ASTM F2259-03

Standard Test Method for Determining the Chemical Composition and Sequence in Alginate by Proton Nuclear Magnetic Resonance (¹H NMR) Spectroscopy

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 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

• Status: Historical
• Publication Date: 09-Apr-2003
• ICS: 11.120.20 - Wound dressings and compresses
• ICS: 71.040.50 - Physicochemical methods of analysis
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.42 - Biomaterials and Biomolecules for TEMPs

Relations

• Referred By: ASTM F2064-17 - Standard Guide for Characterization and Testing of Alginates as Starting Materials Intended for Use in Biomedical and Tissue Engineered Medical Product Applications
• Effective Date: 10-Apr-2003
• Referred By: ASTM F2027-16 - Standard Guide for Characterization and Testing of Raw or Starting Materials for Tissue-Engineered Medical Products
• Effective Date: 10-Apr-2003

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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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
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 F 2259; 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.
1. Scope USP 24-NF19 <761> Nuclear Magnetic Resonance
1.1 This test method covers the determination of the com-
3. Terminology
position and monomer sequence of alginate intended for use in
3.1 Definitions:
biomedical and pharmaceutical applications as well as in
3.1.1 alginate, n—a polysaccharide substance extracted
Tissue Engineered Medical Products (TEMPs) by high-
from brown algae, mainly occurring in the cell walls and
resolution proton NMR ( H NMR). A guide for the character-
intercellular spaces of brown seaweed and kelp. Its main
ization of alginate has been published as Guide F 2064.
function is to contribute to the strength and flexibility of the
1.2 Alginate, a linear polymer composed of b-D-
seaweed plant. Sodium alginate, and in particular calcium
mannuronate (M) and its C-5 epimer a-L-guluronate (G)
cross-linked alginate gels are used in Tissue Engineered
linked by b-(1—>4) glycosidic bonds, is characterized by
Medical Products (TEMPs) as biomedical matrices, controlled
calculating parameters such as mannuronate/guluronate (M/G)
drug delivery systems, and for immobilizing living cells.
ratio,guluronicacidcontent(G-content),andaveragelengthof
3.1.2 degradation, n—change in the chemical structure,
blocks of consecutive G monomers (that is, N ). Knowledge
G>1
physical properties, or appearance of a material. Degradation
of these parameters is important for an understanding of the
ofpolysaccharidesoccursviacleavageoftheglycosidicbonds.
functionality of alginate in TEMP formulations and applica-
It is important to note that degradation is not synonymous with
tions. This test method will assist end users in choosing the
decomposition. Degradation is often used as a synonym for
correct alginate for their particular application. Alginate may
depolymerization when referring to polymers.
have utility as a scaffold or matrix material for TEMPs, in cell
3.1.3 depolymerization, n—reduction in the length of a
and tissue encapsulation applications, and in drug delivery
polymer chain to form shorter polymeric units.
formulations.
1.3 This standard does not purport to address all of the
4. Significance and Use
safety concerns, if any, associated with its use. It is the
4.1 The composition and sequential structure of alginate
responsibility of the user of this standard to establish appro-
determines the functionality of alginate in an application. For
priate safety and health practices and determine the applica-
instance, the gelling properties of an alginate are highly
bility of regulatory limitations prior to use.
dependent upon the monomer composition and sequential
structure of the polymer. Gel strength will depend upon the
2. Referenced Documents
guluronic acid content (F ) and also the average number of
G
2.1 ASTM Standards:
consecutive guluronate moieties in G-block structures (N ).
G>1
E 386 Practice for Data Presentation Relating to High-
4.2 Chemical composition and sequential structure of algi-
Resolution Nuclear Magnetic Resonance (NMR) Spectros-
1 13
2 nate can be determined by H- and C-nuclear magnetic reso-
copy
nance spectroscopy (NMR).Ageneral description of NMR can
F 2064 Guide for Characterization and Testing ofAlginates
be found in <761> of the USP24-NF19. The NMR methodol-
as Starting Materials Intended for Use in Biomedical and
ogy and assignments are based on data published by Grasdalen
Tissue-Engineered Medical Products Application
5,6,7
et al. (1979, 1981, 1983). The NMR technique has made it
2.2 United States Pharmacopeia Document:
Available from U.S. Pharmacopeia (USP), 12601Twinbrook Pkwy., Rockville,
This test method is under the jurisdiction ofASTM Committee F04 on Medical MD 20852.
and Surgical Materials and Devices and is the direct responsibility of Subcommittee Grasdalen, H., Larsen, B., and Smidsrød, O., “A P.M.R. Study of the
F04.42 on Biomaterials and Biomolecules for TEMPs. CompositionandSequenceofUronateResiduesinAlginates.,” Carbohydr. Res.,68,
Current edition approved Apr. 10, 2003. Published May 2003. 1979, pp. 23-31.
2 6 13
Annual Book of ASTM Standards, Vol 03.06. Grasdalen,H.,Larsen,B.,andSmidsrød,O.,“ C-NMRStudiesofMonomeric
Annual Book of ASTM Standards, Vol 13.01. Composition and Sequence in Alginate,” Carbohydr. Res., 89, 1981, pp. 179-191.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F2259–03
possible to determine the monad frequencies F (fraction of 6. Procedure
M
mannuronate units) and F (fraction of guluronate units), the
G
6.1 Sample Preparation:
four nearest neighboring (diad) frequencies F ,F ,F ,
GG MG GM
6.1.1 Prepare 100 mL of a 0.1 % (w/v) alginate solution.
F ,andtheeightnextnearestneighboring(triad)frequencies
MM
6.1.2 Adjust the pH with HCl (1 M, 0.1 M) to pH 5.6, and
F ,F ,F ,F ,F ,F ,F ,F .
GGG GGM MGG MGM MMM MMG GMM GMG
put the alginate sample in a water bath at 100°C for 1 h.
Knowledge of these frequencies enables number averages of
6.1.3 Adjust the pH with HCl (1 M, 0.1 M) to pH 3.8, and
blocklengthstobecalculated.N isthenumberaveragelength
G
put the alginate sample back to the water bath at 100°C for 30
of G-blocks, and N is the number average length of
G>1
min.
G-blocks from which singlets (-MGM-) have been excluded.
6.1.4 AdjustthepHwithNaOH(1M,0.1M)topH7-8,and
Similarly, N is the number average length of M-blocks, and
M
freeze-dry the sample overnight.
N is the number average length of M-blocks from which
M>1
6.1.5 Dissolve the alginate sample in 5 ml 99-99.9 % D O,
13 2
singlets (-GMG-) have been excluded. C NMR must be used
and freeze dry it again.
to determine the M-centered triads and N . This test method
M>1
6.1.6 Dissolve 10 to 12 mg of the sample in 1 mL 99.9 %
describes only the H NMR analysis of alginate. Alginate can
D O.
be well characterized by determining F and N .
G G>1
6.1.7 Add 0.7 mL of the alginate solution to a NMR tube,
4.3 In order to obtain well-resolved NMR spectra, it is
and then add 20 µL 0.3 M TTHA to the same tube.
necessary to reduce the viscosity and increase the mobility of
6.2 Technical Parameters—The most important parameters
the molecules by depolymerization of alginate to a degree of
used for quantitative H NMR analysis of alginate are as
polymerization of about 20 to 50. Acid hydrolysis is used to
follows:
depolymerize the alginate samples. Freeze-drying, followed by
6.2.1 Acquisition:
dissolution in 99 % D O, and another freeze-drying before
2 1
6.2.1.1 H NMR acquisition should be performed at 80°C
dissolution in 99.9 % D O yields samples with low H O
2 2
with sample spinning at 20 Hz using a standard one-
content. TTHA is used as a chelator to prevent traces of
dimensional pulse program.
divalent cations to interact with alginate. While TTHA is a
Nucleus H
more effective chelator, other agents such as EDTAand citrate
Proton spectral width −0.5→9.5 ppm
may be used. Such interactions may lead to line broadening
Number of scans 64
Relaxation delay 2 s
and selective loss of signal intensity.
Proton pulse angle 90°
4.4 Samples are analyzed at a temperature of 80 6 1°C.
Acquisition time 4.096 s
Elevated sample temperature contributes to reducing sample
Number of data points determined by spectral width (in Hz)
sampled and acquisition time; 32768 at
viscosity and r
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