ASTM F2259-03(2008)
(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 F 2064.
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.
General Information
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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information.
Designation:F2259–03 (Reapproved 2008)
Standard Test Method for
Determining the Chemical Composition and Sequence in
Alginate by Proton Nuclear Magnetic Resonance ( H NMR)
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 E386 Practice for Data Presentation Relating to High-
Resolution Nuclear Magnetic Resonance (NMR) Spectros-
1.1 This test method covers the determination of the com-
copy
position and monomer sequence of alginate intended for use in
F2064 Guide for Characterization and Testing of Alginates
biomedical and pharmaceutical applications as well as in
as Starting Materials Intended for Use in Biomedical and
Tissue Engineered Medical Products (TEMPs) by high-
Tissue-Engineered Medical Products Application
resolution proton NMR ( H NMR). A guide for the character-
2.2 United States Pharmacopeia Document:
ization of alginate has been published as Guide F2064.
USP 24-NF19 <761> Nuclear Magnetic Resonance
1.2 Alginate, a linear polymer composed of b-D-
mannuronate (M) and its C-5 epimer a-L-guluronate (G)
3. Terminology
linked by b-(1—>4) glycosidic bonds, is characterized by
3.1 Definitions:
calculating parameters such as mannuronate/guluronate (M/G)
3.1.1 alginate, n—a polysaccharide substance extracted
ratio,guluronicacidcontent(G-content),andaveragelengthof
from brown algae, mainly occurring in the cell walls and
blocks of consecutive G monomers (that is, N ). Knowledge
G>1
intercellular spaces of brown seaweed and kelp. Its main
of these parameters is important for an understanding of the
function is to contribute to the strength and flexibility of the
functionality of alginate in TEMP formulations and applica-
seaweed plant. Sodium alginate, and in particular calcium
tions. This test method will assist end users in choosing the
cross-linked alginate gels are used in Tissue Engineered
correct alginate for their particular application. Alginate may
Medical Products (TEMPs) as biomedical matrices, controlled
have utility as a scaffold or matrix material for TEMPs, in cell
drug delivery systems, and for immobilizing living cells.
and tissue encapsulation applications, and in drug delivery
3.1.2 degradation, n—change in the chemical structure,
formulations.
physical properties, or appearance of a material. Degradation
1.3 The values stated in SI units are to be regarded as
ofpolysaccharidesoccursviacleavageoftheglycosidicbonds.
standard. No other units of measurement are included in this
It is important to note that degradation is not synonymous with
standard.
decomposition. Degradation is often used as a synonym for
1.4 This standard does not purport to address all of the
depolymerization when referring to polymers.
safety concerns, if any, associated with its use. It is the
3.1.3 depolymerization, n—reduction in the length of a
responsibility of the user of this standard to establish appro-
polymer chain to form shorter polymeric units.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
4. Significance and Use
2. Referenced Documents 4.1 The composition and sequential structure of alginate
2 determines the functionality of alginate in an application. For
2.1 ASTM Standards:
instance, the gelling properties of an alginate are highly
dependent upon the monomer composition and sequential
This test method is under the jurisdiction ofASTM Committee F04 on Medical
structure of the polymer. Gel strength will depend upon the
and Surgical Materials and Devices and is the direct responsibility of Subcommittee
guluronic acid content (F ) and also the average number of
G
F04.42 on Biomaterials and Biomolecules for TEMPs.
consecutive guluronate moieties in G-block structures (N ).
Current edition approved May 1, 2008. Published June 2008. Originally G>1
approved in 2003. Last previous edition approved in 2003 as F2259 – 03. DOI:
10.1520/F2259-03R08.
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 Available from U.S. Pharmacopeia (USP), 12601Twinbrook Pkwy., Rockville,
the ASTM website. MD 20852-1790, http://www.usp.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F2259–03 (2008)
NOTE 1—For a sample in 100 % D O, the pH reading on a pH meter is
4.2 Chemical composition and sequential structure of algi-
1 13
0.4 units lower than the true pD, due to an isotope effect on the glass
nate can be determined by H- and C-nuclear magnetic reso-
electrode. The meter reading in such solvents is normally reported
nance spectroscopy (NMR).Ageneral description of NMR can
uncorrected and designated pH*.
be found in <761> of the USP 24-NF19. The NMR method-
ology and assignments are based on data published by Gras- 5.2 Instruments:
, ,
4 5 6
dalen et al. (1979, 1981, 1983). The NMR technique has 5.2.1 Analytical balance (0.1 mg).
made it possible to determine the monad frequencies F (frac- 5.2.2 Laboratory shaking device.
M
tion of mannuronate units) and F (fraction of guluronate 5.2.3 pH meter.
G
units), the four nearest neighboring (diad) frequencies F , 5.2.4 Water bath (100°C).
GG
F ,F ,F , and the eight next nearest neighboring (triad) 5.2.5 Freeze dryer.
MG GM MM
frequencies F ,F ,F ,F ,F ,F ,F , 5.2.6 NMR spectrometer (300 MHz field strength or higher
GGG GGM MGG MGM MMM MMG GMM
F . Knowledge of these frequencies enables number aver- is recommended), capable of maintaining 80 6 1°C sample
GMG
ages of block lengths to be calculated. N is the number temperature during analysis.
G
average length of G-blocks, and N is the number average
G>1
length of G-blocks from which singlets (-MGM-) have been 6. Procedure
excluded. Similarly, N is the number average length of
M
6.1 Sample Preparation:
M-blocks, and N is the number average length of M-blocks
M>1
6.1.1 Prepare 100 mL of a 0.1 % (w/v) alginate solution.
from which singlets (-GMG-) have been excluded. C NMR
6.1.2 Adjust the pH with HCl (1 M, 0.1 M) to pH 5.6, and
must be used to determine the M-centered triads and N .
M>1
put the alginate sample in a water bath at 100°C for 1 h.
This test method describes only the H NMR analysis of
6.1.3 Adjust the pH with HCl (1 M, 0.1 M) to pH 3.8, and
alginate.Alginate can be well characterized by determining F
G
put the alginate sample back to the water bath at 100°C for 30
and N .
G>1
min.
4.3 In order to obtain well-resolved NMR spectra, it is
6.1.4 AdjustthepHwithNaOH(1M,0.1M)topH7-8,and
necessary to reduce the viscosity and increase the mobility of
freeze-dry the sample overnight.
the molecules by depolymerization of alginate to a degree of
6.1.5 Dissolve the alginate sample in 5 ml 99-99.9 % D O,
polymerization of about 20 to 50. Acid hydrolysis is used to
and freeze dry it again.
depolymerize the alginate samples. Freeze-drying, followed by
6.1.6 Dissolve 10 to 12 mg of the sample in 1 mL 99.9 %
dissolution in 99 % D O, and another freeze-drying before
D O.
dissolution in 99.9 % D O yields samples with low H O
2 2
6.1.7 Add 0.7 mL of the alginate solution to a NMR tube,
content. TTHA is used as a chelator to prevent traces of
and then add 20 µL 0.3 M TTHA to the same tube.
divalent cations to interact with alginate. While TTHA is a
6.2 Technical Parameters—The most important parameters
more effective chelator, other agents such as EDTAand citrate 1
used for quantitative H NMR analysis of alginate are as
may be used. Such interactions may lead to line broadening
follows:
and selective loss of signal intensity.
6.2.1 Acquisition:
4.4 Samples are analyzed at a temperature of 80 6 1°C.
6.2.
...
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