1、Designation: F 2605 08Standard Test Method forDetermining the Molar Mass of Sodium Alginate by SizeExclusion Chromatography with Multi-angle Light ScatteringDetection (SEC-MALS)1This standard is issued under the fixed designation F 2605; the number immediately following the designation indicates the
2、 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 determination of the m
3、olarmass of sodium alginate intended for use in biomedical andpharmaceutical applications as well as in tissue engineeredmedical products (TEMPs) by size exclusion chromatographywith multi-angle laser light scattering detection (SEC-MALS).A guide for the characterization of alginate has been publish
4、edas Guide F 2064.1.2 Alginate used in TEMPs should be well characterized,including the molar mass and polydispersity (molar massdistribution) in order to ensure uniformity and correct func-tionality in the final product. This test method will assist endusers in choosing the correct alginate for the
5、ir particularapplication. Alginate may have utility as a scaffold or matrixmaterial for TEMPs, in cell and tissue encapsulation applica-tions, and in drug delivery formulations.1.3 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsi
6、bility 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 2064 Guide for Characterization and Testing of Alginatesas Starting Materials Intended for Us
7、e in Biomedical andTissue-Engineered Medical Products ApplicationF 2315 Guide for Immobilization or Encapsulation of Liv-ing Cells or Tissue in Alginate Gels2.2 United States Pharmacopeia/National Formulary:3Chromatography2.3 National Institute of Standards and Technology:4NIST SP811 Special Publica
8、tion: Guide for the Use of theInternational System of Units3. Terminology3.1 Definitions:3.1.1 alginate, na polysaccharide substance extractedfrom brown algae, mainly occurring in the cell walls andintercellular spaces of brown seaweed and kelp. Its mainfunction is to contribute to the strength and
9、flexibility of theseaweed plant. Sodium alginate, and in particular calciumcross-linked alginate gels are used in tissue engineered medicalproducts (TEMPs) as biomedical scaffolds and matrices, forimmobilizing living cells (see Guide F 2315) and in drugdelivery systems.3.1.2 molar mass average, nthe
10、 given molar mass (Mw)of an alginate 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(iwi
11、5(iNiMi2(iNiMi(1)where:Ni= number of molecules having a specific molar mass Mi,andwi= mass of molecules having a specific molar mass Mi.3.1.2.1 DiscussionIn a polydisperse molecular populationthe relation Mw Mnis always valid. The coefficient Mw/Mnisreferred to as the polydispersity index, and will
12、typically be inthe range 1.5 to 3.0 for commercial alginates.NOTE 1The term molecular weight (abbreviated MW) is obsolete andshould be replaced by the SI (Systme Internationale) equivalent of eitherrelative molecular mass (Mr), which reflects the dimensionless ratio of themass of a single molecule t
13、o an atomic mass unit (see ISO 31-8), or molar1This test method is under the jurisdiction of ASTM 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. Publis
14、hed May 2008.2For referenced ASTM standards, visit the ASTM website, www.astm.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 N
15、ational Formulary, U.S.Pharmaceutical Convention, Inc. (USPC), Rockville, MD.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.1Copyright ASTM International, 100 Barr Harbor
16、 Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.mass (M), which refers to the mass of a mole of a substance and istypically expressed as grams/mole. For polymers and other macromol-ecules, use of the symbols Mw, Mn, and Mzcontinue, referring tomass-average molar mass, number-ave
17、rage 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 composition and sequential structure of alginate, aswell as the molar mass and molar mass distribution, determinesthe functionality of
18、 alginate in an application. For instance, thegelling properties of an alginate are highly dependent upon thecomposition and molar mass of the polymer.4.2 Light scattering is one of very few methods available forthe determination of absolute molar mass and structure, and itis applicable over the bro
19、adest range of molar masses of anymethod. Combining 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
20、 both the number-average andmass-average values for molar mass and size may be obtainedfor most samples. Furthermore, one has the ability to calculatethe distributions of the molar masses and sizes.4.3 Multi-angle laser light scattering (MALS) is a techniquewhere measurements are made simultaneously
21、 over a range ofdifferent angles. MALS detection can be used to obtaininformation on molecular size, since this parameter is deter-mined by the angular variation of the scattered light. Molarmass may in principle be determined by detecting scatteredlight at a single low angle (LALLS). However, advan
22、tages withMALS as compared to LALLS are: (1) less noise at largerangles, (2) the precision of measurements are greatly improvedby detecting at several angles, and (3) the ability to detectangular variation allows determination of size, branching,aggregation, and molecular conformation.4.4 Size exclu
23、sion chromatography uses 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
24、 the pores depends upon 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 av
25、ailable for diffusion, they will suffer retentiondepending on their molecular size, with the smaller moleculeseluting last.4.5 For polyelectrolytes, dialysis against the elution bufferhas been suggested, in order to eliminate Donnan-type artifactsin the molar mass determination by light scattering (
26、1, 2).5However, in the 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
27、the elutionbuffer when it reaches the MALS detector.5. Materials5.1 Chemicals:5.1.1 Alginate sample.5.1.2 Deionized water (Milli-Q Plus or equivalent; conduc-tivity 250 000 300 0.5 0.1AInjected mass = Concentration*200 L.NOTESolid lines indicate solvent/sample flow, dashed lines indicate cabling for
28、 data transfer.FIG. 1 Complete SEC-MALS Set-UpF26050836.3.2.4 Normalize LS-detector responses to correct for dif-ferent sensitivity at different angles. Normalization is per-formed on an isotropic scatterer (low molar mass compound) inthe sample set, and is saved with the data file. For the othersam
29、ples, one reads the normalization performed on an isotropicscatterer from file.6.3.2.5 Check the goodness-of-fit of the LS-detectors usinga 3D-representation of the data or a Debye-plot (in Zimmrepresentation). Do not use LS-detector responses that areclearly non-linear.6.3.2.6 Perform the required
30、calculations for determinationof Mn, Mwand Mw/Mn, using a Zimm representation of theDebye plot (that is, a plot of K*c/R(u) versus sinu2/2) forsolving Eq X2.1.7. Control and Approval of Data7.1 The number average value of Mw, and Mw/Mnfor thereplicates of each sample (4 replicates for pullulan stand
31、ards, 3replicates for alginates) should 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
32、 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 alginate samples should bemanually evaluated. Different curve forms
33、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.7.4 Failure of condition 3 requires reanalysis of the alginatesample
34、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 units of g/mol, for example,160 000 g/mol.NOTESolid line: RI detect
35、or; dashed line: MALS detector; (L) molar mass for each chromatographic data point.FIG. 2 A Chromatogram of Sodium Alginate (Mw160 000 g/mol)F2605084APPENDIXES(Nonmandatory Information)X1. RATIONALEX1.1 The use of naturally occurring biopolymers forbiomedical and pharmaceutical applications and in t
36、issueengineered medical products (TEMPs) is increasing. This testmethod is designed to give guidance in the characterization ofsodium alginate used in such applications.X2. BACKGROUNDX2.1 Alginate is a family of non-branched binary copoly-mers of 1-4 glycosidically linked b-D-mannuronic acid (M)and
37、a-L-guluronic acid (G) residues. The relative amount ofthe two uronic acid monomers and their sequential arrange-ment along the polymer chain vary widely, depending on theorigin of the alginate. The uronic acid residues are distributedalong the polymer chain in a pattern of blocks, where ho-mopolyme
38、ric blocks of G residues (G-blocks), homopolymericblocks of M residues (M-blocks) and blocks with alternatingsequence of M and G units (MG-blocks) co-exist. It has alsobeen shown by NMR spectroscopy that alginate has no regularrepeating unit.X2.2 The principles of SEC-MALS can be summarized asfollow
39、s: 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 proportional tocM, where M is molar mass (or the mass average molar mass,Mw, for
40、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 the heart of thisanalysis:K*c/Ru! 5 1/$Mw*Pu!% 1 2A2c (X2.1)X2.2.1 The excess Rayle
41、igh 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/l04NA,where n0is the refractive index of the solvent, l0is the vacuumwavelength of i
42、ncident light, and NAis Avogadros number.Finally, 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 square radius of themolecules may be determined.X2.2.2 Eq X2.1 is typically solved using a
43、 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 volume element. By extrapolating the Debyeplot to zero angle, the intercept yields the mass d
44、irectly. 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 representation of the Debye plot may bepreferable for macromolecules like alginates and chitosans
45、,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 P. J. Wyatt (3).FIG. X2.1 Structure of AlginateF2605085X2.2.3 No molar mass standards are
46、 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, calcula
47、tion of the massaverage molar mass is given by Eq X2.2:Mw5(iwiMi/(iwi5(iciMi/(ici(X2.2)X3. CONSTANT VALUESX3.1 Calibration and Normalization of MALS-detectorAMALS detector typically incorporates several photoreceptorsin a planar circular arrangement around a flow cell, with laserlight passing down t
48、he bore of the flow cell. Calibration of theMALS detector is required to establish a correct reading,typically for the 90 detector, using a well-defined isotropicscatterer (toluene is commonly employed). Calibration shouldbe done following the instrument manufacturers recom-mended procedures, and ch
49、ecked for consistency and agree-ment with instrument manufacturers expected value. Further-more, normalization of the other photoreceptors to the 90detector is required to reduce errors caused by differences insensitivities of each detector and difference in distance betweenthe sample and each detector. Normalization is typically doneusing an isotropic scatterer in the same solvent as the unknownsamples to be analyzed. Pullulan standards are common in usein aqueous solvents. Bovine serum albumin (BSA) is anothercommonly used isotropic scatter
