1、Designation: F 2605 081Standard 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 th
2、e 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.1NOTESubsection 6.1.5 was editorially corrected in September 2
3、008.1. Scope1.1 This test method covers the determination of the molarmass 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-M
4、ALS).A guide for the characterization of alginate has been publishedas 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 me
5、thod will assist endusers in choosing the correct alginate for their 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 The values stated in SI units are to be regarded asst
6、andard. No other units 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 ap
7、plica-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 Use in Biomedical andTissue-Engineered Medical Products ApplicationF 2315 Guide for Immobilization or Encapsulat
8、ion 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 Publication: Guide for the Use of theInternational System of Units3. Terminology3.1 Definitions:3.1.1 alginate, na po
9、lysaccharide 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 flexibility of theseaweed plant. Sodium alginate, and in particular calciumcross-linked alginate gels are used
10、 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 given molar mass (Mw)of an alginate will always represent an average of all of themolecules in the population
11、. 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 molecules having a specific molar mass Mi,andwi= mass of molecules having
12、 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 typically be inthe range 1.5 to 3.0 for commercial alginates.1This test method is under the jurisdiction of AS
13、TM 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 AS
14、TM 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 Na
15、tional 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.NOTE 1The term molecular w
16、eight (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 to an atomic mass unit (see ISO 31-8), or molarmass (M), which refers to the mass of a m
17、ole 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-average molar mass, and z-averagemolar mass, respectively. For more information regarding proper utiliza-tion of
18、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 alginate in an application. For instance, thegelling properties of an alginate are highly dependent upon thec
19、omposition 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 broadest range of molar masses of anymethod. Combining light scattering detection with size exclu-sion chromatogr
20、aphy (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. Furth
21、ermore, 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 over a range ofdifferent angles. MALS detection can be used to obtaininformation on molecular size, since thi
22、s 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, advantages withMALS as compared to LALLS are: (1) less noise at largerangles, (2) the precision of measurements are
23、 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 exclusion chromatography uses columns, which aretypically packed with polymer particles containing a networkof unif
24、orm 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 average pore sizeo
25、f 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 smaller molecules
26、eluting 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 (1, 2).5However, in the present method, the size exclusion chroma-tography step preceding the light scatter det
27、ection 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 Alginate sample.5.1.2 Deio
28、nized water (Milli-Q Plus or equivalent; conduc-tivity 250 000 300 0.5 0.1AInjected mass = Concentration*200 L.F26050813NOTESolid lines indicate solvent/sample flow, dashed lines indicate cabling for data transfer.FIG. 1 Complete SEC-MALS Set-UpNOTESolid line: RI detector; dashed line: MALS detector
29、; (L) molar mass for each chromatographic data point.FIG. 2 A Chromatogram of Sodium Alginate (Mw160 000 g/mol)F26050814APPENDIXES(Nonmandatory Information)X1. RATIONALEX1.1 The use of naturally occurring biopolymers forbiomedical and pharmaceutical applications and in tissueengineered medical produ
30、cts (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 a-L-guluronic acid (G) residu
31、es. 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-mopolymeric blocks of G residues (G-b
32、locks), 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 asfollows: Samples of polymer are inj
33、ected 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 non-fractionated polydisperse
34、 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 Rayleigh ratio R(u) is the light s
35、catteredby 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 incident light, and NAis Avoga
36、dros 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 Debye plot (thatis, plotting
37、 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 directly. TheDebye plot is als
38、o 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,since only linear fits to ze
39、ro 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 AlginateF26050815X2.2.3 No molar mass standards are required in the analy-sis.
40、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 mola
41、r 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 the bore of the flow cell. Ca
42、libration 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 checked for consistency and ag
43、ree-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 typica
44、lly 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 scatterer for normalization in aque-ous solvent.X3.2 RI-detector Calibration ConstantIn
45、 the experimen-tal setup described herein, the RI-detector is used as anabsolute concentration detector. Depending on the type ofdetector, one may need to calibrate the analog output of thedetector to refractive index (dn/dV). Such calibration can beperformed using a concentration series of samples
46、with knownrefractive index increment (dn/dc), and recording the voltageoutput at each concentration. NaCl is commonly used forcalibration of RI detectors.X3.3 Refractive Index Increment, dn/dcMartinsen et al(4) found a value of dn/dc of 0.150 mL/g for sodium alginatein aqueous 0.1M NaCl at 633 nm. N
47、o uncertainty was reportedfor this value, but it was reported to be in accordance withprevious literature data. For pullulan in aqueous solvent, avalue 0.148 mL/g is suggested according to Nordmeier (5).Values of (dn/dc)(dn/dc measured on dialyzed samples, forexample, at constant chemical potential)
48、 can be determined bymeasuring the refractive index of a concentration series ofsamples on a RI-detector with known calibration constant(dn/dV).X3.4 Second Virial Coeffcient, A2A2is a measure ofmacromolecular self-association and deviation from ideality ofinfinitely dilute solutions. Martinsen et al
49、 (4) found values of1.36*10-3mol.mL.g-2to 7*10-3mol.mL.g-2using wide angleand low angle laser light scattering on sodium alginates ofdifferent sources and chemical composition. A value of 5*10-3mol.mL.g-2appears to be a reasonable average value forsodium alginates, with an uncertainty of 650 %. Nordmeier(5) reported second virial coefficient of 2*10-4mol.mL.g-2forpullulan. A2-values can be determined by light scatteringexperiments in batch mode, and by constructing a classicalZimm plot to fit and extrapolate data to zero concentration andzero
