ASTM F2602-13
(Test Method)Standard Test Method for Determining the Molar Mass of Chitosan and Chitosan Salts by Size Exclusion Chromatography with Multi-angle Light Scattering Detection (SEC-MALS)
Standard Test Method for Determining the Molar Mass of Chitosan and Chitosan Salts by Size Exclusion Chromatography with Multi-angle Light Scattering Detection (SEC-MALS)
SIGNIFICANCE AND USE
4.1 The degree of deacetylation of chitosan, as well at the molar mass and molar mass distribution, determines the functionality of chitosan in an application. For instance, functional and biological effects are highly dependent upon the composition and molar mass of the polymer.
4.2 This test method describes procedures for measurement of molar mass of chitosan chlorides and glutamates, and chitosan base, although it in principle applies to any chitosan salt. The measured molar mass is that for chitosan acetate, since the mobile phase contains acetate as counter ion. This value can further be converted into the corresponding molar mass for the chitosan as a base, or the parent salt form (chloride or glutamate).
4.3 Light scattering is one of very few methods available for the determination of absolute molar mass and structure, and it is applicable over the broadest range of molar masses of any method. Combining light scattering detection with size exclusion chromatography (SEC), which sorts molecules according to size, gives the ability to analyze polydisperse samples, as well as obtaining information on branching and molecular conformation. This means that both the number-average and mass-average values for molar mass and size may be obtained for most samples. Furthermore, one has the ability to calculate the distributions of the molar masses and sizes.
4.4 Multi-angle laser light scattering (MALS) is a technique where measurements of scattered light are made simultaneously over a range of different angles. MALS detection can be used to obtain information on molecular size, since this parameter is determined by the angular variation of the scattered light. Molar mass may in principle be determined by detecting scattered light at a single low angle (LALLS). However, advantages with MALS as compared to LALLS are: (1) less noise at larger angles, (2) the precision of measurements are greatly improved by detecting at several angles, and (3) the ability to det...
SCOPE
1.1 This test method covers the determination of the molar mass of chitosan and chitosan salts intended for use in biomedical and pharmaceutical applications as well as in tissue engineered medical products (TEMPs) by size exclusion chromatography with multi-angle laser light scattering detection (SEC-MALS). A guide for the characterization of chitosan salts has been published as Guide F2103.
1.2 Chitosan and chitosan salts used in TEMPs should be well characterized, including the molar mass and polydispersity (molar mass distribution) in order to ensure uniformity and correct functionality in the final product. This test method will assist end users in choosing the correct chitosan for their particular application. Chitosan may have utility as a scaffold or matrix material for TEMPs, in cell and tissue encapsulation applications, and in drug delivery formulations.
1.3 The values stated in SI units are to be regarded as 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 appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: F2602 − 13
Standard Test Method for
Determining the Molar Mass of Chitosan and Chitosan Salts
by Size Exclusion Chromatography with Multi-angle Light
1
Scattering Detection (SEC-MALS)
This standard is issued under the fixed designation F2602; 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.
3
1. Scope 2.2 United States Pharmacopeia/National Formulary:
<621> Chromatography
1.1 This test method covers the determination of the molar
4
2.3 National Institute of Standards and Technology:
mass of chitosan and chitosan salts intended for use in
NIST SP811 Special Publication: Guide for the Use of the
biomedical and pharmaceutical applications as well as in tissue
International System of Units (SI)
engineered medical products (TEMPs) by size exclusion chro-
matography with multi-angle laser light scattering detection
3. Terminology
(SEC-MALS).Aguideforthecharacterizationofchitosansalts
3.1 Definitions:
has been published as Guide F2103.
3.1.1 chitosan, n—a linear polysaccharide consisting of
1.2 Chitosan and chitosan salts used in TEMPs should be
β(1→4) linked 2-acetamido-2-deoxy-D-glucopyranose (Glc-
well characterized, including the molar mass and polydisper-
NAc) and 2-amino-2-deoxy-D-glucopyranose (GlcN). Chito-
sity(molarmassdistribution)inordertoensureuniformityand
san is a polysaccharide derived by N-deacetylation of chitin.
correct functionality in the final product. This test method will
3.1.2 degree of deacetylation, n—the fraction or percentage
assist end users in choosing the correct chitosan for their
of glucosamine units (GlcN: deacetylated monomers) in a
particular application. Chitosan may have utility as a scaffold
chitosan polymer molecule.
or matrix material for TEMPs, in cell and tissue encapsulation
applications, and in drug delivery formulations. 3.1.3 molar mass average, n—the given molar mass (M) of
a chitosan will always represent an average of all of the
1.3 The values stated in SI units are to be regarded as
molecules in the population. The most common ways to
standard. No other units of measurement are included in this
¯
expressthemolarmassareasthe number average(M )andthe
n
standard.
¯
mass average (M ). The two averages are defined by the
w
1.4 This standard does not purport to address all of the
following equations:
safety concerns, if any, associated with its use. It is the
2
N M w M N M
responsibility of the user of this standard to establish appro- (i i i (i i i (i i i
¯ ¯
M 5 and M 5 5 (1)
n w
priate safety and health practices and determine the applica-
N w N M
(i i (i i (i i i
bility of regulatory limitations prior to use.
where:
N = number of molecules having a specific molar mass M,
2. Referenced Documents
i i
2 and
2.1 ASTM Standards:
w = mass of molecules having a specific molar mass M.
i i
F2103 Guide for Characterization and Testing of Chitosan
Salts as Starting Materials Intended for Use in Biomedical
3.1.3.1 Discussion—In a polydisperse molecular population
and Tissue-Engineered Medical Product Applications ¯ ¯ ¯ ¯
the relation M > M is always valid.The coefficient M /M is
w n w n
referred to as the polydispersity index, and will typically be in
the range 1.5 to 3.0 for commercial chitosans.
1
This test method is under the jurisdiction ofASTM Committee F04 on Medical
NOTE 1—The term molecular weight (abbreviated MW) is obsolete and
and Surgical Materials and Devices and is the direct responsibility of Subcommittee
should be replaced by the SI (Système Internationale) equivalent of either
F04.42 on Biomaterials and Biomolecules for TEMPs.
Current edition approved Aug. 1, 2013. Published September 2013. Originally
ε1
approved in 2008. Last previous edition approved in 2008 as F2602–08 . DOI:
3
10.1520/F2602-13. Available from United States Pharmacopeia and National Formulary, U.S.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or Pharmaceutical Convention, Inc. (USPC), Rockville, MD.
4
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Available from National Institute of Standards and Technology (NIST), 100
Standards volume information, refer to the standard’s Document Summary page on Bureau Dr., Stop 1070, Gaithersburg, MD 20899-1070, http://physics.nist.gov/cuu/
the ASTM website. Units/bibliography.html.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
F2602 − 13
relative molecular mass (M ), which reflects the dimensionless ratio of the
4.6 For polyelectrolytes, dialysis against the
...
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.
´1
Designation: F2602 − 08 F2602 − 13
Standard Test Method for
Determining the Molar Mass of Chitosan and Chitosan Salts
by Size Exclusion Chromatography with Multi-angle Light
1
Scattering Detection (SEC-MALS)
This standard is issued under the fixed designation F2602; 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
ε NOTE—Subsection 6.1.5 was editorially corrected in September 2008.
1. Scope
1.1 This test method covers the determination of the molar mass of chitosan and chitosan salts intended for use in biomedical
and pharmaceutical applications as well as in tissue engineered medical products (TEMPs) by size exclusion chromatography with
multi-angle laser light scattering detection (SEC-MALS). A guide for the characterization of chitosan salts has been published as
Guide F2103.
1.2 Chitosan and chitosan salts used in TEMPs should be well characterized, including the molar mass and polydispersity
(molar mass distribution) in order to ensure uniformity and correct functionality in the final product. This test method will assist
end users in choosing the correct chitosan for their particular application. Chitosan may have utility as a scaffold or matrix material
for TEMPs, in cell and tissue encapsulation applications, and in drug delivery formulations.
1.3 The values stated in SI units are to be regarded as 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 appropriate safety and health practices and determine the applicability of regulatory
limitations prior to use.
2. Referenced Documents
2
2.1 ASTM Standards:
F2103 Guide for Characterization and Testing of Chitosan Salts as Starting Materials Intended for Use in Biomedical and
Tissue-Engineered Medical Product Applications
3
2.2 United States Pharmacopeia/National Formulary:
<621> Chromatography
4
2.3 National Institute of Standards and Technology:
NIST SP811 Special Publication: Guide for the Use of the International System of Units (SI)
3. Terminology
3.1 Definitions:
3.1.1 chitosan, n—a linear polysaccharide consisting of β(1→4) linked 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) and
2-amino-2-deoxy-D-glucopyranose (GlcN). Chitosan is a polysaccharide derived by N-deacetylation of chitin.
3.1.2 degree of deacetylation, n—the fraction or percentage of glucosamine units (GlcN: deacetylated monomers) in a chitosan
polymer molecule.
1
This test method is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.42 on Biomaterials and Biomolecules for TEMPs.
Current edition approved Feb. 1, 2008Aug. 1, 2013. Published May 2008September 2013. Originally approved in 2008. Last previous edition approved in 2008 as
ε1
F2602–08 . DOI: 10.1520/F2602-08E01.10.1520/F2602-13.
2
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.
3
Available from United States Pharmacopeia and National Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville, MD.
4
Available from National Institute of Standards and Technology (NIST), 100 Bureau Dr., Stop 1070, Gaithersburg, MD 20899-1070, http://physics.nist.gov/cuu/Units/
bibliography.html.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
F2602 − 13
3.1.3 molar mass average, n—the given molar mass (M) of a chitosan will always represent an average of all of the molecules
in the population. The most common ways to express the molar mass are as the number average (M¯ ) and the mass average (M¯ ).
n w
The two averages are defined by the following equations:
2
N M w M N M
i i i i i i
(i (i (i
¯ ¯
M 5 and M 5 5 (1)
n w
N w N M
(i i (i i (i i i
where:
N = number of molecules having a specific molar mass M , and
i i
w = mass of molecules having a specific molar mass M .
i i
3.1.3.1 Discussion—
In a polydisperse molecular population the relation M¯ > M¯ is always
...
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