ASTM F2260-03(2008)
(Test Method)Standard Test Method for Determining Degree of Deacetylation in Chitosan Salts by Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy
Standard Test Method for Determining Degree of Deacetylation in Chitosan Salts by Proton Nuclear Magnetic Resonance (<sup>1</sup>H NMR) Spectroscopy
SIGNIFICANCE AND USE
The degree of deacetylation of chitosan salts is an important characterization parameter since the charge density of the chitosan molecule is responsible for potential biological and functional effects.
The degree of deacetylation (% DA) of water-soluble chitosan salts can be determined by 1H nuclear magnetic resonance spectroscopy (1H NMR). Several workers have reported on the NMR determination of chemical composition and sequential arrangement of monomer units in chitin and chitosan. The test method described is primarily based on the work of Vårum et al. (1991), which represents the first publication on routine determination of chemical composition in chitosans by solution state 1H NMR spectroscopy. This test method is applicable for determining the % DA of chitosan chloride and chitosan glutamate salts. It is a simple, rapid, and suitable method for routine use. Quantitative 1H NMR spectroscopy reports directly on the relative concentration of chemically distinct protons in the sample, consequently, no assumptions, calibration curves or calculations other than determination of relative signal intensity ratios are necessary.
In order to obtain well-resolved NMR spectra, depolymerization of chitosans to a number average degree of polymerization (DPn) of ~15 to 30 is required. This reduces the viscosity and increases the mobility of the molecules. Although there are several options for depolymerization of chitosans, the most convenient procedure is that of nitrous acid degradation in deuterated water. The reaction is selective, stoichiometric with respect to GlcN, rapid, and easily controlled (Allan & Peyron, 1995). The reaction selectively cleaves after a GlcN-residue, transforming it into 2,5-anhydro-D-mannose (chitose), consequently, depletion of GlcN after depolymerization is expected. On the other hand, the chitose unit displays characteristic 1H NMR signals the intensity of which may be estimated and utilized in the calculation of % DA, eliminating th...
SCOPE
1.1 This test method covers the determination of the degree of deacetylation in chitosan and chitosan salts 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 chitosan salts has been published as Guide F 2103.
1.2 The test method is applicable for determining the degree of deacetylation (% DA) of chitosan chloride and chitosan glutamate salts and is valid for % DA values from 50 up to and including 99. It is simple, rapid, and suitable for routine use. Knowledge of the degree of deacetylation is important for an understanding of the functionality of chitosan salts in TEMP formulations and applications. This test method will assist end users in choosing the correct chitosan for their particular application. Chitosan salts may have utility in drug delivery applications, as a scaffold or matrix material, and in cell and tissue encapsulation applications.
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: F2260 − 03(Reapproved 2008)
Standard Test Method for
Determining Degree of Deacetylation in Chitosan Salts by
Proton Nuclear Magnetic Resonance ( H NMR)
Spectroscopy
This standard is issued under the fixed designation F2260; 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 terials Having Flat Surfaces
F2103 Guide for Characterization and Testing of Chitosan
1.1 This test method covers the determination of the degree
Salts as Starting Materials Intended for Use in Biomedical
of deacetylation in chitosan and chitosan salts intended for use
and Tissue-Engineered Medical Product Applications
in biomedical and pharmaceutical applications as well as in
2.2 United States Pharmacopeia Document:
Tissue Engineered Medical Products (TEMPs) by high-
1 3
resolution proton NMR ( H NMR). A guide for the character- USP 24-NF19 <761> Nuclear Magnetic Resonance
ization of chitosan salts has been published as Guide F2103.
2.3 European Pharmacopoeia Document:
European Pharmacopoeia Monograph 2002:1774 Chitosan
1.2 Thetestmethodisapplicablefordeterminingthedegree
Chloride
of deacetylation (% DA) of chitosan chloride and chitosan
glutamate salts and is valid for % DAvalues from 50 up to and
3. Terminology
including 99. It is simple, rapid, and suitable for routine use.
Knowledge of the degree of deacetylation is important for an
3.1 Definitions:
understanding of the functionality of chitosan salts in TEMP
3.1.1 chitosan, n—a linear polysaccharide consisting of
formulations and applications. This test method will assist end
β(1→4) linked 2-acetamido-2-deoxy-D-glucopyranose (Glc-
users in choosing the correct chitosan for their particular
NAc) and 2-amino-2-deoxy-D-glucopyranose (GlcN). Chito-
application. Chitosan salts may have utility in drug delivery
san is a polysaccharide derived by N-deacetylation of chitin.
applications, as a scaffold or matrix material, and in cell and
3.1.2 degradation, n—change in the chemical structure,
tissue encapsulation applications.
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
depolymerization when referring to polymers.
1.4 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
3.1.3 degree of deacetylation, n—the fraction or percentage
responsibility of the user of this standard to establish appro-
of glucosamine units (GlcN: deacetylated monomers) in a
priate safety and health practices and determine the applica-
chitosan polymer molecule.
bility of regulatory limitations prior to use.
3.1.4 depolymerization, n—reduction in the length of a
polymer chain to form shorter polymeric units.
2. Referenced Documents
2.1 ASTM Standards:
4. Significance and Use
F386 Test Method for Thickness of Resilient Flooring Ma-
4.1 The degree of deacetylation of chitosan salts is an
important characterization parameter since the charge density
of the chitosan molecule is responsible for potential biological
This test method is under the jurisdiction ofASTM Committee F04 on Medical
and functional effects.
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 F2260 – 03. DOI:
10.1520/F2260-03R08. Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville,
For referenced ASTM standards, visit the ASTM website, www.astm.org, or MD 20852-1790, http://www.usp.org.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Available from European Directorate for the Quality of Medicines (EDQM),
Standards volume information, refer to the standard’s Document Summary page on Publications and Services, European Pharmacopoeia, BP 907, F-67029 Strasbourg,
the ASTM website. France.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2260 − 03 (2008)
4.2 The degree of deacetylation (% DA) of water-soluble 5.1.4 NaOD (sodium deuteroxide), 0.1 M and1MinD O.
chitosan salts can be determined by H nuclear magnetic 5.1.5 NaNO .
resonance spectroscopy ( H NMR). Several workers have 5.1.6 0.15 M TMSP (sodium 3-trimethylsilylpropionate-2,
reported on the NMR determination of chemical composition 2’,3,3’-d)inD O.
4 2
and sequential arrangement of monomer units in chitin and
5.2 Instruments:
chitosan. The test method described is primarily based on the
5.2.1 Analytical balance (0.1 mg).
work of Vårum et al. (1991), which represents the first
5.2.2 Laboratory shaking device.
publication on routine determination of chemical composition
5.2.3 pH meter or pH paper.
in chitosans by solution state H NMR spectroscopy. This test
5.2.4 5 mm NMR tubes.
method is applicable for determining the % DA of chitosan
5.2.5 NMR spectrometer (300 MHz field strength or higher
chloride and chitosan glutamate salts. It is a simple, rapid, and
is recommended although analysis at 100 MHz is possible),
suitable method for routine use. Quantitative H NMR spec-
with variable temperature option, capable of maintaining 90 6
troscopy reports directly on the relative concentration of
1°C sample temperature during analysis, Analog-digital con-
chemically distinct protons in the sample, consequently, no
version (ADC) with minimum 16 bit is recommended.
assumptions, calibration curves or calculations other than
determination of relative signal intensity ratios are necessary.
6. Procedure
4.3 In order to obtain well-resolved NMR spectra, depo-
6.1 Sample Preparation:
lymerization of chitosans to a number average degree of
6.1.1 Dissolve 33 mg chitosan chloride or 47 mg chitosan
polymerization(DP )of~15to30isrequired.Thisreducesthe
n glutamate in 3.3 mL D O by gentle shaking until completely
viscosityandincreasesthemobilityofthemolecules.Although
dissolved.
there are several options for depolymerization of chitosans, the
6.1.2 Add 250 µL of 1 M DCl and shake. Check that the
most convenient procedure is that of nitrous acid degradation
sample pH* is <2.
in deuterated water. The reaction is selective, stoichiometric
6.1.3 Add 100 µLfreshly made NaNO solution (10 mg/mL
with respect to GlcN, rapid, and easily controlled (Allan &
in D O).
Peyron, 1995). The reaction selectively cleaves after a GlcN-
6.1.4 Store the sample at room temperature in the dark for 4
residue, transforming it into 2,5-anhydro-D-mannose (chitose),
h.
consequently, depletion of GlcN after depolymerization is
6.1.5 Use 0.1 M or 1 M NaOD to adjust the sample to pH*
expected. On the other hand, the chitose unit displays charac-
3.8 to 4.2.
teristic H NMR signals the intensity of which may be
6.1.6 Transfer 0.7 mL of the sample solution to an NMR
estimated and utilized in the calculation of % DA, eliminating
tube.
the need for correction factors. Using the intensity of the
6.1.7 Add 5 µL of 0.15 M TMSP for chemical shift
chitose signals, the number average degree of polymerization
referencing.
can easily be calculated as a control of the depolymerization.
NOTE 1—For a sample in 100 % D O, the pH reading on a pH meter is
4.4 Samples are equilibrated and analyzed at a temperature
0.4 units lower than the true pD, due to an isotope effect on the glass
electrode. The meter reading in such solvents is normally reported
of 90 6 1°C. Elevated sample temperature contributes to
uncorrected and designated pH*.
reducing sample viscosity and repositions the proton signal of
residualwatertoanareaoutsidethatofinterest.Whilesamples 6.2 Technical Parameters—The most important parameters
are not stored at 90°C but only analyzed at this elevated used for quantitative H NMR analysis of the degree of
temperature, the NMR tubes should be sealed with a stopper to deacetylation in chitosan salts are as follows:
avoid any evaporation. At a sample pH* of 3.8-4.3 (see 6.1.5 6.2.1 Acquisition:
below), artifactual deacetylation of the sample does not occur 6.2.1.1 H NMR acquisition should be performed at 90°C
with sample spinnin
...
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