1、Designation: F2602 13Standard Test Method forDetermining the Molar Mass of Chitosan and Chitosan Saltsby Size Exclusion Chromatography with Multi-angle LightScattering Detection (SEC-MALS)1This standard is issued under the fixed designation F2602; the number immediately following the designation ind
2、icates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers the determination
3、 of the molarmass of chitosan and chitosan salts intended for use inbiomedical and pharmaceutical applications as well as in tissueengineered medical products (TEMPs) by size exclusion chro-matography with multi-angle laser light scattering detection(SEC-MALS).Aguide for the characterization of chit
4、osan saltshas been published as Guide F2103.1.2 Chitosan and chitosan salts used in TEMPs should bewell characterized, including the molar mass and polydisper-sity (molar mass distribution) in order to ensure uniformity andcorrect functionality in the final product. This test method willassist end u
5、sers in choosing the correct chitosan for theirparticular application. Chitosan may have utility as a scaffoldor matrix material for TEMPs, in cell and tissue encapsulationapplications, and in drug delivery formulations.1.3 The values stated in SI units are to be regarded asstandard. No other units
6、of measurement are included in thisstandard.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulat
7、ory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2F2103 Guide for Characterization and Testing of ChitosanSalts as Starting Materials Intended for Use in Biomedicaland Tissue-Engineered Medical Product Applications2.2 United States Pharmacopeia/National Formulary:3Chromatograph
8、y2.3 National Institute of Standards and Technology:4NIST SP811 Special Publication: Guide for the Use of theInternational System of Units (SI)3. Terminology3.1 Definitions:3.1.1 chitosan, na linear polysaccharide consisting of(14) linked 2-acetamido-2-deoxy-D-glucopyranose (Glc-NAc) and 2-amino-2-d
9、eoxy-D-glucopyranose (GlcN). Chito-san is a polysaccharide derived by N-deacetylation of chitin.3.1.2 degree of deacetylation, nthe fraction or percentageof glucosamine units (GlcN: deacetylated monomers) in achitosan polymer molecule.3.1.3 molar mass average, nthe given molar mass (M) ofa chitosan
10、will always represent an average of all of themolecules in the population. The most common ways toexpress the molar mass are as the number average (Mn) and themass average (Mw). The two averages are defined by thefollowing equations:Mn5(iNiMi(iNiand Mw5(iwiMi(iwi5(iNiMi2(iNiMi(1)where:Ni= number of
11、molecules having a specific molar mass Mi,andwi= mass of molecules having a specific molar mass Mi.3.1.3.1 DiscussionIn a polydisperse molecular populationthe relation Mw Mnis always valid. The coefficient Mw/Mnisreferred to as the polydispersity index, and will typically be inthe range 1.5 to 3.0 f
12、or commercial chitosans.NOTE 1The term molecular weight (abbreviated MW) is obsolete andshould be replaced by the SI (Systme Internationale) equivalent of either1This test method is under the jurisdiction of ASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibi
13、lity of SubcommitteeF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved Aug. 1, 2013. Published September 2013. Originallyapproved in 2008. Last previous edition approved in 2008 as F2602081. DOI:10.1520/F2602-13.2For referenced ASTM standards, visit the ASTM website, www.astm
14、.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from United States Pharmacopeia and National Formulary, U.S.Pharmaceutical Convention, Inc. (USPC), Rockville, MD.
15、4Available from National Institute of Standards and Technology (NIST), 100Bureau Dr., Stop 1070, Gaithersburg, MD 20899-1070, http:/physics.nist.gov/cuu/Units/bibliography.html.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1relative
16、molecular mass (Mr), which reflects the dimensionless ratio of themass of a single molecule to an atomic mass unit (see ISO 31-8), or molarmass (M), which refers to the mass of a mole of a substance and istypically expressed as grams/mole. For polymers and othermacromolecules, use of the symbols Mw,
17、 Mn, and Mzcontinue, referringto mass-average molar mass, number-average molar mass, and z-averagemolar mass, respectively. For more information regarding proper utiliza-tion of SI units, see NIST SP811.4. Significance and Use4.1 The degree of deacetylation of chitosan, as well at themolar mass and
18、molar mass distribution, determines thefunctionality of chitosan in an application. For instance,functional and biological effects are highly dependent upon thecomposition and molar mass of the polymer.4.2 This test method describes procedures for measurementof molar mass of chitosan chlorides and g
19、lutamates, andchitosan base, although it in principle applies to any chitosansalt. The measured molar mass is that for chitosan acetate,since the mobile phase contains acetate as counter ion. Thisvalue can further be converted into the corresponding molarmass for the chitosan as a base, or the paren
20、t salt form (chlorideor glutamate).4.3 Light scattering is one of very few methods available forthe determination of absolute molar mass and structure, and itis applicable over the broadest range of molar masses of anymethod. Combining light scattering detection with size exclu-sion chromatography (
21、SEC), which sorts molecules accordingto size, gives the ability to analyze polydisperse samples, aswell as obtaining information on branching and molecularconformation. This means that both the number-average andmass-average values for molar mass and size may be obtainedfor most samples. Furthermore
22、, one has the ability to calculatethe distributions of the molar masses and sizes.4.4 Multi-angle laser light scattering (MALS) is a techniquewhere measurements of scattered light are made simultane-ously over a range of different angles. MALS detection can beused to obtain information on molecular
23、size, since thisparameter is determined by the angular variation of thescattered light. Molar mass may in principle be determined bydetecting 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 mea
24、sure-ments are greatly improved by detecting at several angles, and(3) the ability to detect angular variation allows determinationof size, branching, aggregation, and molecular conformation.4.5 Size exclusion chromatography uses columns, which aretypically packed with polymer particles containing a
25、 networkof uniform pores into which solute and solvent molecules candiffuse. While in the pores, molecules are effectively trappedand removed from the flow of the mobile phase. The averageresidence time in the pores depends upon the size of the solutemolecules. Molecules that are larger than the ave
26、rage pore sizeof the packing are excluded and experience virtually noretention; these are eluted first, in the void volume of thecolumn. Molecules, which may penetrate the pores will have alarger volume available for diffusion, they will suffer retentiondepending on their molecular size, with the sm
27、aller moleculeseluting last.4.6 For polyelectrolytes, dialysis against the elution bufferhas been suggested, in order to eliminate Donnan-type artifactsin the molar mass determination by light scattering (1, 2).5However, in the present method, the size exclusion chroma-tography step preceding the li
28、ght scatter detection is anefficient substitute for a dialysis step. The sample is separatedon SEC columns with large excess of elution buffer for 30 to 40min, and it is therefore in full equilibrium with the elutionbuffer when it reaches the MALS detector.5. Materials5.1 Chemicals:5.1.1 Chitosan or
29、 chitosan salt sample.5.1.2 Deionized water (Milli-Q Plus or equivalent; conduc-tivity 100 0.5 0.1AInjected mass = Concentration*200 L.TABLE 2 Suggestions for Concentration and Injected Mass ofChitosan Glutamate Samples for SEC-MALSApparent Viscosityas Chitosan Glutamate(mPas)Concentration forInject
30、ion(mg/mL)Injected MassA(mg)50 0.75 0.15AInjected mass = Concentration*200 L.TABLE 3 Suggestions for Concentration and Injected Mass ofChitosan Base Samples for SEC-MALSApparent Viscosityas Chitosan Acetate(mPas)Concentration forInjection(mg/mL)InjectedMassA(mg)500 0.375 0.075AInjected mass = Concen
31、tration*200 L.F2602 1336.4.2.6 Perform the required calculations for determinationof Mn, Mwand Mw/Mn, using a Zimm representation of theDebye plot (that is, a plot of K*c/R() versus sin2/2) forsolving Eq X2.1.6.4.3 Conversion of Mw-valuesThe mobile phase is 0.2mol/L ammonium acetate, and the Mwdeter
32、mined according tothis method is that of chitosan acetate Mw(Ac). This massaverage molar mass can be converted to that of chitosanchloride Mw(Cl), glutamate Mw(G) or base Mw(B) accord-ing to the following equations:MwG! 5 MwAc!*%DA*MG!112 %DA!*MA!/%DA*MAc!11 2 %DA!*MA!(2)MwCl! 5 MwAc!*%DA*MCl!112 %D
33、A!*MA!/%DA*MAc!11 2 %DA!*MA!(3)NOTE 1Solid lines indicate solvent/sample flow, dashed lines indicate cabling for data transfer.FIG. 1 Complete SEC-MALS Set-UpNOTE 1Solid line: RI detector; dashed line: MALS detector; () molar mass for each chromatographic data point.FIG. 2 A Chromatogram of Chitosan
34、 Chloride (Mwof 360 000 g/mol, as acetate)F2602 134MwB! 5 MwAc!*%DA*MB!112 %DA!*MA!/%DA*MAc!11 2 %DA!*MA! (4)where:%DA = degree of deacetylation,M(G) = molar mass of deacetylated chitosan residue inglutamate form = 308 g/mol,M(Cl) = molar mass of deacetylated chitosan residue inchloride form = 197 g
35、/mol,M(Ac) = molar mass of deacetylated chitosan residue inacetate form = 221 g/mol,M(B) = molar mass of uncharged deacetylated chitosanresidue (base) = 161 g/mol, andM(A) = molar mass of acetylated chitosan residue = 203g/mol.7. Control and Approval of Data7.1 The mass average molar mass, Mw, and M
36、w/Mnfor thereplicates of each sample (4 replicates for pullulan standards, 3replicates for chitosans) should be calculated. Standard devia-tions forwshould be calculated.7.2 For approving the data the following conditions apply:7.2.1 Condition 1Mwof pullulan standards (using at least3 replicates) sh
37、ould be within 610 % of the stated value fromthe manufacturer.7.2.2 Condition 2Relative standard deviation (RSD, forexample, standard deviation divided by mean value) forpullulan standards should be less than 610 %.7.2.3 Condition 3Reproducibility in the detector re-sponses for the 3 replicates of c
38、hitosan samples should bemanually evaluated. Different curve forms may indicate col-umn overload, and reanalysis at lower concentration should beconsidered.7.3 If condition 1 or 2 fails, the entire sample set needsreanalysis. The system should be inspected for possible faultsbefore the reanalysis.7.
39、4 Failure of condition 3 requires reanalysis of the chitosansample in question, only.8. Precision and Reporting Results8.1 The precision/relative standard error (RSE) of themethod is 10 %, as shown in method validation.8.2 Data on Mwshould be reported rounded off to thenearest whole ten thousand in
40、units of g/mol, for example260 000 g/mol.8.3 Mwvalues should be reported for chitosans as theacetate form.Additionally, for convenience, the Mwcan also bereported calculated as chitosan base.APPENDIXES(Nonmandatory Information)X1. RATIONALEX1.1 The use of naturally occurring biopolymers for bio-medi
41、cal and pharmaceutical applications and in tissue engi-neered medical products (TEMPs) is increasing. This testmethod is designed to give guidance in characterizing chitosanand chitosan salts used in such applications.X2. BACKGROUNDX2.1 Chitosan is a linear, binary polysaccharide consistingof (14) l
42、inked 2-acetamido-2-deoxy-D-glucopyranose (Gl-cNAc; acetylated unit) and 2-amino-2-deoxy-D-glucopyranose(GlcN; deacetylated unit). The two different monosaccharidesdiffer only by the substitution at carbon 2; GlcNAc contains anN-acetylated amino group, whereas GlcN contains only theamino-group (it i
43、s said to be deacetylated). Thus, the degree ofdeacetylation (in %) is a measure of the fraction of GlcN-unitsin the chitosan chain.X2.2 The principles of SEC-MALS can be summarized asfollows: Samples of polymer are injected into the mobile phaseand separated according to size on the SEC columns. Fo
44、r agiven concentration c (g/mL) of the solute, the scattered lightsignal as measured by the MALS detector is proportional tocM, where M is molar mass (or the mass average molar mass,Mw, for non-fractionated polydisperse samples). Using aconcentration (for example, refractive index) detector to mea-s
45、ure c, one may determine the molar mass in each volumefraction eluted from the columns. Solving Eq X2.1 is the heartof this analysis:K*c/R! 5 1/$Mw*P!%12A2c (X2.1)X2.2.1 The excess Rayleigh ratio R() is the light scatteredby the solution at an angle in excess of that scattered by puresolvent, divide
46、d by the incident light intensity.A2is the secondvirial coefficient. K* is equal to 42n02(dn/dc)2/04NA, wheren0is the refractive index of the solvent, 0is the vacuumwavelength of incident light, and NAis Avogadros number.Finally, P() is a form factor which depends on the structure ofthe scattering m
47、olecules and describes the angular dependenceF2602 135of the scattered light, from which the mean square radius of themolecules may be determined.X2.2.2 Eq X2.1 is typically solved using a Debye plot (thatis, plotting R()/(K*ci) versus sin2(/2), where the sin2(/2)term results from an expansion of P(
48、), for each volumeelement eluted from the SEC columns assuming monodisper-sity within each volume element. By extrapolating the Debyeplot to zero angle, the intercept yields the mass directly. TheDebye plot is also commonly performed using a Zimmrepresentation (that is, plotting (K*ci)/R() versus si
49、n2(/2),from which the intercept yields the inverse of the molar mass(1/Mi). The Zimm representation of the Debye plot may bepreferable for macromolecules like alginates and chitosans,since only linear fits to zero angle are normally required. Acomprehensive review of light scattering and absolute charac-terization of macromolecules, including experimentalprocedures, have been reported by P. J. Wyatt (3).X2.2.3 No molar mass standards are required in the anal
