ASTM E1821-96

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Designation: E 1821 – 96
Standard Test Method for
Determination of Carbohydrates in Biomass by Gas
Chromatography
This standard is issued under the fixed designation E 1821; 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 (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
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 responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica-
1.1 This test method describes the determination of struc-
bility of regulatory limitations prior to use. See Section 8 for
tural carbohydrates present in a biomass sample, expressed as
specific hazards statements.
the percent mass of an oven-dried sample basis of each
anhydrosugar.
2. Referenced Documents
1.2 Sample materials suitable for this procedure include
2.1 ASTM Standards:
hard and softwoods, herbaceous materials, such as sericea and
D 1193 Specification for Reagent Water
switchgrass, agricultural residues, such as corn stover, wheat
E 1690 Test Method for Determination of Ethanol Extrac-
straw, and bagasse, wastepaper, such as boxboard, office waste,
tives in Biomass
and newsprint, acid or alkaline-pretreated biomass, washed
E 1721 Test Method for Determination of Acid-Insoluble
free of any residual acid or alkali, and the solid fraction of
Residue in Biomass
fermentation residues.
E 1756 Test Method for Determination of Total Solids in
1.3 The options for the types of samples to be analyzed in
Biomass
this procedure are:
E 1757 Practice for Preparation of Biomass for Composi-
1.3.1 Prepared Biomass Samples:
tional Analysis
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
3. Terminology
sample.
3.1 Descriptions of Terms Specific to This Standard:
1.3.1.2 45°C Dried Material—Results are reported as the
3.1.1 anhydrosugars, n—the nominal repeating unit of a
percent by mass, based on the oven-dried mass of the 45°C
polysaccharide. When polysaccharides undergo acid hydroly-
dried sample.
sis, each repeating unit adds a single molecule of water to form
1.3.1.3 Freeze Dried Material—Results are reported as the
the free monosaccharide that is analyzed. The extra weight
percent by mass, based on the oven-dried mass of the freeze
from this water of hydrolysis must be corrected for in calcu-
dried sample.
lating the actual mass percent of the polysaccharide in the
1.3.2 Extractives-Free Sample—Results are reported as the
original biomass sample.
percent by mass, based on the oven-dried mass of the extracted
3.1.2 as received biomass, n—material as it is received in its
sample.
field or process collected state.
1.4 This standard method is generally not suitable for
3.1.3 extractives-free biomass—air-dried solids left after
samples that contain soluble, nonstructural carbohydrates un-
biomass has been treated according to Test Method E 1690.
less they are removed prior to analysis.
3.1.4 oven-dried mass, n—the moisture-free mass of any
1.5 This standard does not purport to address all of the
biomass sample (as received, prepared, extractives-free, etc.)
safety concerns, if any, associated with its use. It is the
dried at 105°C as described in Test Method E 1756.
This test method is under the jurisdiction of ASTM Committee E-48 on
Biotechnology and is the direct responsibility of Subcommittee E48.05 on Biomass
Conversion. Annual Book of ASTM Standards, Vol 11.01.
Current edition approved June 10, 1996. Published August 1996. Annual Book of ASTM Standards, Vol 11.05.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
E 1821
3.1.5 prepared biomass, n—as received biomass material c)—this is the ratio of the detector response for monosaccha-
that has been treated according to Practice E 1757 in order to ride c versus the detector response for the internal standard
raise the total solids content above 85 %, based on an oven- (inositol) for a given injection of the specimen.
dried solids weight. 3.2.21 V —87 mL, volume of hydrolysis solution.
f
3.2.22 %T —percentage by mass, of total solids of the
3.1.6 structural carbohydrates, n—polysaccharides that
cannot be removed by extraction with solvents and are liber- specimen prepared by drying at 45°C, as described by Practice
E 1757.
ated from the biomass solids with dilute acid hydrolysis. For
the purpose of this test method, the monosaccharides that are 3.2.23 %T —percentage by mass, of total solids of the
specimen, dried at 105°C, as determined by Test Method
considered present are arabinose, xylose, mannose, galactose,
and glucose. E 1756.
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 E 1756.
sugar on an extractives-free, oven-dried mass basis.
3.2.25 %T —percentage by mass, of total solids of the
3.2.2 %Anhydro —the percent by mass of the anhydro-
ext
whole
extracted specimen determined at 105°C as described by Test
sugar, on an oven-dried mass basis.
Method E 1756.
3.2.3 AR (Amount Ratio)—ratio of the concentration
c
3.2.26 %T —percentage by mass, of total solids of the
(amount) of monosaccharide c to the concentration (amount) of fd
specimen prepared by freeze drying, as described by Practice
internal standard in the specimen.
E 1757.
3.2.4 area —reported area counts for the monosaccharide c
c
3.2.27 %T —percentage, by mass, of total solids of the
peak in the chromatogram, as integrated by the electronic prep
specimen prepared by freeze drying,% T , or by drying at
integrator. fd
45°C, %T , as determined by Practice E 1757.
3.2.5 area —reported area counts for the internal standard 45
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
LF
5. Interferences
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 —concentration of monosaccharide c in specimen 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 in Test Method E 1690.
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 5 0.88 (m/z 132/150); for
5.3 Test specimens not suitable for analysis by this proce-
mannose, galactose and glucose, k 5 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.
I
are unsuitable for this procedure since they may undergo
3.2.16 m —mass of monosaccharide in solution, cor-
corr degradation to materials that are not quantified in this proce-
rected for hydrolysis losses, in mg.
dure, and hence are lost.
3.2.17 RR —averaged response ratio of monosaccharide c
avg
6. Apparatus
to the internal standard (inositol) in the calibration standard.
Derived from multiple injections of the same calibration
6.1 Analytical Balance, readable to 0.1 mg.
standard.
6.2 Autoclave, capable of maintaining 121 6 3°C.
3.2.18 RR —response ratio of monosaccharide c to the
6.3 Convection Ovens, temperature controlled to 45 6 3°C
s
internal standard (inositol) in the specimen.
and 105 6 3°C.
3.2.19 RR —response ratio of monosaccharide c to the 6.4 Desiccator, containing anhydrous calcium sulfate.
STD
internal standard (inositol) in the calibration standard.
6.5 Gas Chromatograph, equipped with electronic integra-
3.2.20 RRF (Relative Response Factor of monosaccharide tor, capillary split injection port, flame ionization detector with
E 1821
make-up gas, 250 μm 3 15 m fused-silica capillary column and dilute to 100 mL. Store at 4°C and discard after four
coated with 50 % cyanopropylphenyl methylpolysiloxane, 0.25 weeks.
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
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 4
mL of ;3 M ammonium hydroxide (NH OH) solution. Use an
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
ammonium hydroxide (NH OH) solution. Prepare immediately
reagents conform to the specifications of the Committee on
before use. Discard after 6 h. This quantity is sufficient for 50
Analytical Reagents of the American 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
mL water. Allow to cool to room temperature before diluting to
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.
of 96 wt % sulfuric acid (H SO ) to 300 mL of water cooled in
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 D 1193.
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 (;3 M)—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.
7.2.3 Vacuum Adaptor for Filtering Crucibles.
7.1.7 Monosaccharide Stock A Solution—Combine the fol-
7.2.4 Vials,13 3 32 mm crimp-top style with
lowing monosaccharides. Weigh each monosaccharide in the
polytetrafluoroethylene-faced rubber septum and aluminum
following nominal amounts (record each actual mass to the
crimp seals or equivalent.
nearest 0.1 mg). Dissolve in water and dilute to 100 mL. Store
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
anhydride, or potassium hydroxide to contact skin or clothing.
Galactose (C H O ) 90–110 mg
6 12 6
Glucose (C H O ) 1900–2100 mg
They are corrosive. Wear protective clothing.
6 12 6
8.2 After the autoclave step, the glass bottles are hot and
7.1.8 Monosaccharide Stock B Solution—Prepare in man-
may be pressurized. Handle with caution.
ner identical to monosaccharide stock A solution.
8.3 Solutions of potassium borohydride will evolve sponta-
7.1.9 Dichloromethane, (CH Cl ).
2 2
neously hydrogen gas on standing. To prevent pressurization,
7.1.10 Inositol Solution (20 mg/mL)—Dissolve 5.000 6
do not seal bottles. Ensure adequate ventilation around such
0.0025 g of inositol (C H O , 98 + wt %) in water and dilute
6 12 6
solutions to avert the accumulation of flammable hydrogen gas.
to 250 mL. Store at 4°C and discard after one week.
Wet potassium borohydride is highly flammable.
7.1.11 Loss Factor Standard Stock Solution—Combine to-
8.4 Methylene chloride is a very volatile solvent and is both
gether each of the following monosaccharides. Weigh each
toxic and a suspected carcinogen. Handle only with adequate
monosaccharide in the following nominal amounts (record
ventilation.
each actual weight to the nearest 0.1 mg). Dissolve in water
9. Sampling, Test Specimens and Test Units
DBy-225 is a trad
...