1、Designation: F 2602 08Standard 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 F 2602; the number immediately following the designation i
2、ndicates 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 (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers the determinat
3、ion 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 c
4、hitosan saltshas been published as Guide F 2103.1.2 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 e
5、nd users 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 This standard does not purport to address all of thesafety concerns, if
6、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 regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2F 2103 Guide for Characterization and Testing of
7、 ChitosanSalts as Starting Materials Intended for Use in Biomedicaland Tissue-Engineered Medical Product Applications2.2 United States Pharmacopeia/National Formulary:3Chromatography2.3 National Institute of Standards and Technology:4NIST SP811 Special Publication: Guide for the Use of theInternatio
8、nal System of Units (SI)3. Terminology3.1 Definitions:3.1.1 chitosan, na linear polysaccharide consisting ofb(14) linked 2-acetamido-2-deoxy-D-glucopyranose(GlcNAc) and 2-amino-2-deoxy-D-glucopyranose (GlcN).Chitosan is a polysaccharide derived by N-deacetylation ofchitin.3.1.2 degree of deacetylati
9、on, 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 will always represent an average of all of themolecules in the population. The most common ways toexpress the molar mass ar
10、e 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 molecules having a specific molar mass Mi,andwi= mass of molecules having a specific molar mass Mi.3.1.3.1 DiscussionIn a p
11、olydisperse 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 for commercial chitosans.NOTE 1The term molecular weight (abbreviated MW) is obsolete andshould be replaced by the SI (Systm
12、e Internationale) equivalent of eitherrelative 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 is1This test method is under the jurisdiction of A
13、STM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved Feb. 1, 2008. Published May 2008.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact A
14、STM 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.4Available from N
15、ational Institute of Standards and Technology (NIST), 100Bureau Dr., Stop 1070, Gaithersburg, MD 20899-1070, http:/physics.nist.gov/cuu/Units/bibliography.html.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.typically expressed as gr
16、ams/mole. For polymers and other macromol-ecules, use of the symbols Mw, Mn, and Mzcontinue, referring tomass-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.
17、1 The degree of deacetylation of chitosan, as well at themolar mass and 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 descr
18、ibes procedures for measurementof molar mass of chitosan chlorides and glutamates, 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 i
19、nto the corresponding molarmass for the chitosan as a base, or the parent 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. Co
20、mbining light scattering detection with size exclu-sion chromatography (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 val
21、ues for molar mass and size may be obtainedfor most samples. Furthermore, 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 simulta-neously over a range of differe
22、nt angles. MALS detection canbe used to obtain information on molecular 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 compare
23、d to LALLS are:(1) less noise at larger angles, (2) the precision of measure-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 use
24、s columns, which aretypically packed with polymer particles containing a 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
25、 the size of the solutemolecules. Molecules that are larger than the average 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,
26、they will suffer retentiondepending on their molecular size, with the smaller 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
27、present method, the size exclusion chroma-tography step preceding the light 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
28、it reaches the MALS detector.5. Materials5.1 Chemicals:5.1.1 Chitosan or 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-
29、MALSApparent Viscosityas Chitosan Glutamate(mPas)Concentration forInjection(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 fo
30、rInjection(mg/mL)Injected MassA(mg)500 0.375 0.075AInjected mass = Concentration*200 L.F26020836.4.3 Conversion of Mw-valuesThe mobile phase is 0.2mol/L ammonium acetate, and the Mwdetermined according tothis method is that of chitosan acetate Mw(Ac). This massaverage molar mass can be converted to
31、that of chitosanchloride Mw(Cl), glutamate Mw(G) or base Mw(B) accord-ing to the following equations:MwG! 5 MwAc! * %DA*MG!1 1 %DA! *MA!/%DA*MAc! 1 1 %DA! *MA!(2)MwCl! 5 MwAc! * %DA*MCl!1 1 %DA! *MA!/%DA*MAc! 1 1 %DA! *MA!(3)NOTESolid lines indicate solvent/sample flow, dashed lines indicate cabling
32、 for data transfer.FIG. 1 Complete SEC-MALS Set-UpNOTESolid line: RI detector; dashed line: MALS detector; (L) molar mass for each chromatographic data point.FIG. 2 A Chromatogram of Chitosan Chloride (Mwof 360 000 g/mol, as acetate)F2602084MwB! 5 MwAc! * %DA*MB!1 1 %DA! *MA!/%DA*MAc! 1 1 %DA! *MA!(
33、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/mol,M(Ac) = molar mass of deacetylated chitosan residue inacetate form = 221 g/mol,M(B) = molar mass of un
34、charged 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 Mw/Mnfor thereplicates of each sample (4 replicates for pullulan standards, 3replicates for chitosans) shoul
35、d be calculated. Standard devia-tions for Mwshould be calculated.7.2 For approving the data the following conditions apply:7.2.1 Condition 1Mwof pullulan standards (using at least3 replicates) should be within 610 % of the stated value fromthe manufacturer.7.2.2 Condition 2Relative standard deviatio
36、n (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 chitosan samples should bemanually evaluated. Different curve forms may indicate col-umn overload, and rea
37、nalysis 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.7.4 Failure of condition 3 requires reanalysis of the chitosansample in question, only.8. Precision and Rep
38、orting 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 units of g/mol, for example260 000 g/mol.8.3 Mwvalues should be reported for chitosans as theacetate form
39、.Additionally, for convenience, the Mwcan also bereported calculated as chitosan base.APPENDIXES(Nonmandatory Information)X1. RATIONALEX1.1 The use of naturally occurring biopolymers forbiomedical and pharmaceutical applications and in tissueengineered medical products (TEMPs) is increasing. This te
40、stmethod is designed to give guidance in characterizing chitosanand chitosan salts used in such applications.X2. BACKGROUNDX2.1 Chitosan is a linear, binary polysaccharide consistingof b(14) linked 2-acetamido-2-deoxy-D-glucopyranose(GlcNAc; acetylated unit) and 2-amino-2-deoxy-D-glucopyranose (GlcN
41、; deacetylated unit). The two differentmonosaccharides differ only by the substitution at carbon 2;GlcNAc contains an N-acetylated amino group, whereas GlcNcontains only the amino-group (it is said to be deacetylated).Thus, the degree of deacetylation (in %) is a measure of thefraction of GlcN-units
42、 in 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. For agiven concentration c (g/mL) of the solute, the scattered lightsignal as measured by the MALS detector is
43、proportional tocM, where M is molar mass (or the mass average molar mass,Mw, for non-fractionated polydisperse samples). Using a con-centration (for example, refractive index) detector to measurec, one may determine the molar mass in each volume fractioneluted from the columns. Solving Eq X2.1 is th
44、e heart of thisanalysis:K*c/Ru! 5 1/$Mw*Pu!% 1 2A2c (X2.1)X2.2.1 The excess Rayleigh ratio R(u) is the light scatteredby the solution at an angle u in excess of that scattered by puresolvent, divided by the incident light intensity.A2is the secondvirial coefficient. K* is equal to 4p2n02(dn/dc)2/l04
45、NA,where n0is the refractive index of the solvent, l0is the vacuumwavelength of incident light, and NAis Avogadros number.F2602085Finally, P(u) is a form factor which depends on the structure ofthe scattering molecules and describes the angular dependenceof the scattered light, from which the mean s
46、quare radius of themolecules may be determined.X2.2.2 Eq X2.1 is typically solved using a Debye plot (thatis, plotting R(u)/(K*ci) versus sin2(u/2), where the sin2(u/2)term results from an expansion of P(u), for each volumeelement eluted from the SEC columns assuming monodisper-sity within each volu
47、me 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(u) versus sin2(u/2),from which the intercept yields the inverse of the molar mass(1/Mi). The Zimm represen
48、tation 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 experimental proce-dures, have been reported by
49、P. J. Wyatt (3).X2.2.3 No molar mass standards are required in the analy-sis. Only a set of fundamental or measured constants (p,n0,(dn/dc), l0,NA,A2) and a set of experimentally measuredvalues (c,R(u), P(u) are required to calculate the molar mass.X2.3 Once the sample has been fractionated and ciand Mihave been determined in each fraction, calculation of the massaverage molar mass is given by Eq X2.2:Mw5(iwiMi/(iwi5(iciMi/(ici(X2.2)X3. CONSTANT VALUESX3.1 Calibration and Normalizat
