1、Designation: D 5296 05Standard Test Method forMolecular Weight Averages and Molecular WeightDistribution of Polystyrene by High Performance Size-Exclusion Chromatography1This standard is issued under the fixed designation D 5296; the number immediately following the designation indicates the year of
2、original 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. Scope*1.1 This test method covers the determination of molecularwei
3、ght (MW) averages and the distribution of molecularweights for linear, soluble polystyrene by liquid high-performance size-exclusion chromatography (HPSEC). Thistest method is not absolute and requires the use of commer-cially available narrow molecular weight distribution (MWD)polystyrene standards
4、 for calibration. This test method isapplicable for samples containing molecular weight compo-nents that have elution volumes falling within the elutionvolume range defined by polystyrene standards (that is, mo-lecular weights generally from 2000 to 2 000 000 gmol1).1.2 The HPSEC is differentiated f
5、rom traditional size-exclusion chromatography SEC (also referred to as gel perme-ation chromatography (GPC) in that the number of theoreticalplates per metre with an HPSEC system is about ten timesgreater than that for traditional SEC (see Terminology D 883and Practice D 3016).2The HPSEC systems emp
6、loy low-volume liquid chromatography components and columnspacked with relatively small (generally 3 to 20 m) mi-croporous particles. High-performance liquid chromatographyinstrumentation and automated data handling systems for dataacquisition and processing are required.1.3 The values stated in SI
7、units are to be regarded as thestandard.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
8、limitations prior to use. Specific precau-tionary statements are given in Section 9.NOTE 1There is no similar or equivalent ISO standard.2. Referenced Documents2.1 ASTM Standards:3D 883 Terminology Relating to PlasticsD 2857 Test Method for Dilute Solution Viscosity of Poly-mersD 3016 Practice for U
9、se of Liquid Exclusion Chromatogra-phy Terms and RelationshipsE 685 Practice for Testing Fixed-Wavelength PhotometricDetectors Used in Liquid ChromatographyE 691 Practice for Conducting an Interlaboratory Test Pro-gram to Determine the Precision of Test Methods3. Terminology3.1 DefinitionsFor defini
10、tions of technical terms pertain-ing to plastics used in this test method see Terminology D 883.4. Summary of Test Method4.1 In this test method a dilute solution of a polystyrenesample is injected into a liquid mobile phase containing thesame solvent used to prepare the polymer solution. The mobile
11、phase transports the polymer into and through a chromato-graphic column (or set of columns connected in series) packedwith a solid or semirigid, porous substrate which separates thepolymer molecules according to their size in solution. Startingfrom injection, a detector continuously monitors the elu
12、ate as afunction of elution volume (or time). Upon emerging from thecolumn(s), the size-separated molecules are detected andrecorded according to their concentration. Through calibration,the elution volumes (or times) are converted to molecularweights, and various molecular weight parameters for the
13、sample are calculated from the molecular weight/concentrationdata.1This test method is under the jurisdiction ofASTM Committee D20 on Plasticsand is the direct responsibility of Subcommittee D20.70 on Analytical Methods.Current edition approved March 1, 2005. Published March 2005. Originallyapproved
14、 in 1992. Last previous edition approved in 1997 as D 5296 - 97.2See also AMD Bibliography and Bibliography Supplements AMD 40-S1, 40-S2,and 40-S3 on Size Exclusion Chromatography, available fromASTM Headquarters.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Cu
15、stomer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1*A Summary of Changes section appears at the end of this standard.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohock
16、en, PA 19428-2959, United States.5. Significance and Use5.1 General UtilityThe molecular weight (MW) and mo-lecular weight distribution (MWD) are fundamental character-istics of a polymer sample. They may be used for a widevariety of correlations for fundamental studies, processing, orproduct applic
17、ations. For example, the observed MWD may becompared to one predicted from assumed kinetics or mecha-nisms for a polymerization reaction. Differences between thevalues will allow alteration of theory or experimental design.Similarly, the strength, melt flow, and other properties of apolymer sample m
18、ay be dependent on MW and MWD.Determinations of MW and MWD are used for quality controlof polymers.5.2 LimitationsBecause of the need for specific calibra-tion of the polymer type under study, and because of thespecific nature of polymer/solvent/column-packing interac-tions, this test method is vali
19、d only for polystyrene andnon-exclusion effects are to be avoided. However, many of theprinciples of the method may be applied in generating HPSECmethods for other polymer systems, for example, using theprinciples of universal calibration. (see Practice D 3016).6. Units and Symbols6.1 Units and symb
20、ols related to function are given in Table1.6.2 Equivalencies used in this test method are as follows:Common Unit/Symbol SI Unit or Symbol1 mLmin1= 1.667 3 108m3s11 3 107dyncm2= 145 psi = 1 MPa7. Apparatus7.1 IntroductionLiquid high-performance size-exclusionchromatography (HPSEC) is a specific form
21、 of liquid chroma-tography and is differentiated from traditional SEC in thatHPSEC uses columns with about ten times the number oftheoretical plates per metre. The principal distinguishing fea-ture of HPSEC is the column packing material that is discussedas follows.7.2 Essential ComponentsThe essent
22、ial components ofinstrumentation are a solvent reservoir, solvent pumping sys-tem, sample injector, packed column(s), solute detector, lowdead-volume liquid chromatography tubing and fittings, wastecontainer, recorder, and an automated data-handling system.Any component may be used that meets safety
23、 and perfor-mance requirements specified as follows.7.2.1 The interrelationships of the components are shownschematically in Fig. 1. For instruments that have injector,column(s), detector, or other components operated aboveambient temperature, the use of a degasser located in thesolvent reservoir or
24、 between the reservoir and pumping systemis recommended to remove air from the solvent. Typicallaboratory glassware and an analytical balance are also needed.NOTE 2Anumber of systems and components for performing HPSECare available commercially.7.3 Solvent ReservoirThe solvent reservoir must holdsuf
25、ficient solvent to ensure consistency of composition for anumber of runs or analyses. The reservoir should isolate thesolvent from the atmosphere, permit control of the environ-ment in contact with the solvent, and be completely inert to thesolvent employed. In addition, some means of agitation (for
26、example, magnetic stirring) is recommended to ensure uniformcomposition.7.4 Solvent Pumping SystemThe principal requirement ofa pumping system is production of a relatively constant andpulseless flow of solvent through the columns. In general, therate of flow should be adjustable between 0.1 and 5.0
27、 mL/minand back-pressures should not exceed limits specified by thecolumn manufacturer (for example, 28 MPa). If the elutionvolume is not being measured directly or corrected for system-atic changes, the precision in the flow rate must be at least60.3 % as measured under the conditions and over the
28、timeinterval required for running a typical analysis.7.5 Sample InjectorThe purpose of an injection system isto generate a sharply defined zone of solution containing thesample when introducing the sample into the flow stream. Avalve and loop assembly or any of a number of commerciallyavailable high
29、-performance liquid chromatography automaticinjection systems can be used for this purpose. Requirementsinclude minimal contribution to band spreading, injector abilityto operate at the back-pressure generated by the columns,repeatability of injection volume, and no carryover.7.6 ColumnsStainless st
30、eel columns with uniform andhighly polished inside walls are usually selected for HPSEC.Columns with lengths ranging from 15 to 50 cm and specialend fittings, frits, and connectors designed to minimize deadvolume and mixing are recommended. Micro-particulate, semi-rigid organic gels, and rigid solid
31、, porous packing materials areused for HPSEC. Generally, the packing materials have narrowparticle size distributions with particle sizes in the range from3 to 20 m. Packing materials also are available in a variety ofshapes and pore sizes. Columns may be packed with particlesof relatively uniform p
32、ore size or with a “mixed bed” ofparticles to produce a broad range of pore sizes for polymerseparation. If a set of columns is used, it is recommended thatthe columns be connected starting from the injector outlet inTABLE 1 Units and Symbols Related to FunctionFunctionCommon Unit/SymbolSI Unit/Symb
33、olBasic property definition Molecular weight (Daltons) gmol1Solvent flow rate mLmin1m3s1Sample weight (mass) mgASample solution volume L, mLAPore size AParticle Size mAElution volume L, mLAElution time sAChromatogram peakheightsmmAColumn back pressure dyncm2(psi) Nm2or pascal (Pa)ASame as common uni
34、t. FIG. 1 Schematic of an HPSEC SystemD5296052order of columns having the smallest to those having thelargest packing pore size.NOTE 3Column packing materials and packed HPSEC columns areavailable commercially from a number of manufacturers. Users of this testmethod are advised to follow manufacture
35、rs guidelines and recommen-dations for the care and use of their HPSEC columns. For example,manufacturers guidelines may override the preceding recommendationfor ordering the placement of columns in a column set because of concernabout the fragility of smaller pore size packing materials.7.7 Detecto
36、rsThe purpose of the detector is to continu-ously monitor the concentration of solute eluting from thechromatographic column(s). Consequently, the detector mustbe sufficiently sensitive and respond linearly to the soluteconcentration. Additionally, the detector must not appreciablydistort the concen
37、tration gradient in the emerging stream. Thisrequirement imposes severe limitations on the volume ofsolution available for detection. For example, use of detectorswith cell volumes greater than 15 L generally will not beaccepted with this test method. Most detectors employed forHPSEC are based upon
38、photometric measurements (refractiveindex, UV-visible, fluorescence and infrared absorbance). Prac-tice E 685 serves as a guide for testing the performance ofphotometric detectors used in high-performance liquid chro-matographic systems. Other detectors also may be used. Thedifferential refractomete
39、r has moderate sensitivity and generalutility. It provides a signal proportional to the difference inrefractive index (DRI) between the solvent and the columneluate. The detector should respond to 107to 108DRI unitwith cell volumes # 10 L.NOTE 4The change in the specific refractive index increment (
40、dn/dc)of polystyrene is negligible at molecular weights greater than about 5000gmol1. No appreciable error in molecular weight averages will beintroduced with this detector for polystyrene as long as its number-averagemolecular weight, Mn, is greater than 5000 gmol1. The principaldisadvantage of the
41、 differential refractometer is that precise control oftemperature, pressure, and flow rate is required to maintain a stable signalfor an appropriate level of sensitivity. For example, most organic liquidshave a temperature coefficient of 104RI units per K. Consequently, thetemperature within the RI
42、detector cell must be controlled to within 104C.NOTE 5Benzoyl peroxide is commonly used as a free radical initiatorfor styrene in the synthesis of polystyrene. The presence of smallconcentrations of initiator fragments containing strong chromophores,such as the benzoate group resulting from the deco
43、mposition of benzoylperoxide, as polymer end groups can significantly alter the ultraviolet(UV) absorption characteristics of polystyrene.4Since the relative con-centration of such end groups increases with decreasing polymer MW, therelationship between detector response and polymer concentration (m
44、olarabsorptivity in the Beer-Lambert law) may change with MW. Photometricdetectors (UV and fluorescence) are particularly sensitive to the presenceof strong chromophoric end groups. Choice of detector and selection ofwavelength are important to ensure a MW-independent concentrationresponse. Failure
45、to do so may result in erroneous MW-averages and adistorted MWD.7.8 Tubing and FittingsAll tubing between the sampleinjector and the detector should be no greater than 0.25-mm0.010-in. internal diameter and of sufficient thickness for useat pressures up to 42 MPa. Connecting column tubing shouldbe k
46、ept as short as possible and all fittings and connectors mustbe designed to prevent mixing and have low dead volumes.7.9 Recorder/PlotterEither a recording potentiometerwith a full-scale response of at least2soraprinting deviceconnected to a data handling system may be used to plot thechromatographi
47、c data. Pen response and signal-to-noise ratioshould be chosen so that the concentration signal is notappreciably perturbed.7.10 Data Handling SystemsMeans must be provided fordetermining chromatographic peak heights or integrated areasegments at prescribed intervals under the HPSEC chromato-gram an
48、d for handling and reporting the data. This can best beaccomplished by means of a computer or a real-time dataacquisition system with either off-line or on-line data process-ing.NOTE 6Data acquisition and handling systems for HPSEC have notbeen standardized. However, it is noted that a number of dif
49、ferentmanufacturers now provide chromatography data systems that includeHPSEC software. Also, some users have developed their own specializedHPSEC computer software.7.11 Other ComponentsSpecial solvent line filters, pres-sure monitors, pulse dampers, flowmeters, thermostated ovens,syphon counters, plotters, raw data storage systems, software,and so forth are oftentimes incorporated with the essentialcomponents previously listed.7.12 HPSEC SystemAny satisfactory combination of theabove components that will meet the performanc
