ASTM E1758-95e1

Standard Test Method for Determination of Carbohydrates in Biomass by High Performance Liquid Chromatography

SCOPE
1.1 This test method covers the determination of carbohydrates present in a biomass sample, expressed as the percent, by mass, of each sugar on a 105°C dried mass basis.
Note 1—The percent sugar must be corrected for the water of hydrolysis before calculating the actual mass percent of the polysaccharide in the original biomass sample.
1.2 Sample materials suitable for this procedure include hard and soft woods, herbaceous materials (such as switchgrass and sericea), agricultural residues (such as corn stover, wheat straw, and bagasse), wastepaper (such as office waste, boxboard, and newsprint), acid or alkaline-pretreated biomass (washed free of any residual acid or alkali), and the solid fraction of fermentation residues. All results are reported relative to the 105°C oven-dried mass of the sample.
1.3 The options for the types of samples to be analyzed in this test method are as follows:
1.3.1 Prepared Biomass Samples:
1.3.1.1 Air Dried (%Tad)—The percent, by mass, of total solids of the air-dried sample.
1.3.1.2 45°C Dried (%T45—The percent, by mass, of total solids of the 45°C dried sample.
1.3.1.3 Freeze Dried (%Tfd )—The percent, by mass, of total solids of the freeze dried sample.
1.3.2 Extractives-Free Sample (%Text)—The percent, by mass, of total solids of the extracted sample determined at 105°C.
1.4 The values stated in SI units are to be regarded as the standard.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Specific precautionary statements are given in Note 3 and Note 4.

General Information

• Status: Historical
• Publication Date: 09-Nov-2001
• ICS: 65.040.20 - Buildings and installations for processing and storage of agricultural produce
• Technical Committee: E48 - Bioenergy and Industrial Chemicals from Biomass
• Drafting Committee: E48.05 - Biomass Conversion

Relations

• Replaced By: ASTM E1758-01 - Standard Test Method for Determination of Carbohydrates in Biomass by High Performance Liquid Chromatography
• Effective Date: 10-Nov-2001

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
e1
Designation: E 1758 – 95
Standard Test Method for
Determination of Carbohydrates in Biomass by High
Performance Liquid Chromatography
This standard is issued under the fixed designation E 1758; 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.
e NOTE—Paragraphs 11.4 and 11.7 were updated editorially in October 1996.
INTRODUCTION
The carbohydrates making up a major portion of biomass samples are polysaccharides constructed
primarily of glucose, xylose, arabinose, galactose, and mannose subunits. The polysaccharides present
in a biomass sample can be hydrolyzed to their component sugar monomers by sulfuric acid in a
two-stage hydrolysis process. These monosaccharides can then be quantitated by ion-moderated
partition HPLC.
1. Scope bility of regulatory limitations prior to use. Specific precau-
tionary statements are given in Note 2 and Note 4.
1.1 This test method covers the determination of carbohy-
drates present in a biomass sample, expressed as the percent,
2. Referenced Documents
by mass, of each sugar on a 105°C dried mass basis.
2.1 ASTM Standards:
1.2 Sample materials suitable for this procedure include
D 1193 Specification for Reagent Water
hard and soft woods, herbaceous materials (such as switchgrass
E 1690 Test Method for the Determination of Ethanol
and sericea), agricultural residues (such as corn stover, wheat
Extractives in Biomass
straw, and bagasse), wastepaper (such as office waste, box-
E 1721 Test Method for the Determination of Acid-
board, and newsprint), acid or alkaline-pretreated biomass
Insoluble Residue in Biomass
(washed free of any residual acid or alkali), and the solid
E 1756 Test Method for the Determination of Total Solids in
fraction of fermentation residues. All results are reported
Biomass
relative to the 105°C oven-dried mass of the sample.
E 1757 Practice for Preparation of Biomass for Composi-
1.3 The options for the types of samples to be analyzed in
tional Analysis
this test method are as follows:
1.3.1 As Received Samples:
3. Terminology
1.3.1.1 Air Dried (%T )—The percent, by mass, of total
ad
3.1 Definitions of Terms Specific to This Standard:
solids of the air-dried sample.
3.1.1 as received biomass—the biomass material as it is
1.3.1.2 45°C Dried (%T )—The percent, by mass, of total
received in its field or process collected state.
solids of the 45°C dried sample.
3.1.2 oven-dried mass—the moisture-free mass of a biom-
1.3.1.3 Freeze Dried (%T )—The percent, by mass, of total
fd
ass sample dried at 105°C as described in Test Method E 1756.
solids of the freeze dried sample.
3.1.3 prepared biomass—material that has been treated
1.3.2 Extractives-Free Sample (%T )—The percent, by
ext
according to Practice E 1757 in order to raise the total solids
mass, of total solids of the extracted sample determined at
content above 85 %, by mass, based on an oven-dried solids
105°C.
mass.
1.4 The values stated in SI units are to be regarded as the
3.2 Abbreviations—Abbreviations of standards used in the
standard.
procedure, and definitions of terms used in the calculations are
1.5 This standard does not purport to address all of the
as follows:
safety concerns, if any, associated with its use. It is the
3.2.1 C —known concentration of sugar recovery standard
responsibility of the user of this standard to establish appro-
before hydrolysis, in mg/mL.
priate safety and health practices and determine the applica-
3.2.2 C —concentration of sugar recovery standard de-
tected by HPLC after hydrolysis, in mg/mL.
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 Oct. 10, 1995. Published December 1995. Annual Book of ASTM Standards, Vol 11.05.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
E 1758
3.2.3 C —concentration of sugar in hydrolyzed sample 6.4 Desiccator, using anhydrous calcium sulfate.
corr
corrected for hydrolysis, in mg/mL. 6.5 Guard Columns, cartridges appropriate for the column
3.2.4 C —concentration of sugar in hydrolyzed sample used.
spl
detected by HPLC, in mg/mL.
NOTE 1—Deashing guard column cartridges from BioRad, of the ionic
3.2.5 CVS (calibration verification standard)—standards + −
form H /CO , are an option when using an HPX-87P column, or
used in determining the quality of the calibration curve as well
equivalent. These cartridges are effective in eliminating baseline ramping.
as the quality of the standard reagents used in preparing the
6.6 Hewlett Packard Model 1090 HPLC, or equivalent,
calibration standards.
with refractive index detector.
3.2.6 m —initial mass of sample, in mg.
4 4
6.7 HPLC Columns, BioRad HPX-87C or HPX-87P, or
3.2.7 % extractives—the percentage, by mass, of extractives
both, or equivalent.
in the extracted sample as described in Test Method E 1690.
6.8 Water Bath, set at 30 6 1°C.
3.2.8 %R —percent recovery of sugar recovery standard,
srs
as determined in 13.2.
7. Reagents and Materials
3.2.9 %sugar —the percentage, by mass, of
extractives-free
7.1 Chemicals:
sugar on an extractives-free 105°C dry weight basis, as
7.1.1 Purity of Reagents—Reagent grade chemicals shall be
determined in 13.6.1.
used in all tests. Unless otherwise indicated, it is intended that
3.2.10 % sugar —the corrected mass percent
whole sample
all reagents conform to the specifications of the Committee on
sugar value on an extractives-free basis corrected to an as
Analytical Reagents of the American Chemical Society where
received (whole sample) 105°C dry mass basis.
such specifications are available. Other grades may be used,
3.2.11 %T —percentage, by mass, of total solids of the
provided it is first ascertained that the reagent is of sufficiently
sample prepared by drying at 45°C, as described by Practice
high purity to permit its use without lessening the accuracy of
E 1757.
the determination.
3.2.12 %T —percentage, by mass, of total solids in the
7.1.2 Purity of Water—Unless otherwise indicated, refer-
sample, dried at 105°C, as determined by Test Method E 1756.
ences to water shall be understood to mean reagent water as
3.2.13 %T —percentage, by mass, of total solids of the
ad
defined by Type 1 of Specification D 1193.
air-dried sample determined at 105°C as described by Test
7.1.3 Calcium Carbonate.
Method E 1756.
7.1.4 High-Purity Sugars (98 % +, By Mass)—Two sets of
3.2.14 %T —percentage, by mass, of total solids of the
ext
glucose, xylose, galactose, arabinose, and mannose, meeting
extracted sample determined at 105°C as described by Test
the requirements described above, dried at 45°C. The sugars
Method E 1756.
are used in preparing calibration standards, calibration verifi-
3.2.15 %T —percentage, by mass, of total solids of the
fd
cation standards (CVS), and sugar recovery standards (SRS).
sample prepared by freeze drying, as described by Test Method
The sugars used in preparing the calibration standards should
E 1756.
be from a source (manufacturer or lot) other than that used in
3.2.16 %T —percentage, by mass, of total solids of the
prep
preparing the CVS. Either set of sugars may be used for
sample prepared by freeze drying, %T , or by drying at 45°C,
fd
preparing the SRS solutions used in determining sugar recov-
%T , as determined by Practice E 1757.
eries after hydrolysis.
3.2.17 SRS (sugar recovery standards)—standards used to
7.1.5 Sulfuric Acid Solution (72 % w/w or 12.00 6 0.02
determine sugar recoveries after hydrolysis.
M)—Slowly add 665 mL of concentrated sulfuric acid (H SO )
2 4
3.2.18 V —volume of filtrate, 87.0 mL.
F
to 300 mL of water while cooling in an ice bath and stirring.
Allow to come to room temperature. Adjust the relative density
4. Significance and Use
to 1.6389 6 0.0012 at 15.6°C/15.6°C.
4.1 The percentage, by mass, of sugar content is used in
7.2 Materials:
conjunction with other assays to determine the total composi-
7.2.1 Autosampler Vials, with crimp top seals to fit.
tion of biomass samples.
7.2.2 Disposable Syringes, 3 mL.
5. Interferences 7.2.3 Disposable Syringe Filters, nylon, 0.2 μm.
7.2.4 Glass Serum Bottles, crimp top style, 125 mL, with
5.1 Samples with high protein content may result in the
rubber stoppers and aluminum seals to fit.
percentage, by mass, of sugar values being biased low, as a
consequence of protein binding with some monosaccharides.
5.2 Test specimens not suitable for analysis by this proce-
dure include alkaline and acid-pretreated biomass samples that
BioRad Aminext, HPX-87C and Aminext HPX-87P, available from BioRad,
Main Office, 3300 Regatta Boulevard, Richmond, CA 94804 has been found suitable
have not been washed. Unwashed pretreated biomass samples
for this purpose.
containing free acid or alkali may change visibly on heating.
Available from Hewlett-Packard, HP Analytical Direct, 2850 Centerville Road,
Wilmington, DE 19808.
6. Apparatus
Reagent Chemicals, American Chemical Society Specifications, American
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
6.1 Analytical Balance, readable to 0.1 mg.
listed by the American Chemical Society, see Analar Standards for Laboratory
6.2 Autoclave, capable of maintaining 121 6 3°C.
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
6.3 Convection Ovens, temperature control to 45 6 3 and
and National Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville,
105 6 3°C. MD.
E 1758
8. Hazards 11.2.2 If Test Method B of this practice is used (drying at
45°C), record the total solids calculated in this practice, %T ,
8.1 Handle the sulfuric acid carefully to avoid contact with
as %T .
skin or clothing, as it is corrosive. prep
11.2.3 If Test Method C of this practice is used (freeze
8.2 The glass bottles are hot and may be pressurized after
drying), record the total solids calculated in this practice, %T ,
the autoclave step. Use caution when handling. fd
as %T .
prep
9. Sampling, Test Specimens, and Test Units
11.3 If extractives-free material is used, determine the total
solids content of the extractive-free material by Test Method
9.1 Test specimens suitable for analysis by this procedure
E 1756 and record this value as %T .
are:
ext
11.4 Weigh 300 6 10 mg of the prepared or extractives-free
9.1.1 As-received biomass, and
sample to the nearest 0.1 mg and place in 16 3 100 mm glass
9.1.2 Extractives-free material prepared according to Test
test tube. Record as m , the initial mass of sample in grams.
Method E 1690.
NOTE 2—Warning: 72 % w/w sulfuric acid is very corrosive and
10. Calibration and Standardization
should be handled by trained personnel only.
10.1 Prepare a series of three to six sugar standards in
11.5 Add 3.00 6 0.01 mL (4.92 6 0.01 g) of 72 % w/w
deionized water at concentrations appropriate for preparing
H SO to the test tube containing the sample and stir for 1 min
2 4
calibration curves to quantitate each sugar of interest. An
or until thoroughly mixed.
HPX-87C column, or equivalent, is used to analyze glucose,
11.6 Place the test tube containing the sample into the water
xylose, and arabinose. If mannose and galactose are also to be
bath controlled to 30 6 1°C and hydrolyze for 2 h. Stir
determined, an HPX-87P column, or equivalent, must be used
approximately every 15 min to ensure the sample is completely
instead. Typically, the concentrations of these sugar standards
mixed and wet.
cover the range starting at the detection limit of the instrument
and extending up to 4.0 mg/mL. NOTE 3—The hydrolysis time may be reduced to1hifthe dried sample
has been milled and sieved to pass through a 20-mesh sieve and is retained
10.2 Prepare an independent CVS, as described in 8.1.2, for
on a 80-mesh sieve.
each set of calibration standards, using sugars obtained from a
source other than that used in preparing the calibration stan-
11.7 Weigh out 300 6 10 mg of each high purity sugar
dards. The CVS will contain precisely known amounts of each
standard (dried at 45°C), described in 8.1.4, to the nearest 0.1
sugar contained in the calibration standards, at a concentration
mg and place each in its own individual 16 3 100 mm glass
in the middle of the validated range of the calibration curve.
test tube. Add acid and hydrolyze these sugars as described in
The CVS will be analyzed after each calibration curve and at
the previous two steps. These SRS’s will be taken through the
regular intervals in the HPLC sequence, as dictated by good
remaining steps in the procedure in parallel with the samples.
laboratory practice. The CVS is used in confirming the quality
The calculated recovery of the SRS will be used to correct for
of the calibration curve(s) and the standard reagents used in
losses caused by the destruction of sugars during the hydrolysis
preparing the calibration standards. An additional benefit is
process.
obtained by bracketing groups of samples in the sequence with
11.8 Transfer each hydrolyzate to a glass bottle and dilute to
the CVS, assuring the analyst of the quality of the calibration
4 % w/w acid concentration by adding 84.00 6 0.04 mL water.
curve throughout the run.
Be careful to transfer all the residual solids along with the
hydrolysis liquor. The total mass added to the tared bottle is
11. Procedure
89.22 g (0.3 g sample, 4.92 g 72 % w/w H SO , and 84.00 g
2 4
11.1 An overview of the overall analytical sequence is as
deionized water). Because the relative density of the 4 % w/w
follows:
acid solution is 1.0250, the total volume of solution, V , is 87.0
F
11.1.1 Hydrolysis of sample with 72 % sulfuric acid,
mL.
11.1.2 Hydrolyzate dilution and autoclaving,
11.9 Stopper the bottles and crimp the aluminum seals into
11.1.3 Filtration of insolubles if separate analysis is desired,
place in preparation for the next step.
11.1.4 Neutralization of hydrolyzate,
11.10 Set the autoclave to a liquid vent cycle to prevent loss
11.1.5 Filtration of sample prior to HPLC analysis,
of sample from the bottle in the event of a loose crimp seal.
11.1.6 HPLC analysis of sugar standards, CVS, SRS, and
Autoclave the sample in the sealed bottle for1hat121 6 3°C.
hydrolyzate samples, and
NOTE 4—Warning: Handle the sealed bottle with caution after the
11.1.7 Calculation of sugar contents.
autoclave step, as it may have become pressurized.
11.2 For samples used as received (as received-biomass),
11.11 After c
...