ASTM F2259-10(2012)e1

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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
´1
Designation: F2259 − 10 (Reapproved 2012)
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.
ε NOTE—Editorial changes were made to subsections 2.2 and 4.2 in November 2012.
1. Scope 2. Referenced Documents
1.1 This test method covers the determination of the com- 2.1 ASTM Standards:
position and monomer sequence of alginate intended for use in E386 Practice for Data Presentation Relating to High-
biomedical and pharmaceutical applications as well as in Resolution Nuclear Magnetic Resonance (NMR) Spec-
Tissue Engineered Medical Products (TEMPs) by high- troscopy
resolution proton NMR ( H NMR). A guide for the character- F2064 Guide for Characterization and Testing of Alginates
ization of alginate has been published as Guide F2064. as Starting Materials Intended for Use in Biomedical and
Tissue Engineered Medical Product Applications
1.2 Alginate, a linear polymer composed of β-D-
2.2 United States Pharmacopeia Document:
mannuronate(M)anditsC-5epimerα-L-guluronate(G)linked
USP 35-NF30 <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
1.3 The values stated in SI units are to be regarded as
flexibility of the seaweed plant. Alginate is classified as a
standard. No other units of measurement are included in this
hydrocolloid. The most commonly used alginate is sodium
standard.
alginate. Sodium alginate and, in particular, calcium cross-
1.4 This standard does not purport to address all of the linked alginate gels are used in Tissue Engineered Medical
safety concerns, if any, associated with its use. It is the
Products (TEMPs) as biomedical matrices, controlled drug
responsibility of the user of this standard to establish appro- delivery systems, and for immobilizing living cells.
priate safety and health practices and determine the applica-
3.1.2 degradation, n—change in the chemical structure,
bility of regulatory limitations prior to use.
physical properties, or appearance of a material. Degradation
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
Current edition approved Oct. 1, 2012. Published November 2012. Originally the ASTM website.
approved in 2003. Last previous edition approved in 2010 as F2259 – 10. DOI: Available from U.S. Pharmacopeia (USP), 12601Twinbrook Pkwy., Rockville,
10.1520/F2259-10R12E01. 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
F2259 − 10 (2012)
ofpolysaccharidesoccursviacleavageoftheglycosidicbonds. 4.4 Samples are analyzed at a temperature of 80 6 1°C.
It is important to note that degradation is not synonymous with Elevated sample temperature contributes to reducing sample
decomposition. Degradation is often used as a synonym for
viscosity and repositions the proton signal of residual water to
depolymerization when referring to polymers.
an area outside that of interest.
3.1.3 depolymerization, n—reduction in the length of a
5. Materials
polymer chain to form shorter polymeric units.
5.1 Chemicals:
4. Significance and Use
5.1.1 Alginate sample.
4.1 The composition and sequential structure of alginate
5.1.2 Deionized water (Milli-Q Plus or equivalent; conduc-
determines the functionality of alginate in an application. For
tivity <10 µS/cm).
instance, the gelling properties of an alginate are highly
5.1.3 HCl (1M, 0.1 M).
dependent upon the monomer composition and sequential
structure of the polymer. Gel strength will depend upon the
5.1.4 NaOH (1M, 0.1 M).
guluronic acid content (F ) and also the average number of
G
5.1.5 D O (99-99.9 %, 99.9 %).
consecutive guluronate moieties in G-block structures (N ).
G>1
5.1.6 TTHA(triethylenetetraminehexaacetic acid) (0.3 M in
4.2 Chemical composition and sequential structure of alg-
D O, adjust pH* to 5-5.5 using DCl or NaOD).
1 13
inate can be determined by H- and C-nuclear magnetic
NOTE 1—For a sample in 100 % D O, the pH reading on a pH meter is
resonancespectroscopy(NMR).AgeneraldescriptionofNMR
0.4 units lower than the true pD, due to an isotope effect on the glass
can be found in <761> of the USP 35-NF30. The NMR
electrode. The meter reading in such solvents is normally reported
methodology and assignments are based on data published by
uncorrected and designated pH*.
4,5,6
Grasdalen et al. (1979, 1981, 1983). The NMR technique
5.2 Instruments:
has made it possible to determine the monad frequencies F
M
(fraction of mannuronate units) and F (fraction of guluronate
5.2.1 Analytical balance (0.1 mg).
G
units), the four nearest neighboring (diad) frequencies F ,
GG
5.2.2 Laboratory shaking device.
F ,F ,F , and the eight next nearest neighboring (triad)
MG GM MM
5.2.3 pH meter.
frequencies F ,F ,F ,F ,F ,F ,F ,
GGG GGM MGG MGM MMM MMG GMM
5.2.4 Water bath (100°C).
F . Knowledge of these frequencies enables number aver-
GMG
5.2.5 Freeze dryer.
ages of block lengths to be calculated. N is the number
G
average length of G-blocks, and N is the number average 5.2.6 NMR spectrometer (300 MHz field strength or higher
G>1
length of G-blocks from which singlets (-MGM-) have been
is recommended), capable of maintaining 80 6 1°C sample
excluded. Similarly, N is the number average length of
temperature during analysis.
M
M-blocks, and N is the number average length of M-blocks
M>1
from which singlets (-GMG-) have been excluded. C NMR
6. Procedure
must be used to determine the M-centered triads and N .
M>1
6.1 Sample Preparation:
This test method describes only the H NMR analysis of
6.1.1 Prepare 100 mL of a 0.1 % (w/v) alginate solution.
alginate.Alginate can be well characterized by determining F
G
and N . 6.1.2 Adjust the pH with HCl (1 M, 0.1 M) to pH 5.6, and
G>1
put the alginate sample in a water bath at 100°C for 1 h.
4.3 In order to obtain well-resolved NMR spectra, it is
necessary to reduce the viscosity and increase the mobility of 6.1.3 Adjust the pH with HCl (1 M, 0.1 M) to pH 3.8, and
put the alginate sample back to the water bath at 100°C for 30
the molecules by depolymerization of alginate to a degree of
polymerization of about 20 to 50. Acid hydrolysis is used to min.
depolymerize the alginate samples. Freeze-drying, followed by
6.1.4 AdjustthepHwithNaOH(1M,0.1M)topH7-8,and
dissolution in 99 % D O, and another freeze-drying before
freeze-dry the sample overnight.
dissolution in 99.9 % D O yields samples with low H O
2 2
6.1.5 Dissolve the alginate sample in 5 ml 99-99.9 % D O,
content. TTHA is used as a chelator to prevent traces of
and
...