referenced Documents
this procedure are:
2.1 ASTM Standards:
1.3.1 Prepared Biomass Samples:
D1193Specification for Reagent Water
1.3.1.1 Air 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 air-dried
tives in Biomass
sample.
E1721Test Method for Determination of Acid-Insoluble
1.3.1.2 45°C Dried Material—Results are reported as the
Residue in Biomass
percent by mass, based on the oven-dried mass of the 45°C
E1756Test Method for Determination of Total Solids in
dried sample.
Biomass
1.3.1.3 Freeze Dried Material—Results are reported as the
E1757Practice for Preparation of Biomass for Composi-
percent by mass, based on the oven-dried mass of the freeze
tional Analysis
dried sample.
1.3.2 Extractives-Free Sample—Results are reported as the
3. Terminology
percentbymass,basedontheoven-driedmassoftheextracted
3.1 Definitions of Terms Specific to This Standard:
sample.
3.1.1 anhydrosugars, n—the nominal repeating unit of a
polysaccharide. When polysaccharides undergo acid
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
in1996.Lastpreviouseditionapprovedin2008asE1821-08.DOI:10.1520/E1821- Standards volume information, refer to the standard’s Document Summary page on
08R15. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1821 − 08 (2015)
hydrolysis,eachrepeatingunitaddsasinglemoleculeofwater arabinose and xylose, k =0.88 (m⁄z 132/150); for mannose,
to form the free monosaccharide that is analyzed. The extra galactose and glucose, k=0.90 (m/z 162/180).
weight from this water of hydrolysis must be taken in to
3.2.14 LF—loss factor for monosaccharide c. Used to cor-
account whencalculating the actual mass percent of the poly-
rect for the amount of monosaccharide lost through degrada-
saccharide 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
3.1.4 oven-dried mass, n—the moisture-free mass of any
to the internal standard (inositol) in the calibration standard.
biomass sample (as received, prepared, extractives-free, etc.)
Derived from multiple injections of the same calibration
dried at 105°C as described in Test Method E1756.
standard.
3.1.5 prepared biomass, n—as received biomass material
3.2.18 RR —response ratio of monosaccharide c to the
s
that has been treated according to Practice E1757 in order to internal standard (inositol) in the specimen.
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:
specimen prepared by drying at 45°C, as described by Practice
3.2.1 %Anhydro —the percent by mass of the anhydro-
ext
E1757.
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
whole
specimen, dried at 105°C, as determined by Test Method
anhydrosugar, on an oven-dried mass basis.
E1756.
3.2.3 AR (Amount Ratio)—ratio of the concentration
c 3.2.24 %T —percentage by mass, of total solids of the
ad
(amount)ofmonosaccharide ctotheconcentration(amount)of
air-dried specimen determined at 105°C as described by Test
internal standard in the specimen.
Method E1756.
3.2.4 area —reported area counts for the monosaccharide c
3.2.25 %T —percentage by mass, of total solids of the
c
ext
peak in the chromatogram, as integrated by the electronic
extracted specimen determined at 105°C as described by Test
integrator.
Method E1756.
3.2.5 area —reported area counts for the internal standard
3.2.26 %T —percentage by mass, of total solids of the
IS
fd
peak in the chromatogram, as integrated by the electronic
specimen prepared by freeze drying, as described by Practice
integrator.
E1757.
3.2.6 C —average concentration of monosaccharide c in 3.2.27 %T —percentage, by mass, of total solids of the
avg
prep
specimen s, in mg/mL, averaged across multiple injections of
specimen prepared by freeze drying,% T , or by drying at
fd
specimen s. 45°C, %T , as determined by Practice E1757.
3.2.7 C —original concentration of monosaccharide c in
LF
4. Significance and Use
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
IS
tion with other assays to determine the total composition of
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
3.2.12 %extractives—the percentage by mass of extractives degrade the liberated monosaccharides into materials that are
in the extracted specimen as described in Test Method E1690. not quantified by this procedure, again biasing the results low.
3.2.13 k—constantusedtoconvertthemassofmonosaccha- 5.2 Incomplete neutralization and removal of acetic acid
ride to the mass of anhydrosugar from which it is derived. For from the methylene chloride extract prior to GC analysis can
E1821 − 08 (2015)
result in ghost peaks appearing in the chromatogram or 7.1.4 Acetic Anhydride ((CH CO) O).
3 2
carryover of monosaccharides from one injection to the next
7.1.5 Ammonium Hydroxide, (NH OH), concentrated
(owing to buildup of monosaccharides in the injection port),
(28–30 wt% NH ).
leading to erroneous quantitation.
7.1.6 Ammonium Hydroxide Solution (;3 M)—Dilute5.0 6
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
have not been washed. Unwashed pretreated biomass samples 7.1.7 Monosaccharide Stock A Solution—Combine the fol-
containing free acid or alkali may change visibly on heating. lowing monosaccharides. Weigh each monosaccharide in the
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
6. Apparatus
Galactose (C H O ) 90–110 mg
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 6 3°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
6.4 Desiccator, containing anhydrous calcium sulfate.
0.0025 g of inositol (C H O , 98+wt%) in water and dilute
6 12 6
to 250 mL. Store at 4°C and discard after one week.
6.5 Gas Chromatograph, equipped with electronic
integrator, capillary split injection port, flame ionization detec- 7.1.11 Loss Factor Standard Stock Solution—Combine to-
gether each of the following monosaccharides. Weigh each
tor with make-up gas, 250 µm×15 m fused-silica capillary
column coated with 50 % cyanopropylphenyl monosaccharide in the following nominal amounts (record
each actual weight to the nearest 0.1 mg). Dissolve in water
methylpolysiloxane, 0.25 µm film thickness (DB-225 or
equivalent). and dilute to 100 mL. Store at 4°C and discard after four
weeks.
6.6 Ice Bath.
Arabinose (C H O ) 900–1100 mg
5 10 5
6.7 Ultrasonic Bath.
Mannose (C H O ) 900–1100 mg
6 12 6
Galactose (C H O ) 900–1100 mg
6 12 6
6.8 Vortex Mixer, or equivalent method to rapidly mix
Xylose (C H O ) 900–1100 mg
5 10 5
solutions in a test tube.
Glucose (C H O ) 900–1100 mg
6 12 6
6.9 Water Bath, setable to 30 6 1°C and 40 6 1°C. 7.1.12 1-Methylimidazole, ((C H N −)(CH )).
3 3 2 3
7.1.13 Potassium Borohydride Solution, (0.15 g/mL)—
7. Reagents and Materials
Dissolve 7.50 6 0.05 g potassium borohydride (KBH)in40
7.1 Chemicals:
mLof ;3Mammoniumhydroxide(NH OH)solution.Usean
7.1.1 Purity of Reagents—Use reagent grade chemicals in ultrasonic bath to get the salt to dissolve in a reasonable
all tests. Unless otherwise indicated, it is intended that all
amount of time. Dilute to 50.0 6 0.1 mL with ;3M
reagents conform to the specifications of the Committee on ammoniumhydroxide(NH OH)solution.Prepareimmediately
Analytical Reagents of theAmerican Chemical Society where
before use. Discard after 6 h. This quantity is sufficient for 50
such specifications are available. Monosaccharides used to specimens and calibration standards.
prepare the monosaccharide stock solutions and loss factor
7.1.14 Potassium Hydroxide Solution (3.5 M)—Dissolve
standard solutions shall be 98+mass% purity. Other chemical
58.06 0.5 g of potassium hydroxide (KOH, 85 wt%) in 200
grades may be substituted, provided it is first ascertained that
mLwater.Allowtocooltoroomtemperaturebeforedilutingto
the reagent is of sufficiently high purity to permit its use
250 mL with water.
without lessening the accuracy of the determination.
7.1.15 Sulfuric Acid Solution (12 M)—Slowly add 665 mL
7.1.2 Purity of Water—Unless otherwise indicated, refer-
of96wt%sulfuricacid(H SO )to300mLofwatercooledin
2 4
ences to water mean reagent water as defined by Type 1 of
an ice bath with stirring. Allow solution to come to room
Specification D1193.
temperature and dilute to 1 L. Check the concentration by
7.1.3 Acetic Acid (CH COOH), glacial.
titration and adjust the concentration to 12.0 6 0.1 M (24.0 6
0.2 N).
DB-225 is a trademark of Agilent Technologies, Inc., 5301 Stevens Creek
7.2 Materials:
Boulevard, Santa Clara CA 95051.
7.2.1 Glass Filtering Crucibles, 50 mL, medium porosity,
Reagent Chemicals, American Chemical Society Specifications , American
nominal pore size of 10 µm.
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
listed by the American Chemical Society, see Analar Standards for Laboratory
7.2.2 Glass Serum Bottles, 125 mL, crimp-top style with
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
rubber stoppers and aluminum seals to fit.
and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,
MD. 7.2.3 Vacuum Adaptor for Filtering Crucibles.
E1821 − 08 (2015)
7.2.4 Vials, 13
...
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: E1821 − 08 E1821 − 08 (Reapproved 2015)
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. A number 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
the kind and amount of the structural carbohydrates made from arabinose, 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
1.1 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 and health practices and determine the applicability of regulatory
limitations prior to use. See Section 8 for specific hazards statements.
2. Referenced Documents
2.1 ASTM Standards:
D1193 Specification for Reagent Water
E1690 Test Method for Determination of Ethanol Extractives in Biomass
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 May 1, 2008June 1, 2015. Published May 2008July 2015. Originally approved in 1996. Last previous edition approved in 20072008 as
E1821-01(2007).E1821-08. DOI: 10.1520/E1821-08.10.1520/E1821-08R15.
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
E1821 − 08 (2015)
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
3. Terminology
3.1 Definitions of Terms Specific to This Standard:
3.1.1 anhydrosugars, n—the nominal repeating unit of a polysaccharide. When polysaccharides undergo acid hydrolysis, each
repeating unit adds a single molecule of water to form the free monosaccharide that is analyzed. The extra weight from this water
of hydrolysis must be taken in to account whencalculating the actual mass percent of the polysaccharide in the original biomass
sample.
3.1.2 as received biomass, n—material as it is received in its field or process collected state.
3.1.3 extractives-free biomass—air-dried solids left after biomass has been treated according to Test Method E1690.
3.1.4 oven-dried mass, n—the moisture-free mass of any biomass sample (as received, prepared, extractives-free, etc.) dried at
105°C as described in Test Method E1756.
3.1.5 prepared biomass, n—as received biomass material that has been treated according to Practice E1757 in order to raise the
total solids content above 85 %, based on an oven-dried solids weight.
3.1.6 structural carbohydrates, n—polysaccharides that cannot be removed by extraction with solvents and are liberated from
the biomass solids with dilute acid hydrolysis. For the purpose of this test method, the monosaccharides that are considered present
are arabinose, xylose, mannose, galactose, and glucose.
3.2 Abbreviations:
3.2.1 %Anhydro —the percent by mass of the anhydrosugar on an extractives-free, oven-dried mass basis.
ext
3.2.2 %Anhydro —the percent by mass of the anhydrosugar, on an oven-dried mass basis.
whole
3.2.3 AR (Amount Ratio)—ratio of the concentration (amount) of monosaccharide c to the concentration (amount) of internal
c
standard in the specimen.
3.2.4 area —reported area counts for the monosaccharide c peak in the chromatogram, as integrated by the electronic integrator.
c
3.2.5 area —reported area counts for the internal standard peak in the chromatogram, as integrated by the electronic integrator.
IS
3.2.6 C —average concentration of monosaccharide c in specimen s, in mg/mL, averaged across multiple injections of
avg
specimen s.
3.2.7 C —original concentration of monosaccharide c in loss factor sample, in mg/mL.
LF
3.2.8 C —concentration of internal standard (inositol) in the calibration standards and specimen, in mg/mL.
IS
3.2.9 C —concentration of monosaccharide c in specimen s, measured by gas chromatography (GC), in mg/mL.
s
3.2.10 C —concentration of monosaccharide c in the calibration standard, in mg/mL.
STD
3.2.11 CV (coeffıcient of variation)—the estimated standard deviation divided by the average value measured.
3.2.12 %extractives—the percentage by mass of extractives in the extracted specimen as described in Test Method E1690.
3.2.13 k—constant used to convert the mass of monosaccharide to the mass of anhydrosugar from which it is derived. For
arabinose and xylose, k = 0.88 (m ⁄z 132/150); for mannose, galactose and glucose, k = 0.90 (m/z 162/180).
3.2.14 LF—loss factor for monosaccharide c. Used to correct for the amount of monosaccharide lost through degradation during
acid hydrolysis of biomass.
3.2.15 m —initial mass of the biomass specimen, in mg.
I
3.2.16 m —mass of monosaccharide in solution, corrected for hydrolysis losses, in mg.
corr
3.2.17 RR —averaged response ratio of monosaccharide c to the internal standard (inositol) in the calibration standard.
avg
Derived from multiple injections of the same calibration standard.
3.2.18 RR —response ratio of monosaccharide c to the internal standard (inositol) in the specimen.
s
3.2.19 RR —response ratio of monosaccharide c to the internal standard (inositol) in the calibration standard.
STD
3.2.20 RRF (Relative Response Factor of monosaccharide c)—this is the ratio of the detector response for monosaccharide c
versus the detector response for the internal standard (inositol) for a given injection of the specimen.
3.2.21 V —87 mL, volume of hydrolysis solution.
f
3.2.22 %T —percentage by mass, of total solids of the specimen prepared by drying at 45°C, as described by Practice E1757.
3.2.23 %T —percentage by mass, of total solids of the specimen, dried at 105°C, as determined by Test Method E1756.
3.2.24 %T —percentage by mass, of total solids of the air-dried specimen determined at 105°C as described by Test Method
ad
E1756.
E1821 − 08 (2015)
3.2.25 %T —percentage by mass, of total solids of the extracted specimen determined at 105°C as described by Test Method
ext
E1756.
3.2.26 %T —percentage by mass, of total solids of the specimen prepared by freeze drying, as described by Practice E1757.
fd
3.2.27 %T —percentage, by mass, of total solids of the specimen prepared by freeze drying,% T , or by drying at 45°C,
prep fd
%T , as determined by Practice E1757.
4. Significance and Use
4.1 The structural carbohydrate content is used in conjunction with other assays to determine the total composition of biomass
samples.
5. Interferences
5.1 The results of structural carbohydrate analysis are affected by incomplete hydrolysis of biomass or hydrolysis conditions that
are too severe. Incomplete hydrolysis will bias the results low because dimeric and oligomeric carbohydrates are not quantified.
Hydrolysis conditions that are too severe degrade the liberated monosaccharides into materials that are not quantified by this
procedure, again biasing the results low.
5.2 Incomplete neutralization and removal of acetic acid from the methylene chloride extract prior to GC analysis can result in
ghost peaks appearing in the chromatogram or carryover of monosaccharides from one injection to the next (owing to buildup of
monosaccharides in the injection port), leading to erroneous quantitation.
5.3 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.
5.4 Materials containing nonstructural carbohydrates also are unsuitable for this procedure since nonstructural carbohydrates
may undergo degradation to materials that are not quantified in this procedure.
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 controlled to 45 6 3°C and 105 6 3°C.
6.4 Desiccator, containing anhydrous calcium sulfate.
6.5 Gas Chromatograph, equipped with electronic integrator, capillary split injection port, flame ionization detector with
make-up gas, 250 μm × 15 m fused-silica capillary column coated with 50 % cyanopropylphenyl methylpolysiloxane, 0.25 μm film
thickness (DB-225 or equivalent).
6.6 Ice Bath.
6.7 Ultrasonic Bath.
6.8 Vortex Mixer, or equivalent method to rapidly mix solutions in a test tube.
6.9 Water Bath, setable to 30 6 1°C and 40 6 1°C.
7. Reagents and Materials
7.1 Chemicals:
7.1.1 Purity of Reagents—Use reagent grade chemicals 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. Monosaccharides used to prepare the monosaccharide stock solutions and loss factor standard
solutions shall be 98+ mass % purity. Other chemical grades may be substituted, 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 mean reagent water as defined by Type 1 of Specification
D1193.
7.1.3 Acetic Acid (CH COOH), glacial.
7.1.4 Acetic Anhydride ((CH CO) O).
3 2
7.1.5 Ammonium Hydroxide, (NH OH), concentrated (28–30 wt % NH ).
4 3
DB-225 is a trademark of Agilent Technologies, Inc., 5301 Stevens Creek Boulevard, Santa Clara CA 95051.
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. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.
E1821 − 08 (2015)
7.1.6 Ammonium Hydroxide Solution (;3 M)—Dilute 5.0 6 0.1 mL of concentrated ammonium hydroxide (NH OH) with
20.06 0.1 mL of water. Prepare fresh before each use.
7.1.7 Monosaccharide Stock A Solution—Combine the following monosaccharides. Weigh each monosaccharide in the
following nominal amounts (record each actual mass to the nearest 0.1 mg). Dissolve in water and dilute to 100 mL. Store at 4°C
and discard after four weeks.
Arabinose (C H O ) 90–110 mg
5 10 5
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 12 6
Glucose (C H O ) 1900–2100 mg
6 12 6
7.1.8 Monosaccharide Stock B Solution—Prepare in manner identical to monosaccharide stock A solution.
7.1.9 Dichloromethane, (CH Cl ).
2 2
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 to 250
6 12 6
mL. Store at 4°C and discard after one week.
7.1.11 Loss Factor Standard Stock Solution—Combine together each of the following monosaccharides. Weigh each
monosaccharide in the following nominal amounts (record each actual weight to the nearest 0.1 mg). Dissolve in water and dilute
to 100 mL. Store at 4°C and discard after four weeks.
Arabinose (C H O ) 900–1100 mg
5 10 5
Mannose (C H O ) 900–1100 mg
6 12 6
Galactose (C H O ) 900–1100 mg
6 12 6
Xylose (C H O ) 900–1100 mg
5 10 5
Glucose (C H O ) 900–1100 mg
6 12 6
7.1.12 1-Methylimidazole, ((C H N −)(CH )).
3 3 2 3
7.1.13 Potassium Borohydride Solution, (0.15 g/mL)—Dissolve 7.50 6 0.05 g potassium borohydride (KBH ) in 40 mL of ;3
M ammonium hydroxide (NH OH) solution. Use an ultrasonic bath to get the salt to dissolve in a reasonable amount of time. Dilute
to 50.0 6 0.1 mL with ;3 M ammonium hydroxide (NH OH) solution. Prepare immediately before use. Discard after 6 h. This
quantity is sufficient for 50 specimens and calibration standards.
7.1.14 Potassium Hydroxide Solution (3.5 M)—Dissolve 58.06 0.5 g of potassium hydroxide (KOH, 85 wt %) in 200 mL water.
Allow to cool to room temperature before diluting to 250 mL with water.
7.1.15 Sulfuric Acid Solution (12 M)—Slowly add 665 mL of 96 wt % sulfuric acid (H SO ) to 300 mL of water c
...
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