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Significance and Use
4.1 The degree of deacetylation of chitosan, as well at the molar mass and molar mass distribution, determines the functionality of chitosan in an application. For instance, functional and biological effects are highly dependent upon the composition and molar mass of the polymer.
4.2 This test method describes procedures for measurement of molar mass of chitosan chlorides and glutamates, and chitosan base, although it in principle applies to any chitosan salt. The measured molar mass is that for chitosan acetate, since the mobile phase contains acetate as counter ion. This value can further be converted into the corresponding molar mass for the chitosan as a base, or the parent salt form (chloride or glutamate).
4.3 Light scattering is one of very few methods available for the determination of absolute molar mass and structure, and it is applicable over the broadest range of molar masses of any method. Combining light scattering detection with size exclusion chromatography (SEC), which sorts molecules according to size, gives the ability to analyze polydisperse samples, as well as obtaining information on branching and molecular conformation. This means that both the number-average and mass-average values for molar mass and size may be obtained for most samples. Furthermore, one has the ability to calculate the distributions of the molar masses and sizes.
4.4 Multi-angle laser light scattering (MALS) is a technique where measurements of scattered light are made simultaneously over a range of different angles. MALS detection can be used to obtain information on molecular size, since this parameter is determined by the angular variation of the scattered light. Molar mass may in principle be determined by detecting scattered light at a single low angle (LALLS). However, advantages with MALS as compared to LALLS are: (1) less noise at larger angles, (2) the precision of measurements are greatly improved by detecting at several angles, and (3) the ability to detect angular variation allows determination of size, branching, aggregation, and molecular conformation.
4.5 Size exclusion chromatography uses columns, which are typically packed with polymer particles containing a network of uniform pores into which solute and solvent molecules can diffuse. While in the pores, molecules are effectively trapped and removed from the flow of the mobile phase. The average residence time in the pores depends upon the size of the solute molecules. Molecules that are larger than the average pore size of the packing are excluded and experience virtually no retention; these are eluted first, in the void volume of the column. Molecules, which may penetrate the pores will have a larger volume available for diffusion, they will suffer retention depending on their molecular size, with the smaller molecules eluting last.
4.6 For polyelectrolytes, dialysis against the elution buffer has been suggested, in order to eliminate Donnan-type artifacts in the molar mass determination by light scattering (1, 2).5 However, in the present method, the size exclusion chromatography step preceding the light scatter detection is an efficient substitute for a dialysis step. The sample is separated on SEC columns with large excess of elution buffer for 30 to 40 min, and it is therefore in full equilibrium with the elution buffer when it reaches the MALS detector.
1.1 This test method covers the determination of the molar mass of chitosan and chitosan salts intended for use in biomedical and pharmaceutical applications as well as in tissue engineered medical products (TEMPs) by size exclusion chromatography with multi-angle laser light scattering detection (SEC-MALS). A guide for the characterization of chitosan salts has been published as Guide F2103.
1.2 Chitosan and chitosan salts used in TEMPs should be well characterized, including the molar mass and polydispersity (molar mass distribution) in order to ensure uniformity and correct functionality in the final product. This test method will assist end users in choosing the correct chitosan for their particular application. Chitosan 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.
2. Referenced Documents (purchase separately) The documents listed below are referenced within the subject standard but are not provided as part of the standard.
National Institute of Standards and TechnologyNIST SP811 Special Publication: Guide for the Use of the International System of Units (SI)
F2103 Guide for Characterization and Testing of Chitosan Salts as Starting Materials Intended for Use in Biomedical and Tissue-Engineered Medical Product Applications
United States Pharmacopeia/National Formulary<621> Chromatography
ICS Number Code 11.120.10 (Medicaments)
ASTM F2602-13, Standard Test Method for Determining the Molar Mass of Chitosan and Chitosan Salts by Size Exclusion Chromatography with Multi-angle Light Scattering Detection (SEC-MALS), ASTM International, West Conshohocken, PA, 2013, www.astm.orgBack to Top