ASTM F2259-10
(Test Method)Standard Test Method for Determining the Chemical Composition and Sequence in Alginate by Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy
Standard Test Method for Determining the Chemical Composition and Sequence in Alginate by Proton Nuclear Magnetic Resonance (<sup>1</sup>H NMR) Spectroscopy
SIGNIFICANCE AND USE
The composition and sequential structure of alginate determines the functionality of alginate in an application. For instance, the gelling properties of an alginate are highly dependent upon the monomer composition and sequential structure of the polymer. Gel strength will depend upon the guluronic acid content (FG) and also the average number of consecutive guluronate moieties in G-block structures (NG>1).
Chemical composition and sequential structure of alginate can be determined by 1H- and 13C-nuclear magnetic resonance spectroscopy (NMR). A general description of NMR can be found in 761> of the USP24-NF19. The NMR methodology and assignments are based on data published by Grasdalen et al. (1979, 1981, 1983). , , The NMR technique has made it possible to determine the monad frequencies FM (fraction of mannuronate units) and FG (fraction of guluronate units), the four nearest neighboring (diad) frequencies FGG, FMG, FGM, FMM, and the eight next nearest neighboring (triad) frequencies FGGG, FGGM, FMGG, FMGM, FMMM, FMMG, FGMM, FGMG. Knowledge of these frequencies enables number averages of block lengths to be calculated. NG is the number average length of G-blocks, and NG>1 is the number average length of G-blocks from which singlets (-MGM-) have been excluded. Similarly, NM is the number average length of M-blocks, and NM>1 is the number average length of M-blocks from which singlets (-GMG-) have been excluded. 13C NMR must be used to determine the M-centered triads and NM>1. This test method describes only the 1H NMR analysis of alginate. Alginate can be well characterized by determining FG and NG>1.
In order to obtain well-resolved NMR spectra, it is necessary to reduce the viscosity and increase the mobility of the molecules by depolymerization of alginate to a degree of polymerization of about 20 to 50. Acid hydrolysis is used to depolymerize the alginate samples. Freeze-drying, followed by dissolution in 99 % D2O, and another freeze-drying before dissolut...
SCOPE
1.1 This test method covers the determination of the composition and monomer sequence of alginate intended for use in biomedical and pharmaceutical applications as well as in Tissue Engineered Medical Products (TEMPs) by high-resolution proton NMR (1H NMR). A guide for the characterization of alginate has been published as Guide F2064.
1.2 Alginate, a linear polymer composed of β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G) linked by β-(1>4) glycosidic bonds, is characterized by calculating parameters such as mannuronate/guluronate (M/G) ratio, guluronic acid content (G-content), and average length of blocks of consecutive G monomers (that is, NG>1). Knowledge of these parameters is important for an understanding of the functionality of alginate in TEMP formulations and applications. This test method will assist end users in choosing the correct alginate for their particular application. Alginate 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: F2259 − 10
StandardTest Method for
Determining the Chemical Composition and Sequence in
1
Alginate by Proton Nuclear Magnetic Resonance ( H NMR)
1
Spectroscopy
This standard is issued under the fixed designation F2259; 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 2. Referenced Documents
2
2.1 ASTM Standards:
1.1 This test method covers the determination of the com-
E386 Practice for Data Presentation Relating to High-
position and monomer sequence of alginate intended for use in
Resolution Nuclear Magnetic Resonance (NMR) Spec-
biomedical and pharmaceutical applications as well as in
troscopy
Tissue Engineered Medical Products (TEMPs) by high-
F2064 Guide for Characterization and Testing of Alginates
1
resolution proton NMR ( H NMR). A guide for the character-
as Starting Materials Intended for Use in Biomedical and
ization of alginate has been published as Guide F2064.
Tissue-Engineered Medical Products Application
1.2 Alginate, a linear polymer composed of β-D-
2.2 United States Pharmacopeia Document:
3
mannuronate(M)anditsC-5epimerα-L-guluronate(G)linked
USP 24-NF19 <761> Nuclear Magnetic Resonance
by β-(1—>4) glycosidic bonds, is characterized by calculating
parameters such as mannuronate/guluronate (M/G) ratio, gu-
3. Terminology
luronic acid content (G-content), and average length of blocks
3.1 Definitions:
of consecutive G monomers (that is, N ). Knowledge of
G>1
3.1.1 alginate, n—polysaccharide obtained from some of
these parameters is important for an understanding of the
the more common species of marine algae, consisting of an
functionality of alginate in TEMP formulations and applica-
insoluble mix of calcium, magnesium, sodium, and potassium
tions. This test method will assist end users in choosing the
salts.
correct alginate for their particular application. Alginate may
3.1.1.1 Discussion—Alginate exists in brown algae as its
have utility as a scaffold or matrix material for TEMPs, in cell
most abundant polysaccharide, mainly occurring in the cell
and tissue encapsulation applications, and in drug delivery
walls and intercellular spaces of brown seaweed and kelp.
formulations.
Alginate’s main function is to contribute to the strength and
flexibility of the seaweed plant. Alginate is classified as a
1.3 The values stated in SI units are to be regarded as
hydrocolloid. The most commonly used alginate is sodium
standard. No other units of measurement are included in this
alginate. Sodium alginate and, in particular, calcium cross-
standard.
linked alginate gels are used in Tissue Engineered Medical
1.4 This standard does not purport to address all of the
Products (TEMPs) as biomedical matrices, controlled drug
safety concerns, if any, associated with its use. It is the delivery systems, and for immobilizing living cells.
responsibility of the user of this standard to establish appro-
3.1.2 degradation, n—change in the chemical structure,
priate safety and health practices and determine the applica-
physical properties, or appearance of a material. Degradation
bility of regulatory limitations prior to use.
ofpolysaccharidesoccursviacleavageoftheglycosidicbonds.
It is important to note that degradation is not synonymous with
decomposition. Degradation is often used as a synonym for
depolymerization when referring to polymers.
1 2
This test method is under the jurisdiction ofASTM Committee F04 on Medical For referenced ASTM standards, visit the ASTM website, www.astm.org, or
and Surgical Materials and Devices and is the direct responsibility of Subcommittee contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
F04.42 on Biomaterials and Biomolecules for TEMPs. Standards volume information, refer to the standard’s Document Summary page on
CurrenteditionapprovedJune1,2010.PublishedJuly2010.Originallyapproved the ASTM website.
3
in 2003. Last previous edition approved in 2008 as F2259 – 03 (2008). DOI: Available from U.S. Pharmacopeia (USP), 12601Twinbrook Pkwy., Rockville,
10.1520/F2259-10. MD 20852-1790, http://www.usp.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
F2259 − 10
3.1.3 depolymerization, n—reduction in the length of a 5. Materials
polymer chain to form shorter polymeric units.
5.1 Chemicals:
5.1.1 Alginate sample.
4. Significance and Use
5.1.2 Deionized water (Milli-Q Plus or equivalent; conduc-
4.1 The composition and sequential structure of alginate
tivity <10 µS/cm).
determines the functionality of algi
...
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:F2259–03 (Reapproved 2008) Designation:F2259–10
Standard Test Method for
Determining the Chemical Composition and Sequence in
1
Alginate by Proton Nuclear Magnetic Resonance ( H NMR)
1
Spectroscopy
This standard is issued under the fixed designation F2259; 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 determination of the composition and monomer sequence of alginate intended for use in
biomedical and pharmaceutical applications as well as inTissue Engineered Medical Products (TEMPs) by high-resolution proton
1
NMR ( H NMR). A guide for the characterization of alginate has been published as Guide F2064.
1.2 Alginate, a linear polymer composed of b-D-mannuronate (M) and its C-5 epimer a-L-guluronate (G) linked by b-(1—>4)
glycosidic bonds, is characterized by calculating parameters such as mannuronate/guluronate (M/G) ratio, guluronic acid content
(G-content),andaveragelengthofblocksofconsecutiveGmonomers(thatis,N ).Knowledgeoftheseparametersisimportant
G>1
for an understanding of the functionality of alginate inTEMPformulations and applications.This test method will assist end users
in choosing the correct alginate for their particular application. Alginate 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:
E386 Practice for Data Presentation Relating to High-Resolution Nuclear Magnetic Resonance (NMR) Spectroscopy
F2064 Guide for Characterization and Testing of Alginates as Starting Materials Intended for Use in Biomedical and
Tissue-Engineered Medical Products Application
2.2 United States Pharmacopeia Document:
3
USP 24-NF19 <761> Nuclear Magnetic Resonance
3. Terminology
3.1 Definitions:
3.1.1 alginate, n—a polysaccharide substance extracted from brown algae, mainly occurring in the cell walls and intercellular
spaces of brown seaweed and kelp. Its main function is to contribute to the strength and flexibility of the seaweed plant. Sodium
alginate, and in particular calcium cross-linked alginate gels are used in Tissue Engineered Medical Products (TEMPs) as
biomedical matrices, controlled drug delivery systems, and for immobilizing living cells. —polysaccharide obtained from some
of the more common species of marine algae, consisting of an insoluble mix of calcium, magnesium, sodium, and potassium salts.
3.1.1.1 Discussion—Alginate exists in brown algae as its most abundant polysaccharide, mainly occurring in the cell walls and
intercellular spaces of brown seaweed and kelp. Alginate’s main function is to contribute to the strength and flexibility of the
seaweed plant.Alginate is classified as a hydrocolloid.The most commonly used alginate is sodium alginate. Sodium alginate and,
in particular, calcium cross-linked alginate gels are used in Tissue Engineered Medical Products (TEMPs) as biomedical matrices,
controlled drug delivery systems, and for immobilizing living cells.
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 May 1, 2008. Published June 2008. Originally approved in 2003. Last previous edition approved in 2003 as F2259–03. DOI:
10.1520/F2259-03R08.
Current edition approved June 1, 2010. Published July 2010. Originally approved in 2003. Last previous edition approved in 2008 as F2259 – 03 (2008). DOI:
10.1520/F2259-10.
2
For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM 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 U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville, MD 20
...
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