ASTM E1821-01(2007)
(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
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 SamplesAir Dried MaterialResults are reported as the percent by mass, based on the oven-dried mass of the air-dried sample.45C Dried Material
Results are reported as the percent by mass, based on the oven-dried mass of the 45C dried sample.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 SampleResults 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 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.
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Designation:E1821–01 (Reapproved 2007)
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 (e) 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
1.5 This standard does not purport to address all of the
the percent mass of an oven-dried sample basis of each
safety concerns, if any, associated with its use. It is the
anhydrosugar.
responsibility of the user of this standard to establish appro-
1.2 Sample materials suitable for this procedure include
priate safety and health practices and determine the applica-
hard and softwoods, herbaceous materials, such as sericea and
bility of regulatory limitations prior to use. See Section 8 for
switchgrass, agricultural residues, such as corn stover, wheat
specific hazards statements.
straw,andbagasse,wastepaper,suchasboxboard,officewaste,
and newsprint, acid or alkaline-pretreated biomass, washed
2. Referenced Documents
free of any residual acid or alkali, and the solid fraction of
2.1 ASTM Standards:
fermentation residues.
D1193 Specification for Reagent Water
1.3 The options for the types of samples to be analyzed in
E1690 Test Method for Determination of Ethanol Extrac-
this procedure are:
tives in Biomass
1.3.1 Prepared Biomass Samples:
E1721 Test Method for Determination of Acid-Insoluble
1.3.1.1 Air Dried Material—Results are reported as the
Residue in Biomass
percent by mass, based on the oven-dried mass of the air-dried
E1756 Test Method for Determination of Total Solids in
sample.
Biomass
1.3.1.2 45°C Dried Material—Results are reported as the
E1757 Practice for Preparation of Biomass for Composi-
percent by mass, based on the oven-dried mass of the 45°C
tional Analysis
dried sample.
1.3.1.3 Freeze Dried Material—Results are reported as the
3. Terminology
percent by mass, based on the oven-dried mass of the freeze
3.1 Descriptions of Terms Specific to This Standard:
dried sample.
3.1.1 anhydrosugars, n—the nominal repeating unit of a
1.3.2 Extractives-Free Sample—Results are reported as the
polysaccharide. When polysaccharides undergo acid hydroly-
percentbymass,basedontheoven-driedmassoftheextracted
sis,eachrepeatingunitaddsasinglemoleculeofwatertoform
sample.
the free monosaccharide that is analyzed. The extra weight
from this water of hydrolysis must be taken in to account
This test method is under the jurisdiction of ASTM Committee E48 on
Biotechnology and is the direct responsibility of Subcommittee E48.05 on Biomass For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Conversion. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Current edition approved Nov. 15, 2007. Published January 2008. Originally Standards volume information, refer to the standard’s Document Summary page on
approved in 1996. Last previous edition approved in 2001 as E1821-01. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E1821–01 (2007)
whencalculating the actual mass percent of the polysaccharide 3.2.17 RR —averagedresponseratioofmonosaccharide c
avg
in the original biomass sample. to the internal standard (inositol) in the calibration standard.
Derived from multiple injections of the same calibration
3.1.2 as received biomass, n—materialasitisreceivedinits
field or process collected state. standard.
3.2.18 RR —response ratio of monosaccharide c to the
3.1.3 extractives-free biomass—air-dried solids left after
s
internal standard (inositol) in the specimen.
biomass has been treated according to Test Method E1690.
3.2.19 RR —response ratio of monosaccharide c to the
3.1.4 oven-dried mass, n—the moisture-free mass of any
STD
internal standard (inositol) in the calibration standard.
biomass sample (as received, prepared, extractives-free, etc.)
3.2.20 RRF (Relative Response Factor of monosaccharide
dried at 105°C as described in Test Method E1756.
c)—this is the ratio of the detector response for monosaccha-
3.1.5 prepared biomass, n—as received biomass material
ride c versus the detector response for the internal standard
that has been treated according to Practice E1757 in order to
(inositol) for a given injection of the specimen.
raise the total solids content above 85%, based on an oven-
3.2.21 V—87 mL, volume of hydrolysis solution.
dried solids weight. f
3.2.22 %T —percentage by mass, of total solids of the
3.1.6 structural carbohydrates, n—polysaccharides that
specimen prepared by drying at 45°C, as described by Practice
cannot be removed by extraction with solvents and are liber-
E1757.
ated from the biomass solids with dilute acid hydrolysis. For
3.2.23 %T —percentage by mass, of total solids of the
the purpose of this test method, the monosaccharides that are 105
specimen, dried at 105°C, as determined by Test Method
considered present are arabinose, xylose, mannose, galactose,
E1756.
and glucose.
3.2.24 %T —percentage by mass, of total solids of the
3.2 Abbreviations: ad
air-dried specimen determined at 105°C as described by Test
3.2.1 %Anhydro —the percent by mass of the anhydro-
ext
Method E1756.
sugar on an extractives-free, oven-dried mass basis.
3.2.25 %T —percentage by mass, of total solids of the
ext
3.2.2 %Anhydro —the percent by mass of the anhydro-
whole
extracted specimen determined at 105°C as described by Test
sugar, on an oven-dried mass basis.
Method E1756.
3.2.3 AR (Amount Ratio)—ratio of the concentration
c
3.2.26 %T —percentage by mass, of total solids of the
fd
(amount)ofmonosaccharide ctotheconcentration(amount)of
specimen prepared by freeze drying, as described by Practice
internal standard in the specimen.
E1757.
3.2.4 area —reported area counts for the monosaccharide c
c
3.2.27 %T —percentage, by mass, of total solids of the
prep
peak in the chromatogram, as integrated by the electronic
specimen prepared by freeze drying,% T , or by drying at
fd
integrator.
45°C, %T , as determined by Practice E1757.
3.2.5 area —reported area counts for the internal standard
IS
peak in the chromatogram, as integrated by the electronic
4. Significance and Use
integrator.
4.1 The structural carbohydrate content is used in conjunc-
3.2.6 C —average concentration of monosaccharide c in
avg
tion with other assays to determine the total composition of
specimen s, in mg/mL, averaged across multiple injections of
biomass samples.
specimen s.
3.2.7 C —original concentration of monosaccharide c in
5. Interferences
LF
loss factor sample, in mg/mL.
5.1 The results of structural carbohydrate analysis are af-
3.2.8 C —concentration of internal standard (inositol) in
IS
fected by incomplete hydrolysis of biomass or hydrolysis
the calibration standards and specimen, in mg/mL.
conditions that are too severe. Incomplete hydrolysis will bias
3.2.9 C —concentrationofmonosaccharide cinspecimen s,
s
the results low because dimeric and oligomeric carbohydrates
measured by gas chromatography (GC), in mg/mL.
are not quantified. Hydrolysis conditions that are too severe
3.2.10 C —concentration of monosaccharide c in the
STD
degrade the liberated monosaccharides into materials that are
calibration standard, in mg/mL.
not quantified by this procedure, again biasing the results low.
3.2.11 CV (coeffıcient of variation)—the estimated standard
5.2 Incomplete neutralization and removal of acetic acid
deviation divided by the average value measured.
from the methylene chloride extract prior to GC analysis can
3.2.12 %extractives—the percentage by mass of extractives
result in ghost peaks appearing in the chromatogram or
in the extracted specimen as described inTest Method E1690.
carryover of monosaccharides from one injection to the next
3.2.13 k—constant used to convert the mass of monosac-
(owing to buildup of monosaccharides in the injection port),
charide to the mass of anhydrosugar from which it is derived.
leading to erroneous quantitation.
For arabinose and xylose, k =0.88 (m/z 132/150); for man-
5.3 Test specimens not suitable for analysis by this proce-
nose, galactose and glucose, k=0.90 (m/z 162/180).
dure include alkaline and acid-pretreated biomass samples that
3.2.14 LF—loss factor for monosaccharide c. Used to cor-
have not been washed. Unwashed pretreated biomass samples
rect for the amount of monosaccharide lost through degrada-
containing free acid or alkali may change visibly on heating.
tion during acid hydrolysis of biomass.
5.4 Materials containing nonstructural carbohydrates also
3.2.15 m—initial mass of the biomass specimen, in mg. are unsuitable for this procedure since nonstructural carbohy-
I
3.2.16 m —mass of monosaccharide in solution, cor- drates may undergo degradation to materials that are not
corr
rected for hydrolysis losses, in mg. quantified in this procedure.
E1821–01 (2007)
6. Apparatus 7.1.9 Dichloromethane, (CH Cl ).
2 2
7.1.10 Inositol Solution (20 mg/mL)—Dissolve 5.000 6
6.1 Analytical Balance, readable to 0.1 mg.
0.0025 g of inositol (C H O , 98+wt%) in water and dilute
6.2 Autoclave, capable of maintaining 121 6 3°C. 6 12 6
to 250 mL. Store at 4°C and discard after one week.
6.3 Convection Ovens, temperature controlled to 45 6 3°C
7.1.11 Loss Factor Standard Stock Solution—Combine to-
and 105 6 3°C.
gether each of the following monosaccharides. Weigh each
6.4 Desiccator, containing anhydrous calcium sulfate.
monosaccharide in the following nominal amounts (record
6.5 Gas Chromatograph, equipped with electronic integra-
each actual weight to the nearest 0.1 mg). Dissolve in water
tor,capillarysplitinjectionport,flameionizationdetectorwith
and dilute to 100 mL. Store at 4°C and discard after four
make-up gas, 250 µm 315 m fused-silica capillary column
weeks.
coatedwith50%cyanopropylphenylmethylpolysiloxane,0.25
µm film thickness (DBy-225 or equivalent). Arabinose (C H O ) 900–1100 mg
5 10 5
Mannose (C H O ) 900–1100 mg
6 12 6
6.6 Ice Bath.
Galactose (C H O ) 900–1100 mg
6 12 6
6.7 Ultrasonic Bath.
Xylose (C H O ) 900–1100 mg
5 10 5
Glucose (C H O ) 900–1100 mg
6.8 Vortex Mixer, or equivalent method to rapidly mix 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 200
standard solutions shall be 98+mass% purity. Other chemical
mLwater.Allowtocooltoroomtemperaturebeforedilutingto
grades may be substituted, provided it is first ascertained that
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
7.1.2 Purity of Water—Unless otherwise indicated, refer-
an ice bath with stirring. Allow solution to come to room
ences to water mean reagent water as defined by Type 1 of
temperature and dilute to 1 L. Check the concentration by
Specification D1193.
titration and adjust the concentration to 12.06 0.1 M (24.0 6
7.1.3 Acetic Acid (CH COOH), glacial.
0.2 N).
7.1.4 Acetic Anhydride ((CH CO) O).
3 2
7.2 Materials:
7.1.5 Ammonium Hydroxide, (NH OH), concentrated
7.2.1 Glass Filtering Crucibles, 50 mL, medium porosity,
(28–30 wt% NH ).
nominal pore size of 10 µm.
7.1.6 Ammonium Hydroxide Solution (;3M)—Dilute 5.0
7.2.2 Glass Serum Bottles, 125 mL, crimp-top style with
6 0.1 mL of concentrated ammonium hydroxide (NH OH)
rubber stoppers and aluminum seals to fit.
with 20.06 0.1 mL of water. Prepare fresh before each use.
7.1.7 Monosaccharide Stock A Solution—Combine the fol- 7.2.3 Vacuum Adaptor for Filtering Crucibles.
lowing monosaccharides. Weigh each monosaccharide in the 7.2.4 Vials,13 3 32 mm crimp-top style with
following nominal amounts (record each actual mass to the polytetrafluoroethylene-faced rubber septum and aluminum
nearest 0.1 mg). Dissolve in water and dilute to 100 mL. Store crimp seals or equivalent.
at 4°C and discard after four weeks.
8. Hazards
Arabinose (C H O ) 90–110 mg
5 10 5
Xylose (C H O ) 650–750 mg
5 10 5
8.1 Do not permit sulfuric acid, glacial acetic acid, acetic
Mannose (C H O ) 90–110 mg
6 12 6
Galactose (C H O ) 90–110 mg
anhydride, or potassium hydroxide to contact skin or clothing.
6 12 6
Glucose (C H O ) 1900–2100 mg
6 12 6
They are corrosive. Wear protective clothing.
7.1.8 Monosaccharide Stock B Solution—Prepare in man- 8.2 After the autoclave step, the glass bottles are hot and
ner identical to monosaccharide stock A solution. may be pressurized. Handle with caution.
8.3 Solutions of potassium borohydride will spontaneously
evolvehydrogengasonstanding.Topreventpressurization,do
DBy-225isatrademarkofJ&WScientificInc.,91BlueRavineRoad,Folsom,
not seal bottles. Ensure adequate ventilation around such
CA 95630.
solutionstoaverttheaccumulationofflammablehydrogengas.
Reagent Chemicals, American Chemical S
...
This document is not anASTM standard and is intended only to provide the user of anASTM 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:E1821–96 Designation:E1821–01 (Reapproved 2007)
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 (e) 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
1.1 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 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
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 ofASTM Committee E-48 on Biotechnology and is the direct responsibility of Subcommittee E48.05 on Biomass Conversion.
Current edition approved June 10, 1996. Published August 1996.
This test method is under the jurisdiction ofASTM Committee E48 on Biotechnology and is the direct responsibility of Subcommittee E48.05 on Biomass Conversion.
Current edition approved Nov. 15, 2007. Published January 2008. Originally approved in 1996. Last previous edition approved in 2001 as E1821-01.
ForreferencedASTMstandards,visittheASTMwebsite,www.astm.org,orcontactASTMCustomerServiceatservice@astm.org.For Annual Book of ASTM Standards
, Vol 11.01.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.
E1821–01 (2007)
3. Terminology
3.1 Descriptions 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 corrected for 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
thebiomasssolidswithdiluteacidhydrolysis.Forthepurposeofthistestmethod,themonosaccharidesthatareconsideredpresent
are arabinose, xylose, mannose, galactose, and glucose.
3.2 Abbreviations:Abbreviations: 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
c
integrator.
3.2.5 area —reportedareacountsfortheinternalstandardpeakinthechromatogram,asintegratedbytheelectronicintegrator.
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—lossfactorformonosaccharide c.Usedtocorrectfortheamountofmonosaccharidelostthroughdegradationduring
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 —percentagebymass,oftotalsolidsofthespecimenpreparedbydryingat45°C,asdescribedbyPracticeE1757.
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.
3.2.25 %T — percentage by mass, of total solids of the extracted specimen determined at 105°C as described byTest 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.
E1821–01 (2007)
5. Interferences
5.1 Theresultsofstructuralcarbohydrateanalysisareaffectedbyincompletehydrolysisofbiomassorhydrolysisconditionsthat
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 they nonstructural carbohy-
drates may undergo degradation to materials that are not quantified in this procedure, and hence are lost. 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 315 m fused-silica capillary column coated with 50% cyanopropylphenyl methylpolysiloxane, 0.25 µm
film thickness (DBy-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—Unlessotherwiseindicated,referencestowatermeanreagentwaterasdefinedbyType1ofSpecification
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
7.1.6 Ammonium Hydroxide Solution (;3M)—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.
Annual Book of ASTM Standards, Vol 11.05.
DBy-225 is a trademark of J&W Scientific Inc., 91 Blue Ravine Road, Folsom, CA 95630.
DBy-225 is a trademark of J&W Scientific Inc., 91 Blue Ravine Road, Folsom, CA 95630.
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.
E1821–01 (2007)
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)—Dissolve7.50 60.05gpotassiumborohydride(KBH)in40mLof ;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)—Dissolve58.060.5gofpotassiumhydroxide(KOH,85wt%)in200mLwater.
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%
...
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