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

SIGNIFICANCE AND USE
4.1 This guide contains a listing of those characterization parameters that are directly related to the functionality of alginate. This guide can be used as an aid in the selection and characterization of the appropriate alginate for a particular application. This guide is intended to give guidance in the methods and types of testing necessary to properly characterize, assess, and ensure consistency in the performance of a particular alginate. It may have use in the regulation of these devices by appropriate authorities.  
4.2 The alginate covered by this guide may be gelled, extruded, or otherwise formulated into biomedical devices for use in tissue-engineered medical products or drug delivery devices for implantation as determined to be appropriate, based on supporting biocompatibility and physical test data. Recommendations in this guide should not be interpreted as a guarantee of clinical success in any tissue engineered medical product or drug delivery application. Further guidance for immobilizing or encapsulating living cells or tissue in alginate gels can be found in Guide F2315.  
4.3 To ensure that the material supplied satisfies requirements for use in TEMPS, several general areas of characterization should be considered. These are: identity of alginate, physical and chemical characterization and testing, impurities profile, and performance-related tests.
SCOPE
1.1 This guide covers the evaluation of alginates suitable for use in biomedical or pharmaceutical applications, or both, including, but not limited to, Tissue Engineered Medical Products (TEMPs).  
1.2 This guide addresses key parameters relevant for the functionality, characterization, and purity of alginates.  
1.3 As with any material, some characteristics of alginates may be altered by processing techniques (such as molding, extrusion, machining, assembly, sterilization, and so forth) required for the production of a specific part or device. Therefore, properties of fabricated forms of this polymer should be evaluated using test methods that are appropriate to ensure safety and efficacy and are not addressed in this guide.  
1.4 Warning—Mercury has been designated by EPA and many state agencies as a hazardous material that can cause central nervous system, kidney, and liver damage. Mercury, or its vapor, may be hazardous to health and corrosive to materials. Caution should be taken when handling mercury and mercury-containing products. See the applicable product Material Safety Data Sheet (MSDS) for details and EPA’s website (http://www.epa.gov/mercury/faq.htm) for additional information. Users should be aware that selling mercury or mercury-containing products, or both, in your state may be prohibited by state law.  
1.5 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.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 28-Feb-2017
• ICS: 11.040.40 - Implants for surgery, prosthetics and orthotics
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.42 - Biomaterials and Biomolecules for TEMPs

Relations

• Replaces: ASTM F2064-14 - Standard Guide for Characterization and Testing of Alginates as Starting Materials Intended for Use in Biomedical and Tissue Engineered Medical Product Applications
• Effective Date: 01-Mar-2017
• Refers: ASTM F749-20 - Standard Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit
• Effective Date: 01-Feb-2020
• Refers: ASTM F1903-18 - Standard Practice for Testing for Cellular Responses to Particles <emph type="bdit" >in vitro</emph>
• Effective Date: 01-Oct-2018
• Refers: ASTM F1439-03(2018) - Standard Guide for Performance of Lifetime Bioassay for the Tumorigenic Potential of Implant Materials
• Effective Date: 01-Feb-2018
• Refers: ASTM F756-17 - Standard Practice for Assessment of Hemolytic Properties of Materials
• Effective Date: 01-Mar-2017
• Refers: ASTM E2975-16 - Standard Test Method for Calibration or Calibration Verification of Concentric Cylinder Rotational Viscometers
• Effective Date: 01-Dec-2016
• Refers: ASTM F2605-16 - Standard Test Method for Determining the Molar Mass of Sodium Alginate by Size Exclusion Chromatography with Multi-angle Light Scattering Detection (SEC-MALS)
• Effective Date: 01-May-2016
• Refers: ASTM F748-16 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Apr-2016
• Refers: ASTM F895-11(2016) - Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• Effective Date: 01-Apr-2016
• Refers: ASTM E2975-15 - Standard Test Method for Calibration of Concentric Cylinder Rotational Viscometers
• Effective Date: 01-Sep-2015
• Refers: ASTM E2975-14 - Standard Test Method for Calibration of Concentric Cylinder Rotational Viscometers
• Effective Date: 01-Aug-2014
• Refers: ASTM F756-13 - Standard Practice for Assessment of Hemolytic Properties of Materials
• Effective Date: 01-Dec-2013
• Refers: ASTM F1439-03(2013) - Standard Guide for Performance of Lifetime Bioassay for the Tumorigenic Potential of Implant Materials
• Effective Date: 01-Oct-2013
• Refers: ASTM F749-13 - Standard Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit
• Effective Date: 01-Mar-2013
• Refers: ASTM F813-07(2012) - Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices
• Effective Date: 01-Oct-2012

Overview

ASTM F2064-17 is a comprehensive standard guide published by ASTM International for the characterization and testing of alginates as starting materials intended for use in biomedical and tissue-engineered medical products (TEMPs). Alginate, a naturally derived polysaccharide predominantly sourced from brown algae, features unique properties making it valuable for biomedical applications, especially as a component in hydrogels, drug delivery systems, and tissue scaffolds.

This standard provides a framework for identifying critical characterization parameters for alginates, helping manufacturers and researchers select, assess, and ensure consistent performance for biomedical and pharmaceutical use. By following ASTM F2064-17, stakeholders can better evaluate alginate quality, functionality, impurity profile, and suitability for use in advanced medical devices and tissue-engineered constructs.

Key Topics

• Identification of Alginate: Establishes accepted methods for confirming the identity of alginate through chemical and spectral analyses, such as FT-IR and NMR spectroscopy.
• Physical and Chemical Characterization: Outlines procedures for evaluating the molecular composition, molar mass, sequential structure, and viscosity, which are critical to the functionality of alginate in biomedical settings.
• Impurity Profile Assessment: Provides guidance on testing for common impurities such as endotoxins, proteins, heavy metals, and microbiological contaminants, ensuring alginate meets safety and quality requirements.
• Performance-related Tests: Recommends appropriate tests to correlate alginate properties with intended biomedical applications.
• Processing and Stability Considerations: Explains how factors like sterilization, storage, and processing methods may influence alginate properties and thus must be appropriately evaluated.
• Safety and Regulatory Compliance: Highlights the importance of biocompatibility testing and adherence to regulatory guidance, including documentation to support safety and efficacy.

Applications

ASTM F2064-17 is designed to support the use of alginate in a variety of biomedical and pharmaceutical applications, including:

• Tissue Engineered Medical Products (TEMPs): Alginate-based scaffolds provide a supportive matrix for cell immobilization and tissue regeneration.
• Drug Delivery Devices: Alginate’s ability to gel and encapsulate therapeutic agents facilitates controlled and sustained drug release.
• Implantable Biomedical Devices: Alginates can be formulated for implantation, subject to biocompatibility and performance verification.
• Wound Dressings: Due to its moisture-retentive properties and biocompatibility, alginate is used in wound management.
• Cell Encapsulation/Immobilization: As referenced in ASTM F2315, alginate gels are used to encapsulate living cells or tissues for therapeutic applications.

Following ASTM F2064-17 ensures a consistent approach to alginate testing and quality control, streamlining regulatory submissions, supporting device documentation, and enhancing the predictability of clinical performance.

Related Standards

Utilization of ASTM F2064-17 can be complemented by reference to several other key standards and guidance documents, including:

• ASTM F2315: Guide for immobilization or encapsulation of living cells or tissue in alginate gels
• ISO 10993: Biological evaluation of medical devices (Parts relevant to biocompatibility and toxicological assessment)
• USP Monograph USP 35/NF 30: Pertaining to sodium alginate identification and quality
• ANSI/AAMI/ISO 11737: For sterilization and microbiological methods in medical products
• FDA and ICH Guidelines: For heavy metal testing, endotoxin limits, and stability assessment

By integrating ASTM F2064-17 with these related standards, organizations can create robust, compliant, and effective strategies for alginate characterization in biomedical R&D and manufacturing.

Keywords

alginates, biomedical material characterization, tissue engineered medical products, TEMPs, alginate testing, biomedical alginate, medical device standards, alginate quality control, pharmaceutical alginate, hydrocolloids, biocompatibility, medical scaffolds, drug delivery systems, ASTM F2064-17

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: F2064 − 17
Standard Guide for
Characterization and Testing of Alginates as Starting
Materials Intended for Use in Biomedical and Tissue
Engineered Medical Product Applications
This standard is issued under the fixed designation F2064; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Alginatehasfoundusesinavarietyofproductsrangingfromsimpletechnicalapplicationssuchas
viscosifiers to advanced biomedical matrices providing controlled drug delivery from immobilized
living cells. As for most hydrocolloids, the functionality of alginate is related to its chemical and
structural composition. The aim of this guide is to identify key parameters relevant for the
functionalityandcharacterizationofalginatesforthedevelopmentofnewcommercialapplicationsof
alginates for the biomedical and pharmaceutical industries.
1. Scope 1.5 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
1.1 Thisguidecoverstheevaluationofalginatessuitablefor
responsibility of the user of this standard to establish appro-
use in biomedical or pharmaceutical applications, or both,
priate safety and health practices and determine the applica-
including, but not limited to, Tissue Engineered Medical
bility of regulatory limitations prior to use.
Products (TEMPs).
1.6 This international standard was developed in accor-
1.2 This guide addresses key parameters relevant for the
dance with internationally recognized principles on standard-
functionality, characterization, and purity of alginates.
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
1.3 As with any material, some characteristics of alginates
may be altered by processing techniques (such as molding, mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
extrusion, machining, assembly, sterilization, and so forth)
required for the production of a specific part or device.
2. Referenced Documents
Therefore, properties of fabricated forms of this polymer
should be evaluated using test methods that are appropriate to
2.1 ASTM Standards:
ensure safety and efficacy and are not addressed in this guide.
E2975Test Method for Calibration or Calibration Verifica-
tion of Concentric Cylinder Rotational Viscometers
1.4 Warning—Mercury has been designated by EPA and
F619Practice for Extraction of Medical Plastics
many state agencies as a hazardous material that can cause
F748PracticeforSelectingGenericBiologicalTestMethods
central nervous system, kidney, and liver damage. Mercury, or
for Materials and Devices
its vapor, may be hazardous to health and corrosive to
F749Practice for Evaluating Material Extracts by Intracuta-
materials.Cautionshouldbetakenwhenhandlingmercuryand
neous Injection in the Rabbit
mercury-containing products. See the applicable product Ma-
F756Practice for Assessment of Hemolytic Properties of
terial Safety Data Sheet (MSDS) for details and EPA’s website
Materials
(http://www.epa.gov/mercury/faq.htm) for additional informa-
F763Practice for Short-Term Screening of Implant Materi-
tion. Users should be aware that selling mercury or mercury-
als
containingproducts,orboth,inyourstatemaybeprohibitedby
F813Practice for Direct Contact Cell Culture Evaluation of
state law.
Materials for Medical Devices
This guide is under the jurisdiction ofASTM Committee F04 on Medical and
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.42 on Biomaterials and Biomolecules for TEMPs. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved March 1, 2017. Published April 2017. Originally contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
approved in 2000. Last previous edition approved in 2014 as F2064–14. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/F2064-17. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2064 − 17
F895TestMethodforAgarDiffusionCellCultureScreening 2.5 FDA Documents:
for Cytotoxicity FDA Interim Guidance for Human and Veterinary Drug
F981Practice for Assessment of Compatibility of Biomate- Products and Biologicals. Kinetic LAL techniques.
rials for Surgical Implants with Respect to Effect of DHHS, July 15, 1991
Materials on Muscle and Insertion into Bone
2.6 ANSI Documents:
F1251Terminology Relating to Polymeric Biomaterials in
ANSI/AAMI/ISO 11737-1: 2006Sterilization of Medical
Medical and Surgical Devices (Withdrawn 2012)
Devices—Microbiological Methods—Part 1: Estimation
F1439Guide for Performance of Lifetime Bioassay for the
of Bioburden on Product.
Tumorigenic Potential of Implant Materials
ANSI/AAMI/ISO 11737-2: 1998Sterilization of Medical
F1903Practice for Testing For Biological Responses to
Devices—MicrobiologicalMethods—Part2:TestsofSte-
Particles In Vitro
rilityPerformedintheValidationofaSterilizationProcess
F1904Practice for Testing the Biological Responses to
2.7 AAMI Documents:
Particles in vivo
AAMI/ISO 14160—1998Sterilization of Single-Use Medi-
F1905Practice For Selecting Tests for Determining the
cal Devices Incorporating Materials of Animal Origin—
Propensity of Materials to Cause Immunotoxicity (With-
Validation and Routine Control of Sterilization by Liquid
drawn 2011)
Chemical Sterilants
F1906Practice for Evaluation of Immune Responses In
AAMI ST67: 2011Sterilization of Health Care Products—
BiocompatibilityTestingUsingELISATests,Lymphocyte
Requirements and Guidance for Selecting a Sterility
Proliferation, and Cell Migration (Withdrawn 2011)
Assurance Level (SAL) for Products Labeled “Sterile”
F2259Test Method for Determining the Chemical Compo-
AAMI TIR No. 19—1998Guidance for ANSI/AAMI/ISO
sition and Sequence in Alginate by Proton Nuclear Mag-
10993-7: 1995, Biological Evaluation of Medical
netic Resonance ( H NMR) Spectroscopy
Devices—Part 7: Ethylene Oxide Sterilization Residuals
F2315Guide for Immobilization or Encapsulation of Living
2.8 National Institute of Standards and Technology:
Cells or Tissue in Alginate Gels
NIST SP811Special Publication: Guide for the Use of the
F2605Test Method for Determining the Molar Mass of
International System of Units
SodiumAlginatebySizeExclusionChromatographywith
2.9 Other Documents:
Multi-angle Light Scattering Detection (SEC-MALS)
21CFR184.1724 Listing of Specific SubstancesAffirmed as
2.2 USP Document: 10
GRAS–Sodium Alginate
USP Monograph USP 35/NF 30Sodium Alginate
2.3 ISO Documents:
3. Terminology
ISO 31-8Quantities and units — Part 8: Physical chemistry
3.1 Definitions of Terms Specific to This Standard: (see also
and molecular physics
Terminology F1251):
ISO 10993Biological Evaluation of Medical Devices:
3.1.1 alginate, n—a polysaccharide substance containing
ISO 10993-1Biological Evaluation of Medical Devices—
calcium, magnesium, sodium, and potassium salts obtained
Part 1: Evaluation and Testing
from some of the more common species of marine algae.
ISO 10993-3Part 3: Tests for Genotoxicity, Carcinogenicity
Alginate exists in brown algae as the most abundant
and Reproductive Toxicity
polysaccharide, mainly occurring in the cell walls and inter-
ISO 10993-9—Part 9:Framework for Identification and
cellularspacesofbrownseaweedandkelp.Itsmainfunctionis
Quantification of Potential Degradation Products
to contribute to the strength and flexibility of the seaweed
ISO 10993-17—Part 17:Methods for Establishment of Al-
plant. Alginate is classified as a hydrocolloid. The most
lowable Limits for Leachable Substances Using Health-
commonly used alginate is sodium alginate.
Based Risk Assessment
3.1.2 decomposition, n—structural changes of alginates due
ISO 13408-1: 1998:Aseptic Processing of Health Care
to exposure to environmental, chemical or thermal factors,
Products—Part 1: General Requirements.
such as temperatures greater than 180°C. Decomposition can
2.4 ICH Documents:
result in deleterious changes to the alginate.
International Conference on Harmonization (ICH) S2Guid-
ance on Genotoxicity Testing and Data Interpretation for 3.1.3 degradation, n—change in the chemical structure,
physical properties, or appearance of a material. Degradation
Pharmaceuticals Intended for Human Use
International Conference on Harmonization (ICH) Q1A of polysaccharides occurs by means of cleavage of the glyco-
sidic bonds, usually by acid catalyzed hydrolysis. Degradation
ICHHarmonizedTripartite Guidance for StabilityTesting
of New Drug Substances and Products (2003)
Available from U. S. Food and Drug Administration, 5600 Fishers Lane,
The last approved version of this historical standard is referenced on Rockville MD 20857-0001.
www.astm.org. AssociationfortheAdvancementofMedicalInstrumentation1110NorthGlebe
AvailablefromU.S.Pharmacopeia(USP),12601TwinbrookPkwy.,Rockville, Rd., Suite 220, Arlington, VA 22201–4795.
MD 20852. Available from National Institute of Standards and Technology (NIST), 100
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St., Bureau Dr., Stop 1070, Gaithersburg, MD 20899-1070, http://physics.nist.gov/cuu/
4th Floor, New York, NY 10036. Units/bibliography.html.
6 10
Available from ICH Secretariat, c/o IFPMA, 30 rue de St-Jean, P.O. Box 758, Available from Superintendent of Documents, U.S. Government Printing
1211 Geneva 13, Switzerland. Office, Washington, DC 20402.
F2064 − 17
can also occur thermally. It is important to note that degrada- 4. Significance and Use
tion is not synonymous with decomposition. Degradation is
4.1 This guide contains a listing of those characterization
often used as a synonym for depolymerization when referring
parameters that are directly related to the functionality of
to polymers.
alginate. This guide can be used as an aid in the selection and
3.1.4 depolymerization, n—reductioninlengthofapolymer
characterization of the appropriate alginate for a particular
chain to form shorter polymeric units. Depolymerization may application. This guide is intended to give guidance in the
reducethepolymerchaintooligomericormonomericunits,or
methods and types of testing necessary to properly
both. In alginates, hydrolysis of the glycosidic bonds is the characterize,assess,andensureconsistencyintheperformance
primary mechanism.
of a particular alginate. It may have use in the regulation of
these devices by appropriate authorities.
3.1.5 Endotoxin, n—a high-molecular weight lipopolysac-
charide (LPS) complex associated with the cell wall of
4.2 The alginate covered by this guide may be gelled,
gram-negative bacteria that is pyrogenic in humans. Though
extruded, or otherwise formulated into biomedical devices for
endotoxins are pyrogens, not all pyrogens are endotoxins.
use in tissue-engineered medical products or drug delivery
devicesforimplantationasdeterminedtobeappropriate,based
3.1.6 G—abbreviation for α-L-guluronic acid, one of the
on supporting biocompatibility and physical test data. Recom-
two monomers making up the alginate polysaccharide mol-
mendations in this guide should not be interpreted as a
ecule. G-rich alginate has a greater than 50 % content of
guarantee of clinical success in any tissue engineered medical
guluronate residues in the polymer chain. G-block refers to a
product or drug delivery application. Further guidance for
homopolymeric block of G residues.
immobilizing or encapsulating living cells or tissue in alginate
3.1.7 hydrocolloid, n—a water-soluble polymer of colloidal
gels can be found in Guide F2315.
nature when hydrated.
4.3 To ensure that the material supplied satisfies require-
3.1.8 M—abbreviation for ß-D-mannuronic acid, one of the
ments for use in TEMPS, several general areas of character-
two monomers making up the alginate polysaccharide chain.
ization should be considered. These are: identity of alginate,
M-richalginatehasagreaterthan50%contentofmannuronate
physical and chemical characterization and testing, impurities
residues in the polymer chain.
profile, and performance-related tests.
3.1.9 molar mass average, n—the given mass-average mo-
lar mass (Mw) of an alginate will always represent an average
5. Chemical and Physical Test Methods
of all of the molecules in the population. The most common
5.1 Identity of Alginate—The identity of alginates can be
¯
ways to express the Mw are as the number average ~M ! and
n
establishedbyseveralmethodsincluding,butnotlimitedtothe
¯
the weight average ~M !. The two averages are defined by the
w
following:
following equations:
5.1.1 Sodium alginate monograph USP 35/NF30.
5.1.2 Fourier Transform Infrared Spectroscopy (FT-IR)—
N M w M N M
(i i i (i i i (i i i
¯ ¯
M 5 and M 5 5 (1)
Almost all organic chemical compounds absorb infrared radia-
n w
N w N M
(i i (i i (i i i
tion at frequencies characteristic for the functional groups in
the compound. A FT-IR spectrum will show absorption bands
where:
relatingtobondstretchingandbendingandcanthereforeserve
N = number of molecules having a specific molar mass, M,
i i
as a unique fingerprint of a specific compound. Identity of
and
sodium alginate can be assessed by Fourier transform infrared
w = mass of molecules having a specific molar mass, M.
i i
spectroscopy (FT-IR).
¯ ¯
Inapolydispersemolecularpopulationtherelation M >M
w n
5.1.2.1 Alginate as a powder—In attenuated total reflec-
¯ ¯
is always valid. The coefficient M /M is referred to as the
w n
tance (ATR), an infrared beam enters a diamond crystal.
polydispersityindex,andwilltypicallybeintherangefrom1.5
Internal reflection within the crystal creates an evanescent
to 3.0 for commercial alginates.
wave. The wave continues beyond the crystal surface and into
3.1.9.1 Discussion—The term molecular weight (abbrevi- the sample that is held in close contact to the crystal surface.
atedMS)isobsoleteandshouldbereplacedbytheSI(Système
The penetration depth of the beam is of the order of a few
Internationale) equivalent of either relative molecular mass microns.The beam is reflected several times within the crystal
(Mr), which reflects the dimensionless ratio of the mass of a
and carries spectral information from the sample into the
single molecule to an atomic mass unit (see ISO 31-8), or detector. The sample is analyzed as a powder.Apply a powder
molar mass (M), which refers to the mass of a mole of a
sample of alginate to the FT-IR ATR crystal and follow the
substance and is typically expressed as grams/mole. For instrument manufacturer’s procedure for recording spectra.
polymers and other macromolecules, use of the symbols Mw,
RecordtheIRspectrumofthecrystalwithoutsample(CO and
Mn, and Mz continue, referring to mass-average molar mass, H O correction), then record the IR spectrum of the sample
–1
number-average molar mass, and z-average molar mass, re-
using 4 scans at a speed of 0.2 cm /s and a resolution of 4
–1 –1 –1
spectively. For more information regarding proper utilization cm from 4000 cm to 650 cm . A typical FT-IR ATR
of SI units, see NIST SP811. spectrum of sodium alginate is shown in Fig. 1.
3.1.10 pyrogen, n—any substance that produces fever when 5.1.2.2 Alginate film—Cast an alginate film from a 0.25%
administered parenterally. (w/v)solutionofsodiumalginatebydryingapproximately500
F2064 − 17
FIG. 1 Typical FT-IR ATR Spectrum of Sodium Alginate
µLofthesampleontoadisposableIRcardfor3to4hat60°C. notnecessarily,causedifferencesinperformanceofanalginate
–1
Record a background spectrum between 4000 and 400 cm
inaparticularenduse.Thisinformationmaybedeterminedby
–1
1 13
using 128 scans at a resolution of 4 cm . Record the IR the following method: High-resolution H and C-nuclear
spectrumofadriedblankIRcard,thenrecordtheIRspectrum
magnetic resonance spectroscopy (NMR). Sodium alginate
–1
of the sample using 128 scans at a resolution of 4 cm ,%
should be dissolved in D O and partially degraded to a degree
transmission mode. Label the peaks. Typical frequencies
of depolymerization of 20 to 30 using mild acid hydrolysis
–1
(cm ) for sodium alginate are 3375-3390 (b), 1613 (s), 1416
before recording proton or carbon NMR spectra (Grasdalen,
(s), 1320 (w), 1125, 1089, 1031 (s), 948 (m), 903 (m), and 811
H., Larsen, B., and Smidsrød, O., Carbohydr. Res., 68, 23-31,
(m).Thepeakdesignatorsare:sh:sharp;s:strong;m:medium;
1979). Techniques have been developed to determine the
w: weak; and b: broad.
monadfrequenciesF (fractionofguluronateresidues)andF
G M
(fraction of mannuronate residues), the four nearest neighbor-
5.2 Physical and chemical characterization of alginate:
ing(diad)frequencies(F ,F ,F ,andF )andtheeight
5.2.1 The composition and sequential structure of alginate
GG GM MG MM
can be a key functional attribute of any alginate. Variations in next nearest neighboring (triad) frequencies (F ,F ,
GGG GGM
the composition or the sequential structure, or both, may, but F ,F ,F ,F ,F , and F ). A typical
GMM GMG MGM MGG MMG MMM
FIG. 2 Typical H NMR of Sodium Alginate
F2064 − 17
H-NMR spectrum of alginate is shown in Fig. 2. Alginate is (2)Using 0.1 M NaNO (sodium nitrate) as an eluant in
characterized by calculating parameters such as M/G ratio, combination with a Waters Ultrahydrogel 2000 column in
G-content, consecutive number of G monomers (that is, G>1), series with an Ultrahydrogel Linear column.
andaveragelengthofblocksofconsecutiveGmonomers.Test
5.2.2.3 Polydispersity—Depending on the end use and the
Method F2259 gives guidance on determining the chemical
sensitivityoftheapplicationtothemolarmass,thepresenceof
composition and sequence of alginate by proton NMR.
a wide range of alginate fractions may be an issue. In such
5.2.2 Molar mass (molecular weight; typically expressed as cases, calculation of the polydispersity will be important.
grams/mole) of an alginate will define certain performance Typically,thisisbetween1.5and3.0forcommercialalginates.
characteristics such as viscosity or gel strength, or both. As
5.2.2.4 Depending on the final use and the required perfor-
suchanddependingonthesensitivityofaparticularenduseto
mance control, other characterization assays can include, but
these variations, determination of molar mass directly or
are not limited to the following:
indirectly may be necessary. Commercial alginates are poly-
5.2.2.5 Viscosity in Aqueous Solution—Viscosity is defined
disperse with respect to molar mass (M ). Molar mass may be
w
as a liquid’s resistance to flow. The molecular mass of an
expressed as the number average (M ) or the weight average
N
alginate will determine the extent to which it will thicken an
(M ).Molarmassmaybedeterminedbymethodssuchas,but
W
aqueous solution. Therefore, a simple viscosity test may yield
not limited, to the following:
information on the relative differences in molar mass among
5.2.2.1 Molar Mass Determination Based on Intrinsic
alginate samples. To allow comparison between laboratories,
Viscosity—The intrinsic viscosity describes a polymer’s ability
theviscometerusedmustbecalibratedwithtraceablestandards
to form viscous solutions in water and is directly proportional
(see Test Method E2975). The viscosity measured will depend
to the average molar mass of the polymer. The intrinsic
on several parameters related to how the testing is conducted.
viscosity is a characteristic of the polymer under specified
Important parameters to control include, but are not limited to
solvent and temperature conditions; it is independent of con-
the following:
centration. The intrinsic viscosity (η) is directly related to the
(1) Temperature—The temperature at which the measure-
molarmassofapolymerthroughtheMark-Houwink-Sakurada
ment is performed is critical. An increase in temperature will,
a
(MHS) equation: [η]=KM , where K is a constant, M is the
in almost every case, result in a decrease in the viscosity.
viscosity derived average molar mass, and a is an empirical
Consistent and controlled temperature (that is, with a standard
constant describing the conformation of the polymer. For
temperature bath) is critical to achieving reproducible results.
alginate, the exponent (a) is close to unity at an ionic strength
Typically,thetemperatureusedtomeasureviscositycanbe20,
of 0.1 (for example, 0.1 M NaCl). By measuring the intrinsic
25, or 37 °C, or a combination thereof.
viscosity, the viscosity average molar mass can be determined
(2) Alginate Concentration—The moisture content of the
if K and a are accurately known for the sample: log [η] = log alginate must be known in order to prepare correct concentra-
K + a(log M), where M is the molar mass. The intrinsic
tions of alginate.
viscosityisdeterminedbymeasuringtherelativeviscosityina (3) Ionic strength—The viscosity of an alginate solution is
Ubbelohde capillary viscometer. The measurements should be
very sensitive to the ionic environment in which the measure-
performed in a solvent containing 0.1 M NaCl (a non-gelling, ment is made.Although any ion can have an impact, multiva-
monovalent salt) at a constant temperature of 20°C, and at a
lent ions other than magnesium will have the most effect. The
sufficiently low alginate concentration. Automatic operation most important aspect is to keep the ionic content consistent.
and data acquisition are preferred. Typically viscosity measurements are made in deionized water
or a standardized ionic environment such as isotonic saline.
5.2.2.2 Molar Mass and Polydispersity Determination by
(4) Molecular Mass—Viscosity measurements are sensi-
Size Exclusion Chromatography With Multiple Angle Light
tivetothemolecularmassofthealginate.Thefollowingisone
Scattering Detection (SEC-MALS)—As there are no alginate
suggestion concerning the measurement of alginate viscosity,
standardscurrentlyavailable,refractiveindexdetectorscannot
but any appropriate method would apply. To measure the
beadequatelycalibrated.Itisnotsufficienttoonlyusepullulan
apparent viscosity of sodium alginate, prepare a solution in
or other polysaccharide standards as a calibration material.
deionized water with a concen
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM 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: F2064 − 14 F2064 − 17
Standard Guide for
Characterization and Testing of Alginates as Starting
Materials Intended for Use in Biomedical and Tissue
Engineered Medical Product Applications
This standard is issued under the fixed designation F2064; 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.
INTRODUCTION
Alginate has found uses in a variety of products ranging from simple technical applications such as
viscosifiers to advanced biomedical matrices providing controlled drug delivery from immobilized
living cells. As for most hydrocolloids, the functionality of alginate is related to its chemical and
structural composition. The aim of this guide is to identify key parameters relevant for the
functionality and characterization of alginates for the development of new commercial applications of
alginates for the biomedical and pharmaceutical industries.
1. Scope
1.1 This guide covers the evaluation of alginates suitable for use in biomedical or pharmaceutical applications, or both,
including, but not limited to, Tissue Engineered Medical Products (TEMPs).
1.2 This guide addresses key parameters relevant for the functionality, characterization, and purity of alginates.
1.3 As with any material, some characteristics of alginates may be altered by processing techniques (such as molding, extrusion,
machining, assembly, sterilization, and so forth) required for the production of a specific part or device. Therefore, properties of
fabricated forms of this polymer should be evaluated using test methods that are appropriate to ensure safety and efficacy and are
not addressed in this guide.
1.4 Warning—Mercury has been designated by EPA and many state agencies as a hazardous material that can cause central
nervous system, kidney, and liver damage. Mercury, or its vapor, may be hazardous to health and corrosive to materials. Caution
should be taken when handling mercury and mercury-containing products. See the applicable product Material Safety Data Sheet
(MSDS) for details and EPA’s website (http://www.epa.gov/mercury/faq.htm) for additional information. Users should be aware
that selling mercury or mercury-containing products, or both, in your state may be prohibited by state law.
1.5 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.
1.6 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D2196E2975 Test Methods for Rheological Properties of Non-Newtonian Materials by Rotational ViscometerMethod for
Calibration or Calibration Verification of Concentric Cylinder Rotational Viscometers
F619 Practice for Extraction of Medical Plastics
This guide 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 Oct. 1, 2014March 1, 2017. Published February 2015April 2017. Originally approved in 2000. Last previous edition approved in 20002014 as
ε1
F2064 – 00 (2006)F2064 – 14. . DOI: 10.1520/F2064-14.10.1520/F2064-17.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2064 − 17
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
F749 Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit
F756 Practice for Assessment of Hemolytic Properties of Materials
F763 Practice for Short-Term Screening of Implant Materials
F813 Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices
F895 Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
F981 Practice for Assessment of Compatibility of Biomaterials for Surgical Implants with Respect to Effect of Materials on
Muscle and Insertion into Bone
F1251 Terminology Relating to Polymeric Biomaterials in Medical and Surgical Devices (Withdrawn 2012)
F1439 Guide for Performance of Lifetime Bioassay for the Tumorigenic Potential of Implant Materials
F1903 Practice for Testing For Biological Responses to Particles In Vitro
F1904 Practice for Testing the Biological Responses to Particles in vivo
F1905 Practice For Selecting Tests for Determining the Propensity of Materials to Cause Immunotoxicity (Withdrawn 2011)
F1906 Practice for Evaluation of Immune Responses In Biocompatibility Testing Using ELISA Tests, Lymphocyte Proliferation,
and Cell Migration (Withdrawn 2011)
F2259 Test Method for Determining the Chemical Composition and Sequence in Alginate by Proton Nuclear Magnetic
Resonance ( H NMR) Spectroscopy
F2315 Guide for Immobilization or Encapsulation of Living Cells or Tissue in Alginate Gels
F2605 Test Method for Determining the Molar Mass of Sodium Alginate by Size Exclusion Chromatography with Multi-angle
Light Scattering Detection (SEC-MALS)
2.2 USP Document:
USP Monograph USP 35/NF 30 Sodium Alginate
2.3 ISO Documents:
ISO 31-8 Quantities and units — Part 8: Physical chemistry and molecular physics
ISO 10993 Biological Evaluation of Medical Devices:
ISO 10993-1 Biological Evaluation of Medical Devices—Part 1: Evaluation and Testing
ISO 10993-3 Part 3: Tests for Genotoxicity, Carcinogenicity and Reproductive Toxicity
ISO 10993-9—Part 9: Framework for Identification and Quantification of Potential Degradation Products
ISO 10993-17—Part 17: Methods for Establishment of Allowable Limits for Leachable Substances Using Health-Based Risk
Assessment
ISO 13408-1: 1998: Aseptic Processing of Health Care Products—Part 1: General Requirements.
2.4 ICH Documents:
International Conference on Harmonization (ICH) S2 Guidance on Genotoxicity Testing and Data Interpretation for
Pharmaceuticals Intended for Human Use
International Conference on Harmonization (ICH) Q1A ICH Harmonized Tripartite Guidance for Stability Testing of New Drug
Substances and Products (2003)
2.5 FDA Documents:
FDA Interim Guidance for Human and Veterinary Drug Products and Biologicals. Kinetic LAL techniques. DHHS, July 15,
2.6 ANSI Documents:
ANSI/AAMI/ISO 11737-1: 2006 Sterilization of Medical Devices—Microbiological Methods—Part 1: Estimation of Bioburden
on Product.
ANSI/AAMI/ISO 11737-2: 1998 Sterilization of Medical Devices—Microbiological Methods—Part 2: Tests of Sterility
Performed in the Validation of a Sterilization Process
2.7 AAMI Documents:
AAMI/ISO 14160—1998 Sterilization of Single-Use Medical Devices Incorporating Materials of Animal Origin—Validation
and Routine Control of Sterilization by Liquid Chemical Sterilants
AAMI ST67: 2011 Sterilization of Health Care Products—Requirements and Guidance for Selecting a Sterility Assurance Level
(SAL) for Products Labeled “Sterile”
AAMI TIR No. 19—1998 Guidance for ANSI/AAMI/ISO 10993-7: 1995, Biological Evaluation of Medical Devices—Part 7:
Ethylene Oxide Sterilization Residuals
The last approved version of this historical standard is referenced on www.astm.org.
Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville, MD 20852.
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036.
Available from ICH Secretariat, c/o IFPMA, 30 rue de St-Jean, P.O. Box 758, 1211 Geneva 13, Switzerland.
Available from U. S. Food and Drug Administration, 5600 Fishers Lane, Rockville MD 20857-0001.
Association for the Advancement of Medical Instrumentation 1110 North Glebe Rd., Suite 220, Arlington, VA 22201–4795.
F2064 − 17
2.8 National Institute of Standards and Technology:
NIST SP811 Special Publication: Guide for the Use of the International System of Units
2.9 Other Documents:
21CFR184.1724 Listing of Specific Substances Affirmed as GRAS–Sodium Alginate
3. Terminology
3.1 Definitions of Terms Specific to This Standard: (see also Terminology F1251):
3.1.1 alginate, n—a polysaccharide substance containing calcium, magnesium, sodium, and potassium salts obtained from some
of the more common species of marine algae. Alginate exists in brown algae as the most abundant polysaccharide, 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. Alginate is classified as a hydrocolloid. The most commonly used alginate is sodium alginate.
3.1.2 decomposition, n—structural changes of alginates due to exposure to environmental, chemical or thermal factors, such as
temperatures greater than 180°C. Decomposition can result in deleterious changes to the alginate.
3.1.3 degradation, n—change in the chemical structure, physical properties, or appearance of a material. Degradation of
polysaccharides occurs by means of cleavage of the glycosidic bonds, usually by acid catalyzed hydrolysis. Degradation can also
occur thermally. 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.
3.1.4 depolymerization, n—reduction in length of a polymer chain to form shorter polymeric units. Depolymerization may
reduce the polymer chain to oligomeric or monomeric units, or both. In alginates, hydrolysis of the glycosidic bonds is the primary
mechanism.
3.1.5 Endotoxin, n—a high-molecular weight lipopolysaccharide (LPS) complex associated with the cell wall of gram-negative
bacteria that is pyrogenic in humans. Though endotoxins are pyrogens, not all pyrogens are endotoxins.
3.1.6 G—abbreviation for α-L-guluronic acid, one of the two monomers making up the alginate polysaccharide molecule. G-rich
alginate has a greater than 50 % content of guluronate residues in the polymer chain. G-block refers to a homopolymeric block
of G residues.
3.1.7 hydrocolloid, n—a water-soluble polymer of colloidal nature when hydrated.
3.1.8 M—abbreviation for ß-D-mannuronic acid, one of the two monomers making up the alginate polysaccharide chain. M-rich
alginate has a greater than 50% content of mannuronate residues in the polymer chain.
3.1.9 molar mass average, n—the given mass-average molar mass (Mw) of an alginate will always represent an average of all
¯
of the molecules in the population. The most common ways to express the Mw are as the number average ~M ! and the weight
n
¯
average ~M !. The two averages are defined by the following equations:
w
N M w M N M
(i i i (i i i (i i i
¯ ¯
M 5 and M 5 5 (1)
n w
N w N M
(i i (i i (i i i
where:
N = number of molecules having a specific molar mass, M , and
i i
w = mass of molecules having a specific molar mass, M .
i i
In a polydisperse molecular population the relation M¯ > M¯ is always valid. The coefficient M¯ /M¯ is referred to as the
w n w n
polydispersity index, and will typically be in the range from 1.5 to 3.0 for commercial alginates.
Available from National Institute of Standards and Technology (NIST), 100 Bureau Dr., Stop 1070, Gaithersburg, MD 20899-1070, http://physics.nist.gov/cuu/
Units/bibliography.html.
Available from Superintendent of Documents, U.S. Government Printing Office, Washington, DC 20402.
3.1.9.1 Discussion—
The term molecular weight (abbreviated MS) is obsolete and should be replaced by the SI (Système Internationale) equivalent of
either relative molecular mass (Mr), which reflects the dimensionless ratio of the mass of a single molecule to an atomic mass unit
(see ISO 31-8), or molar mass (M), which refers to the mass of a mole of a substance and is typically expressed as grams/mole.
For polymers and other macromolecules, use of the symbols Mw, Mn, and Mz continue, referring to mass-average molar mass,
number-average molar mass, and z-average molar mass, respectively. For more information regarding proper utilization of SI units,
see NIST SP811.
3.1.10 pyrogen, n—any substance that produces fever when administered parenterally.
F2064 − 17
4. Significance and Use
4.1 This guide contains a listing of those characterization parameters that are directly related to the functionality of alginate.
This guide can be used as an aid in the selection and characterization of the appropriate alginate for a particular application. This
guide is intended to give guidance in the methods and types of testing necessary to properly characterize, assess, and ensure
consistency in the performance of a particular alginate. It may have use in the regulation of these devices by appropriate authorities.
4.2 The alginate covered by this guide may be gelled, extruded, or otherwise formulated into biomedical devices for use in
tissue-engineered medical products or drug delivery devices for implantation as determined to be appropriate, based on supporting
biocompatibility and physical test data. Recommendations in this guide should not be interpreted as a guarantee of clinical success
in any tissue engineered medical product or drug delivery application. Further guidance for immobilizing or encapsulating living
cells or tissue in alginate gels can be found in Guide F2315.
4.3 To ensure that the material supplied satisfies requirements for use in TEMPS, several general areas of characterization
should be considered. These are: identity of alginate, physical and chemical characterization and testing, impurities profile, and
performance-related tests.
5. Chemical and Physical Test Methods
5.1 Identity of Alginate—The identity of alginates can be established by several methods including, but not limited to the
following:
5.1.1 Sodium alginate monograph USP 35/NF30.
5.1.2 Fourier Transform Infrared Spectroscopy (FT-IR)—Almost all organic chemical compounds absorb infrared radiation at
frequencies characteristic for the functional groups in the compound. A FT-IR spectrum will show absorption bands relating to
bond stretching and bending and can therefore serve as a unique fingerprint of a specific compound. Identity of sodium alginate
can be assessed by Fourier transform infrared spectroscopy (FT-IR).
5.1.2.1 Alginate as a powder—In attenuated total reflectance (ATR), an infrared beam enters a diamond crystal. Internal
reflection within the crystal creates an evanescent wave. The wave continues beyond the crystal surface and into the sample that
is held in close contact to the crystal surface. The penetration depth of the beam is of the order of a few microns. The beam is
reflected several times within the crystal and carries spectral information from the sample into the detector. The sample is analyzed
as a powder. Apply a powder sample of alginate to the FT-IR ATR crystal and follow the instrument manufacturer’s procedure for
recording spectra. Record the IR spectrum of the crystal without sample (CO and H O correction), then record the IR spectrum
2 2
–1 –1 –1 –1
of the sample using 4 scans at a speed of 0.2 cm /s and a resolution of 4 cm from 4000 cm to 650 cm . A typical FT-IR ATR
spectrum of sodium alginate is shown in Fig. 1.
5.1.2.2 Alginate film—Cast an alginate film from a 0.25 % (w/v) solution of sodium alginate by drying approximately 500 μL
–1
of the sample onto a disposable IR card for 3 to 4 h at 60°C. Record a background spectrum between 4000 and 400 cm using
–1
128 scans at a resolution of 4 cm . Record the IR spectrum of a dried blank IR card, then record the IR spectrum of the sample
–1 –1
using 128 scans at a resolution of 4 cm , % transmission mode. Label the peaks. Typical frequencies (cm ) for sodium alginate
are 3375-3390 (b), 1613 (s), 1416 (s), 1320 (w), 1125, 1089, 1031 (s), 948 (m), 903 (m), and 811 (m). The peak designators are:
sh: sharp; s: strong; m: medium; w: weak; and b: broad.
FIG. 1 Typical FT-IR ATR Spectrum of Sodium Alginate
F2064 − 17
5.2 Physical and chemical characterization of alginate:
5.2.1 The composition and sequential structure of alginate can be a key functional attribute of any alginate. Variations in the
composition or the sequential structure, or both, may, but not necessarily, cause differences in performance of an alginate in a
1 13
particular end use. This information may be determined by the following method: High-resolution H and C-nuclear magnetic
resonance spectroscopy (NMR). Sodium alginate should be dissolved in D O and partially degraded to a degree of
depolymerization of 20 to 30 using mild acid hydrolysis before recording proton or carbon NMR spectra (Grasdalen, H., Larsen,
B., and Smidsrød, O., Carbohydr. Res., 68, 23-31, 1979). Techniques have been developed to determine the monad frequencies F
G
(fraction of guluronate residues) and F (fraction of mannuronate residues), the four nearest neighboring (diad) frequencies (F ,
M GG
F , F , and F ) and the eight next nearest neighboring (triad) frequencies (F , F , F , F , F , F , F ,
GM MG MM GGG GGM GMM GMG MGM MGG MMG
and F ). A typical H-NMR spectrum of alginate is shown in Fig. 2. Alginate is characterized by calculating parameters such
MMM
as M/G ratio, G-content, consecutive number of G monomers (that is, G>1), and average length of blocks of consecutive G
monomers. Test Method F2259 gives guidance on determining the chemical composition and sequence of alginate by proton NMR.
5.2.2 Molar mass (molecular weight; typically expressed as grams/mole) of an alginate will define certain performance
characteristics such as viscosity or gel strength, or both. As such and depending on the sensitivity of a particular end use to these
variations, determination of molar mass directly or indirectly may be necessary. Commercial alginates are polydisperse with
respect to molar mass (M ). Molar mass may be expressed as the number average (M ) or the weight average (M ). Molar mass
w N W
may be determined by methods such as, but not limited, to the following:
5.2.2.1 Molar Mass Determination Based on Intrinsic Viscosity—The intrinsic viscosity describes a polymer’s ability to form
viscous solutions in water and is directly proportional to the average molar mass of the polymer. The intrinsic viscosity is a
characteristic of the polymer under specified solvent and temperature conditions; it is independent of concentration. The intrinsic
a
viscosity (η) is directly related to the molar mass of a polymer through the Mark-Houwink-Sakurada (MHS) equation: [η] = KM ,
where K is a constant, M is the viscosity derived average molar mass, and a is an empirical constant describing the conformation
of the polymer. For alginate, the exponent (a) is close to unity at an ionic strength of 0.1 (for example, 0.1 M NaCl). By measuring
the intrinsic viscosity, the viscosity average molar mass can be determined if K and a are accurately known for the sample: log
[η] = log K + a(log M), where M is the molar mass. The intrinsic viscosity is determined by measuring the relative viscosity in
a Ubbelohde capillary viscometer. The measurements should be performed in a solvent containing 0.1 M NaCl (a non-gelling,
monovalent salt) at a constant temperature of 20°C, and at a sufficiently low alginate concentration. Automatic operation and data
acquisition are preferred.
5.2.2.2 Molar Mass and Polydispersity Determination by Size Exclusion Chromatography With Multiple Angle Light Scattering
Detection (SEC-MALS)—As there are no alginate standards currently available, refractive index detectors can not be adequately
calibrated. It is not sufficient to only use pullulan or other polysaccharide standards as a calibration material. Therefore, the method
of choice is to use refractive index coupled to multiple angle light scattering detection (MALS). For separation of the alginate into
different molar mass fractions, a hydrophilic column with the appropriate pore size is required. Such columns include, but are not
limited to, those mentioned in the techniques as follows: The precision of these techniques must be determined as results can vary
by 10 to 20 %. Typical methods using these techniques include, but are not limited to the following:
FIG. 2 Typical H NMR of Sodium Alginate
F2064 − 17
(1) Using 0.01 M sodium EDTA/0.05 M sodium sulfate, pH 6.0 as the mobile phase with separation using TSK 3000, TSK
5000, and TSK 6000 columns. Test Method F2605 gives guidance in determining the molar mass of sodium alginate by
SEC-MALS.
(2) Using 0.1 M NaNO (sodium nitrate) as an eluant in combination with a Waters Ultrahydrogel 2000 column in series with
an Ultrahydrogel Linear column.
5.2.2.3 Polydispersity—Depending on the end use and the sensitivity of the application to the molar mass, the presence of a wide
range of alginate fractions may be an issue. In such cases, calculation of the polydispersity will be important. Typically, this is
between 1.5 and 3.0 for commercial alginates.
5.2.2.4 Depending on the final use and the required performance control, other characterization assays can include, but are not
limited to the following:
5.2.2.5 Viscosity in Aqueous Solution—Viscosity is defined as a liquid’s resistance to flow. The molecular mass of an alginate
will determine the extent to which it will thicken an aqueous solution. Therefore, a simple viscosity test may yield information on
the relative differences in molar mass among alginate samples. To allow comparison between laboratories, the viscometer used
must be calibrated with traceable standards (see Test MethodsMethod D2196E2975). The viscosity measured will depend on
several parameters related to how the testing is conducted. Important parameters to control include, but are not limited to the
following:
(1) Temperature—The temperature at which the measurement is performed is critical. An increase in temperature will, in
almost every case, result in a decrease in the viscosity. Consistent and controlled temperature (that is, with a standard temperature
bath) is critical to achieving reproducible results. Typically, the temperature used to measure viscosity can be 2
...