1、Designation: F2602 13F2602 18Standard 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 designa
2、tion indicates 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 deter
3、mination of the molar mass of chitosan and chitosan salts intended for use in biomedicaland pharmaceutical applications as well as in tissue engineered medical products (TEMPs) by size exclusion chromatography withmulti-angle laser light scattering detection (SEC-MALS). A guide for the characterizat
4、ion of chitosan salts has been published asGuide 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
5、assistend users in choosing the correct chitosan for their particular application. Chitosan may have utility as a scaffold or matrix materialfor 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. N
6、o 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 responsibilityof the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices
7、 and determine theapplicability of regulatory limitations prior to use.1.5 This international standard was developed in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Development of International Standards, Guides and Recomme
8、ndations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2F2103 Guide for Characterization and Testing of Chitosan Salts as Starting Materials Intended for Use in Biomedical andTissue-Engineered Medical Product Applications2
9、.2 United States Pharmacopeia/National Formulary:3Chromatography2.2 National Institute of Standards and Technology:3NIST SP811 Special Publication: Guide for the Use of the International System of Units (SI)2.3 ISO Document:4ISO 80000-9:2009 Quantities and units Part 9: Physical chemistry and molecu
10、lar physics3. Terminology3.1 Definitions:1 This test method is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved Aug. 1, 2013June 1, 201
11、8. Published September 2013August 2018. Originally approved in 2008. Last previous edition approved in 20082013as F26020813.1. DOI: 10.1520/F2602-13.10.1520/F2602-18.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Bo
12、ok of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.3 Available from United States Pharmacopeia and National Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville, MD.3 Available from National Institute of Standards and Technology (NIS
13、T), 100 Bureau Dr., Stop 1070, Gaithersburg, MD 20899-1070, http:/physics.nist.gov/cuu/Units/bibliography.html.4 Available from International Organization for Standardization (ISO), ISO Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier, Geneva,Switzerland, http:/www.iso.org.
14、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. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior edi
15、tions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.1.1 chitosan, na linear polysaccharide consist
16、ing of (14) linked 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) and2-amino-2-deoxy-D-glucopyranose (GlcN). Chitosan is a polysaccharide derived by N-deacetylation of chitin.3.1.1.1 chitin, na linear polysaccharide consisting of (14) linked 2-acetamido-2-deoxy-D-glucopyranose.3.1.2 degree of deacetyl
17、ation, nthe fraction or percentage of glucosamine units (GlcN: deacetylated monomers) in a chitosanpolymer molecule.3.1.3 molar mass average, nthe given molar mass (M) of a chitosan will always represent an average of all of the moleculesin the population. The most common ways to express the molar m
18、ass are as the number average (Mn) and the mass average (Mw).The two averages are defined by the following equations:Mn 5(iNiMi(iNiand Mw 5(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 Disc
19、ussionIn a polydispersedisperse molecular population the relation Mw Mn is always valid. The coefficientratio Mw /Mn is referredto as the polydispersity index, dispersity, and will typically be in the range 1.5 to 3.0 for commercial chitosans.NOTE 1The term molecular weight (abbreviated MW) is obsol
20、ete and should be replaced by the SI (Systme Internationale) equivalent of eitherrelative molecular mass (Mr), which reflects the dimensionless ratio of the mass of a single molecule to an atomic mass unit (see ISO 31-8),80000-9:2009),or molar mass (M), which refers to the mass of a mole of a substa
21、nce 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 NI
22、ST SP811.4. Significance and Use4.1 The degree of deacetylation of chitosan, as well at the molar mass and molar mass distribution, determines the functionalityof chitosan in an application. For instance, functional and biological effects are highly dependent upon the composition and molarmass of th
23、e polymer.4.2 This test method describes procedures for measurement of molar mass of chitosan chlorides and glutamates, and chitosanbase, although it in principle applies to any chitosan salt. The measured molar mass is that for chitosan acetate, since the mobilephase contains acetate as counter ion
24、. This value can further be converted into the corresponding molar mass for the chitosan asa 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 isapplicable over the broade
25、st range of molar masses of any method. Combining light scattering detection with size exclusionchromatography (SEC), which sorts molecules according to size, gives the ability to analyze polydisperse samples, as well asobtaining information on branching and molecular conformation. This means that b
26、oth the number-average and mass-averagevalues for molar mass and size may be obtained for most samples. Furthermore, one has the ability to calculate the distributionsof the molar masses and sizes.4.4 Multi-angle laser light scattering (MALS) is a technique where measurements of scattered light are
27、made simultaneouslyover a range of different angles. MALS detection can be used to obtain information on molecular size, since this parameter isdetermined by the angular variation of the scattered light. Molar mass may in principle be determined by detecting scattered lightat a single low angle (LAL
28、LS). However, advantages with MALS as compared to LALLS are: (1) less noise at larger angles, (2)the precision of measurements are greatly is improved by detecting at several angles, and (3) the ability to detect angular variationallows determination of size, branching, aggregation, and molecular co
29、nformation.4.5 Size exclusion chromatography uses columns, which are typically packed with polymer particles containing a network ofuniform pores into which solute and solvent molecules can diffuse. While in the pores, molecules are effectively trapped andremoved from the flow of the mobile phase. T
30、he 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 areeluted first, in the void volume of the column. Molecules, which may penetrate the por
31、es will have a larger volume available fordiffusion, they will suffer retention depending on be retained in the column for a time dependent upon their molecular size, withthe smaller molecules eluting last.after larger molecules.F2602 1824.6 For polyelectrolytes, dialysis against the elution buffer
32、has been suggested, in order to eliminate Donnan-type artifacts inthe molar mass determination by light scattering (1, 2).5 However, in the present method, the size exclusion chromatography steppreceding the light scatter detection is an efficient substitute for a dialysis step. The sample is separa
33、ted on SEC columns with largeexcess of elution buffer for 30 to 40 min, and it is therefore in full equilibrium with the elution buffer when it reaches the MALSdetector.5. Materials5.1 Chemicals:5.1.1 Chitosan or chitosan salt sample.5.1.2 Deionized water (Milli-Q Plus or equivalent; conductivity 10
34、0 0.5 0.1A Injected mass = Concentration*200 L.F2602 1836.2.2 Dissolve the chitosan base in 1 % 1 % (volume/volume) acetic acid to a 1 % 1 % (weight/volume) solution by shakingat about 100 min-1 overnight at cool temperature (3 to 8C).6.2.3 Dilute samples in mobile phase (Note2(2 mol/L ammonium acet
35、ate, not stock solution) to the required concentration(Table 3) and shake gently for a few seconds.6.2.4 Filter all samples through a 0.45 m filter, and transfer to HPLC vials.6.3 Chromatography and Data Collection:6.3.1 The complete experimental setup of the SEC-MALS system is shown in Fig. 1. The
36、refractive index detector is placedat the end of the solvent/sample line as it is highly sensitive to pressure changes.6.3.2 Pullulan standards should be injected and analyzed with 2 replicates before and after all chitosan samples (total of 4replicates). Three (3) replicates should be injected for
37、chitosans.6.3.3 A procedure for setting up the chromatography run and collecting the data is given below:6.3.3.1 Use a flow rate of 0.5 mL/min.6.3.3.2 Purge the injector with mobile phase before the sample set is run.TABLE 2 Suggestions for Concentration and Injected Mass ofChitosan Glutamate Sample
38、s for SEC-MALSApparent Viscosityas Chitosan Glutamate(mPas)Concentration forInjection(mg/mL)Injected MassA(mg)50 0.75 0.15A Injected mass = Concentration*200 L.TABLE 3 Suggestions for Concentration and Injected Mass ofChitosan Base Samples for SEC-MALSApparent Viscosityas Chitosan Acetate(mPas)Conce
39、ntration forInjection(mg/mL)InjectedMassA(mg)500 0.375 0.075A Injected mass = Concentration*200 L.NOTE 1Solid lines indicate solvent/sample flow, dashed lines indicate cabling for data transfer.FIG. 1 Complete SEC-MALS Set-UpF2602 1846.3.3.3 Purge the RI-detector for at least 30 min (at 0.5 mL/min)
40、before start of the run.6.3.3.4 Confirm that both the MALS detector and RI detector has a have stable and low baseline level.levels.6.3.3.5 Define the collection set-up as follows:(1) Inject 200 L of sample.(2) After a collection delay of 10 mL (20 min), data should be collected from both detectors
41、every 2 seconds for 40 mL (80min).(3) Use dn/dc = 0.148 mL/g and 0.142 mL/g for pullulans and chitosans, respectively (relevant only for calculations).(4) Use a second virial coefficient of 2*10-4 mol.mL.g-2 and 5*10-3 mol.mL.g-2 for pullulans and chitosans, respectively(relevant only for calculatio
42、ns).6.3.4 After all samples have been run, purge the injector with deionized water to wash off remaining salt from the valves.6.4 Data Analysis:6.4.1 Data analysis follows closely recommended procedures for SEC-MALS data. Generally, the chromatograms are dividedinto a number of volume elements, defi
43、ned by the peak width, the rate of data collection and the flow rate. Concentration Theconcentration of the sample in each volume element (ci) is determined from the RI-detector response using known values of dn/dcand dn/dV (the RI-detector calibration constant). Furthermore, LS-detector response is
44、 divided by c, the molar mass in each volumeelement (Mi) is considered monodisperse, and the mass is determined from a Zimm representation of a Debye plot by extrapolationto zero angle (which is essentially a solution to Eq X2.1 in X2.2). Once the values of ci and Mi are known, calculation of the va
45、riousaverage molar masses is straightforward.6.4.2 In detail, the above procedure consists of the following operations to be performed in a suitable software:6.4.2.1 Define baselines for signals from both detectors.6.4.2.2 Calculate inter-detector delay volume using a monodisperse low-molar mass pul
46、lulan standard.6.4.2.3 Define the peak area of interest.6.4.2.4 Normalize LS-detector responses to correct for different sensitivity at different angles. Normalization is performed onan isotropic scatterer (low molar mass compound) in the sample set, and is saved with the data file. For the other sa
47、mples, onereads the normalization performed on an isotropic scatterer from file.6.4.2.5 Check the goodness-of-fit of the LS-detectors using a 3-D representation of the data or a Debye-plot (in Zimmrepresentation). Do not use LS-detector responses that are clearly non-linear.6.4.2.6 Perform the requi
48、red calculations for determination of Mn, Mw and Mw/Mn, using a Zimm representation of the Debyeplot (that is, a plot of K*c/R() versus sin2/2) for solving Eq X2.1.6.4.3 Conversion of Mw-valuesThe mobile phase is 0.2 mol/L ammonium acetate, and the Mw determined according to thismethod is that of ch
49、itosan acetate Mw(Ac). This mass average molar mass can be converted to that of chitosan chlorideMw(Cl), glutamate Mw(G) or base Mw(B) according to Eq 2-4the following equations:MwG!5MwAc!*%DA*MG!112%DA!*MA!/%DA*MAc!112%DA!*MA! (2)M wG! 5M wAc! *S DDA *MG!11 2 DDA!*MA!DDA *MAc!11 2 DDA!*MA!D (2)NOTE 1Solid line: RI detector; dashed line: MALS detector; () molar mass for each chromatographic data point.FIG. 2 A Chromatogram of Chitosan Chloride (Mw of 360 000 g/mol, as acetate)F2602 185M
