1、Designation: F2605 16Standard 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 F2605; the number immediately following the designation indicates the y
2、ear 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 of the mola
3、rmass (typically expressed as grams/mole) of sodium alginateintended for use in biomedical and pharmaceutical applicationsas well as in tissue-engineered medical products (TEMPs) bysize exclusion chromatography with multi-angle laser lightscattering detection (SEC-MALS). A guide for the character-iz
4、ation of alginate has been published as Guide F2064.1.2 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 cho
5、osing the correct alginate for their particularapplication. Alginate may have utility as a scaffold or matrixmaterial 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 of measurem
6、ent 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 regulatory limitat
7、ions prior to use.2. Referenced Documents2.1 ASTM Standards:2F2064 Guide for Characterization and Testing of Alginatesas Starting Materials Intended for Use in Biomedical andTissue Engineered Medical Product ApplicationsF2315 Guide for Immobilization or Encapsulation of LivingCells or Tissue in Algi
8、nate 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 Units2.4 ISO Standards:5ISO 31-8 Quantities and units- Part 8: Physical chemistryand molecular
9、 physics3. 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 flexibility of theseaweed plant. Sodium alginate, a
10、nd in particular calciumcross-linked alginate gels are used in tissue-engineered medicalproducts (TEMPs) as biomedical scaffolds and matrices, forimmobilizing living cells (see Guide F2315), and in drugdelivery systems.3.1.2 molar mass average, nthe given molar mass (Mw)of an alginate will always re
11、present 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)1This test method is under the jur
12、isdiction 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 May 1, 2016. Published June 2016. Originallyapproved in 2008. Last previous edition approved in 2008
13、 as F2605 081. DOI:10.1520/F2605-16.2For 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 St
14、ates Pharmacopeia and National 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.5Available from Interna
15、tional Organization for Standardization (ISO), ISOCentral Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier,Geneva, Switzerland, http:/www.iso.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1where:Ni= number of mo
16、lecules 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 typically be inthe range 1.5 to 3.0 for
17、 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 to an atomic mass unit (see ISO 31-8), o
18、r 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, Mn, and Mzcontinue, referringto mass-average molar mass, number-average molar mass, and z-averagemolar mass, respectively. For more
19、 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 alginate in an application. For instance, thegelling properties
20、 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 broadest range of molar masses of anymethod. Combining light scatte
21、ring detection with size exclu-sion chromatography (SEC), which sorts molecules accordingto size, gives the ability to analyze polydisperse samples, aswell as to obtain information on branching and molecularconformation. This means that both the number-average andmass-average values for molar mass a
22、nd 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 over a range ofdifferent angles and used to determine the scatt
23、ering at 0,which directly relates to molecular weight. MALS detectioncan be used to obtain information on molecular size, since thisparameter is determined by the angular variation of thescattered light. This can be related to branching, aggregation,and molecular conformation. Molar mass can also be
24、 deter-mined by detecting scattered light at a single low angle (LALS)and assuming that this is not significantly different from thescattering at 0.4.4 Size exclusion chromatography uses columns, which aretypically packed with polymer particles containing a networkof uniform pores into which solute
25、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 sizeof the packing are excluded a
26、nd experience virtually noretention; these are eluted first, in the void volume of thecolumn. Molecules which penetrate the pores will have a largervolume available for diffusion; their retention will depend ontheir molecular size, with the smaller molecules eluting last.4.5 For polyelectrolytes, di
27、alysis against the elution bufferhas been suggested, in order to eliminate Donnan-type artifactsin the molar mass determination by light scattering (1, 2).6However, in the present method, the size exclusion chroma-tography step preceding the light scatter detection is anefficient substitute for a di
28、alysis 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 Deionized water (Milli-Q Plus or equivalent;
29、conduc-tivity 250 000 300 0.5 0.1AInjected mass = Concentration*200 L.F2605 1637.4 Failure of condition 3 requires reanalysis of the alginatesample in question, only.8. Precision and Reporting Results8.1 The precision/relative standard error (RSE) of themethod is 10 %, as shown in method validation.
30、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.NOTE 1Solid lines indicate solvent/sample flow, dashed lines indicate cabling for data transfer.FIG. 1 Complete SEC-MALS SetupNOTE 1Solid line: RI detector; dashed line: MALS de
31、tector; () molar mass for each chromatographic data point.FIG. 2 A Chromatogram of Sodium Alginate (Mw160 000 g mol)F2605 164APPENDIXES(Nonmandatory Information)X1. RATIONALEX1.1 The use of naturally occurring biopolymers forbiomedical and pharmaceutical applications and in tissue-engineered medical
32、 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 -D-mannuronic acid (M)and -L-guluronic acid (G) re
33、sidues. 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
34、(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 nuclear magnetic resonance (NMR) spectros-copy that alginate has no regular repeating unit.X2.2 The principles of SEC-MALS can be summarized as
35、follows: 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
36、, for non-fractionated polydisperse samples). Using aconcentration (for example, refractive index) detector to mea-sure 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 Ray
37、leigh ratio R() is the light scatteredby the solution at an angle in excess of that scattered by puresolvent, divided 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 incid
38、ent light, and NAis Avogadros number.Finally, P() 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 Deby
39、e plot (thatis, plotting R()/(K*ci) versus sin2(/2), where the sin2(/2)term results from an expansion of P(), 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.
40、TheDebye plot is also commonly performed using a Zimmrepresentation (that is, plotting (K*ci)/R() versus sin2(/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
41、 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 Column calibration molecular weight standards arenot required in the analysis.
42、Only a set of fundamental ormeasured constants (,n0,(dn/dc), 0,NA,A2) and a set ofexperimentally measured values (c,R(), P(), detector cali-bration constant) are required to calculate the molar mass.FIG. X2.1 Structure of AlginateF2605 165X2.3 Once the sample has been fractionated and ciand Mihave b
43、een 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 Normalization of MALS-detectorAMALS detector typically incorporates several photoreceptorsin a planar circular arrangement around a f
44、low cell. Calibrationof the MALS detector is required to establish a correct reading,typically for the 90 detector, using a well-defined isotropicscatterer (toluene or a NIST-traceable molecular weight stan-dard can be used). Calibration should be done following theinstrument manufacturers recommend
45、ed procedures, andchecked for consistency and agreement with the instrumentmanufacturers expected value. Furthermore, normalization ofthe other photoreceptors to the 90 detector is required toreduce errors caused by differences in sensitivities of eachdetector and the difference in distance between
46、the sample andeach detector. Normalization is typically done using an isotro-pic scatterer in the same solvent as the unknown samples to beanalyzed. Pullulan standards are commonly used in aqueoussolvents. Bovine serum albumin (BSA) is another commonlyused isotropic scatterer for normalization in aq
47、ueous solvent.X3.2 RI-detector Calibration ConstantIn 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
48、 beperformed using a concentration series of samples 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
49、 for sodium alginatein aqueous 0.1M NaCl at 633 nm. No 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) 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 Coeff
