ASTM E3503 - 25 草本生物质中半纤维素的高效液相色谱测定法

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Designation: E3503 −25
Standard Test Method for
Determination of Hemicellulose in Herbaceous Biomass by
High Performance Liquid Chromatography
1
This standard is issued under the ﬁxed designation E3503; 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.
1. Scope
1.1 This test method covers the quantiﬁcation of hemicel-
lulose present in herbaceous biomass substrates using a dilute
acid incubation period and high performance liquid chroma-
tography (HPLC) analysis. This method can be utilized across
a variety of substrates containing 1 % to 44 % hemicellulosic
content.
2
1.2 This method intends to provide carbohydrate analysis
for evaluation of fermentation efficacy for various biomass
saccharides associated with hemicellulose. Current fermenta-
tion technologies can convert arabinose, galactose, glucose,
mannose, and xylose and will leave uronic acids and acetic
acids unconverted, so they are not included in this assay. It
should be noted that co-quantiﬁcation of xylose, mannose, and
galactose occurs. Additionally, glucose is simultaneously re-
leased from starch, cellulose, and hemicellulose during this
assay and the source cannot be determined, so glucose is not
quantiﬁed in this assay.
1.3 Units—The values stated in SI units are to be regarded
as the standard. No other units of measurement are included in
this standard.
1.4 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 appro-
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
1.5 This international standard was developed in accor-
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
3
E1705 Terminology Relating to Bioenergy and Industrial
Chemicals from Biomass
E1756 Test Method for Determination of Total Solids in
Biomass
E1757 Practice for Preparation of Biomass for Composi-
tional Analysis
2.2 Other Documents:
AOAC 935.29 Moisture in Malt – Gravimetric Method
(AOAC 2005)
4
3. Terminology
3.1 Deﬁnitions of Terms:
3.1.1 For deﬁnitions of terms used in this standard, refer to
Terminology E1705.
NOTE 1—The user is advised that the deﬁnitions used by various
industries, marketers, regulatory bodies, and other ASTM Committees can
differ from those in this test method. When using this test method, it is
critical to use terminology from Committee E48.
3.1.2 arabinose, n—a ﬁve-carbon monosaccharide common
in biomass.
3.1.3 calibration veriﬁcation standard, CVS, n—standards
used in determining the quality of the calibration curve as well
as the quality of the standard reagents used in preparing the
calibration standards.
3.1.4 ﬁve-carbon monosaccharide, n—a simple sugar with
ﬁve carbon atoms also known as a pentose.
3.1.5 galactose, n—six-carbon monosaccharide common in
biomass.
3.1.6 glucose, n—six-carbon monosaccharide common in
biomass.
1
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 Dec. 1, 2025. Published January 2026. DOI: 10.1520/
E3503-25.
2
The Determination of Hemicellulose in Herbaceous Biomass by High Perfor-
mance Liquid Chromatography assay process and conditions are covered by a
pending patent held by Soliton, 5132 S Cliff Ave, Sioux Falls, SD 57108. Interested
parties are invited to submit information regarding the identiﬁcation of an
alternative(s) to this patented item to the ASTM International Headquarters. Your
comments will receive careful consideration at a meeting of the responsible
technical committee which you may attend.
3
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at www.astm.org/contact. For Annual Book of
ASTM Standards volume information, refer to the standard’s Document Summary
page on the ASTM website.
4
Available from AOAC International, 2275 Research Blvd., Suite 300,
Rockville, MD 20850-3250, http://www.aoac.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
1

3.1.7 hemicellulose, n—a polymeric, branched carbohydrate

with mixed ﬁve and six carbon monomeric sugars, typically

with a xylose or mannose backbone.

3.1.7.1 Discussion—The glucan portion of hemicellulose

present in biomass samples is not quantiﬁed under the condi-

tions prescribed in this method. Additionally, hemicellulose as

analyzed by this method will include measurement of any free

monosaccharides present in the herbaceous biomass.

3.1.8 herbaceous biomass, n—non-woody plant materials,

to include grasses, agricultural residues, and grains, which are

fast growing and are harvested or die back at the end of each

growing cycle.

3.1.9 mannose, n—a six-carbon monosaccharide common in

biomass.

3.1.10 monosaccharide, n—any of the class of sugars that

cannot be hydrolyzed to give a simpler sugar.

3.1.11 polysaccharide, n—a carbohydrate whose molecules

consist of a long chain of sugar residues bonded together.

3.1.12 six-carbon monosaccharide, n—a simple sugar with

six carbon atoms also known as a hexose.

3.1.13 xylose, n—a ﬁve-carbon monosaccharide common in

biomass.

4. Signiﬁcance and Use

4.1 This test method consists of a two-step acid hydrolysis

prior to measurement of xylose, galactose, arabinose, and

mannose by HPLC.

4.2 The hydrolysis conditions have been optimized to en-

sure maximum hemicellulose hydrolysis while minimizing

degradation of the monosaccharides.

5. Interferences

5.1 This test method employs non-speciﬁc acid hydrolysis

conditions which do not differentiate between polysaccharide

sources. All polysaccharides comprising xylan, galactan,

arabinan, and mannan that are exposed to the hydrolysis

conditions will be included in the ﬁnal hemicellulose result.

6. Apparatus

6.1 Analytical balance, with precision to 0.1 mg.

6.2 Water bath, capable of maintaining 85 °C 62 °C.

6.3 Ice water bath.

6.4 HPLC system, equipped with refractive index detector.

6.5 Bio-rad HPX-87H HPLC column with Micro-Guard

Cation H pre-column guard ﬁlter, or equivalent carbohydrate

analysis HPLC column.

6.6 Centrifuge, capable of maintaining 5000 rpm with mini-

mum of a 20 cm diameter rotor.

6.7 Vortex mixer, variable speed, capable of reaching

3000 rpm.

7. Reagents and Materials

7.1 Purity of Reagents—Reagent grade materials shall be

used 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.

Other grades may be used,

provided it is ﬁrst ascertained that the reagent is of sufficiently

high purity to permit its use without lessening the accuracy the

determination.

7.1.1 D-(+)-xylose, ≥99.5 % (GC).

7.1.2 D-(-)-arabinose, ≥99.5 % (GC).

7.1.3 Sulfuric Acid, 60 % w/w.

7.1.4 Water, HPLC grade, 0.2 µm ﬁltered (or deionized

18 MΩ⁄cm ﬁltered water).

7.1.5 Axion HPLC Calibration Standard Kit LQC-HPLC-

HEM-K, or equivalent.

7.1.6 Axion HPLC Validation Standard LQC-HPLC-

HEM-V, or equivalent.

7.2 Materials:

7.2.1 Positive displacement automatic pipettors, capable of

multi-dispensing with corresponding compatible tips.

7.2.2 Sample racks, to accommodate tubes with outer diam-

eter (OD) of 16 mm.

7.2.3 Conical polypropylene centrifuge tube, 50 mL, with a

tightly ﬁtting screw cap.

7.2.4 Autosampler vials and caps, 2 mL ﬁll volume.

7.2.5 Disposable syringes, 3 mL, ﬁtted with 0.2 µm nylon

syringe ﬁlters.

8. Hazards

8.1 Handle sulfuric acid carefully to avoid contact with skin

or clothing, as it is corrosive.

8.2 Adhere to the general safety measures that apply to all

chemical substances. For more information regarding the safe

usage and handling of the reagents and components, please

refer to the associated safety data sheets (SDS).

8.3 This test method should not be carried out by anyone

other than a trained and experienced laboratory analyst.

9. Sampling, Test Specimens, and Test Units

9.1 Before analysis, test material should be prepared ac-

cording to Practice E1757.

9.2 Care shall be taken to ensure a representative sample is

used for analysis. The material should be at room temperature

before sampling.

9.3 Prior to calculation, total solids should be analyzed

according to either Test Method E1756 or AOAC 935.29.

10. Calibration and Standardization

10.1 Prepare a series of calibration standards containing at a

minimum xylose and arabinose using the required concentra-

tion range in Table 1. A minimum of ﬁve calibration points and

a calibration validation standard are required.

ACS Reagent Chemicals, Speciﬁcations and Procedures for Reagents and

Standard-Grade Reference Materials, 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. Pharma-

copeial Convention, Inc. (USPC), Rockville, MD.

E3503 − 25

NOTE 2—It is recommended that a calibration validation standard be

analyzed once every ten samples to ensure instrument stability across the

sample set.

10.2 It is recommended that samples be analyzed the day

the hydrolysis assay is completed. If a delay in HPLC analysis

occurs, the samples may be stored in a refrigerator (2 °C to

4 °C) for no longer than three days. Delays longer than three

days will require that the hydrolysis assay be repeated on a

fresh portion of test sample.

10.3 Calibration standards may be prepared or purchased

from several vendors. Prepared standards should be stored in a

refrigerator (2 °C to 4 °C) or according to manufacturer in-

structions.

10.4 Create a calibration curve using a linear regression.

Using the curve, determine the concentration in g/100 mL for

all samples, standard(s), and the reagent blank.

10.5 Calibration veriﬁcation standards and reference mate-

rials must have a % recovery between 95 % to 105 % for all

components of interest for any sample results to be accepted.

11. Procedure

11.1 Acid Hydrolysis Procedure—High Concentration:

11.1.1 Weigh out 500 mg 610 mg of sample into a tared

centrifuge tube and record weight to the nearest 0.1 mg.

11.1.2 A reagent blank and standard(s) must also be pre-

pared with each sample set.

11.1.2.1 D-(+)-xylose should be analyzed as a quality con-

trol standard with each sample set. Recovery of the

D-(+)-xylose should be > 95 % to consider results accurate and

valid. This recovery ensures assay procedures, HPLC

performance, and calculations are being applied correctly.

Recovery of the D-(+)-xylose used in the assay should be

calculated using the formula in 12.1.

NOTE 3—The recovery of the monosaccharide standard is not used to

correct or calculate the hemicellulose result according to 12.2.

11.1.3 Add 2.0 mL of 60 % w/w sulfuric acid to each tube

with a positive displacement automatic pipettor.

11.1.4 Cap tubes and mix each sample vigorously on vortex

mixer until the entire sample goes into suspension.

11.1.5 Incubate samples in an ice water bath for 30 min

total.

11.1.6 Samples must be vortexed, approximately 30 s, at the

halfway point (15 min) during incubation window.

11.1.7 Remove samples from ice water bath after 30 min.

11.2 Acid Hydrolysis Procedure—Low Concentration:

11.2.1 With a positive displacement automatic pipettor, add

15.0 mL of DI water to each tube, cap tubes and vortex each

sample.

11.2.2 Incubate samples in an 85 °C 62 °C water bath for

5 h.

11.2.3 Samples must be vortexed, approximately 30 s, at the

halfway point (2.5 h or 150 min) during incubation window.

11.2.4 Remove samples from water bath at 5 h.

11.2.5 Add 18.0 mL of DI water to each tube, including the

blank and standard, and vortex approximately 30 s to ensure

homogeneity.

11.2.6 Pour supernatant into syringe ﬁtted with syringe ﬁlter

and add sufficient sample to ﬁll HPLC vial.

11.2.6.1 Centrifuging the samples for 5 min at 5000 rpm

may help ﬁltration if desired.

11.3 HPLC Analysis Procedure:

11.3.1 Following ﬁltration and aliquoting, analyze the

standards, blank, and samples via HPLC-RID using the param-

eters listed below in Table 2.

NOTE 4—HPLC parameters may vary if an alternative column is

utilized for the analysis.

12. Calculation

12.1 Calculate the recovery of the xylose standard, in

percent, on a dry weight basis as follows:

% Recovery of Xylose Standard5

X2Y

100

A1R

⁄

D⁄ 100

×100

where:

R= the total volume of liquid added to the standard,

A= the weight of the sample,

D= the % wt total solids content of prepared standard,

X= the % wt/wt xylose of the sample, and

Y= the % wt/wt xylose of the method blank.

TABLE 1 Required Concentration Ranges

Component Required Concentration Range,

g/100 mL

Xylose 0.05 to 1.25

Arabinose 0.01 to 1.25

Calibration Veriﬁcation Mid-point of linear range: suggested

concentration 0.20

TABLE 2 HPLC Analysis Parameters

HPLC Acquisition Parameters Setting Speciﬁcation

Injection Volume 5.0 µL

Mobile Phase 0.005 M Sulfuric Acid

Flow Rate 0.6 mL/min

Autosampler Temperature 8 °C

Guard Column Temperature 65 °C

Column Temperature 65 °C

Detector Temperature 35 °C

Detector Refractive Index

Run Time 30 min

E3503 − 25

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标签： #纤维 #测定 #高效液

摘要：

本规范ASTM E3503 - 25详细规定了利用高效液相色谱（HPLC）技术测定草本生物质中半纤维素含量的标准试验方法。该标准适用于各类草本植物样品，如秸秆、草类及能源作物等，旨在通过精确的色谱分离与定量分析，为生物质原料的组成评价提供可靠依据。文档描述了从样品前处理、水解步骤到色谱条件设置与结果计算的全流程操作要求，有助于确保不同实验室间数据的可比性与重复性。对于从事生物质能源、生物基材料研发及农业废弃物资源化利用的科研人员与质量检测机构，该标准是评估草本生物质中半纤维素含量的关键技术参考，

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