1、Designation: D5296 11Standard Test Method forMolecular Weight Averages and Molecular WeightDistribution of Polystyrene by High Performance Size-Exclusion Chromatography1This standard is issued under the fixed designation D5296; the number immediately following the designation indicates the year ofor
2、iginal 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. Scope*1.1 This test method covers the determination of molecularweight
3、 (MW) averages and the distribution of molecularweights for linear, soluble polystyrene by high-performancesize-exclusion chromatography (HPSEC). This test method isnot absolute and requires the use of commercially availablenarrow molecular weight distribution (MWD) polystyrenestandards for calibrat
4、ion. This test method is applicable forsamples containing molecular weight components that haveelution volumes falling within the elution volume rangedefined by polystyrene standards (that is, molecular weightsgenerally from 2000 to 2 000 000 gmol1).1.2 The HPSEC is differentiated from traditional s
5、ize-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 at least ten timesgreater than that for traditional SEC (see Terminology D883and Practice D3016).2The HPSEC systems employ low-volume l
6、iquid chromatography components and columnspacked with relatively small (generally 3 to 20 m) micropo-rous particles. High-performance liquid chromatography in-strumentation and automated data handling systems for dataacquisition and processing are required.1.3 The values stated in SI units are to b
7、e 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 limitations pr
8、ior to use. Specific precau-tionary statements are given in Section 9.NOTE 1There is no known ISO equivalent to this standard.2. Referenced Documents2.1 ASTM Standards:3D883 Terminology Relating to PlasticsD2857 Practice for Dilute Solution Viscosity of PolymersD3016 Practice for Use of Liquid Exclu
9、sion Chromatogra-phy Terms and RelationshipsE685 Practice for Testing Fixed-Wavelength PhotometricDetectors Used in Liquid ChromatographyE691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test Method3. Terminology3.1 DefinitionsFor definitions of technical terms per
10、tain-ing to plastics used in this test method see Terminology D883.4. 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 mobilephase transports the polymer
11、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 eluate as afunction of elution v
12、olume (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 thesample are calculated from th
13、e molecular weight/concentrationdata.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 are used for a wide variety of1This test method is under the jurisdiction ofASTM Committee D20
14、on Plasticsand is the direct responsibility of Subcommittee D20.70 on Analytical Methods.Current edition approved Sept. 1, 2011. Published September 2011. Originallyapproved in 1992. Last previous edition approved in 2005 as D5296 - 05. DOI:10.1520/D5296-11.2See also AMD Bibliography and Bibliograph
15、y 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 Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the stand
16、ards 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 Conshohocken, PA 19428-2959, United States.correlations for fundamental studies, processing, or productapplications.
17、 For example, the observed MWD is compared toone predicted from assumed kinetics or mechanisms for apolymerization reaction. Differences between the values willallow alteration of theory or experimental design. Similarly, thestrength, melt flow, and other properties of a polymer sampleusually are de
18、pendent on MW and MWD. Determinations ofMW and MWD are used for quality control of 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 valid only
19、for polystyrene andnon-exclusion effects are to be avoided. However, many of theprinciples of the method have been applied in generatingHPSEC methods for other polymer systems, for example, usingthe principles of universal calibration. (see Practice D3016).6. Units and Symbols6.1 Units and symbols r
20、elated 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 of l
21、iquid chroma-tography and is differentiated from traditional SEC in thatHPSEC uses columns with at least 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 essentia
22、l 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 used shall meet the safety and
23、performancerequirements 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 betwee
24、n 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 holdsufficient
25、 solvent to ensure consistency of composition for anumber of runs or analyses. The reservoir shall permit controlof the environment in contact with the solvent, and becompletely inert to the solvent employed. In addition, somemeans of agitation (for example, magnetic stirring) is recom-mended to ens
26、ure uniform composition.7.4 Solvent Pumping SystemThe principal requirement ofa pumping system is production of a constant and pulselessflow of solvent through the columns. In general, the rate offlow shall be adjustable between 0.1 and 5.0 mL/min andback-pressures shall not exceed limits specified
27、by the columnmanufacturer (for example, 28 MPa). If the elution volume isnot being measured directly or corrected for systematicchanges, the precision in the flow rate must be at least 60.3 %as measured under the conditions and over the time intervalrequired for running a typical analysis.7.5 Sample
28、 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-performance liquid chromatography automaticinjection systems i
29、s suitable 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 steel columns with uniform andhighly polished inside walls are us
30、ually 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, porous packing materials areused for HPSEC. Generally, the pa
31、cking 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 are either packed with particlesof relatively uniform pore size or with a “mixed bed” ofparticles to produce a bro
32、ad 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 inorder of columns having the smallest to those having thelargest packing pore size.NOTE 3Column packing materials and packed HPSEC columns area
33、vailable commercially from a number of manufacturers. Users of this testmethod are advised to follow manufacturers guidelines and recommen-dations for the care and use of their HPSEC columns. For example,TABLE 1 Units and Symbols Related to FunctionFunctionCommon Unit/SymbolSI Unit/SymbolBasic prope
34、rty 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 unit.FIG. 1 Sche
35、matic of an HPSEC SystemD5296 112manufacturers 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 DetectorsThe purpose of the detector is to continu-ously monitor t
36、he 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 concentration gradient in the emerging stream. Thisrequirement im
37、poses 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 photometric measurements (refractiveindex, UV-visible, fluo
38、rescence and infrared absorbance). Prac-tice E685 serves as a guide for testing the performance ofphotometric detectors used in high-performance liquid chro-matographic systems. Other detectors with appropriate sensi-tivity are also acceptable. The differential refractometer hasmoderate sensitivity
39、and general utility. It provides a signalproportional to the difference in refractive index (DRI) betweenthe solvent and the column eluate. The detector shall be able torespond to 107to 108DRI unit with cell volumes # 10 L.NOTE 4The change in the specific refractive index increment (dn/dc)of polysty
40、rene 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 differential re
41、fractometer 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 detector cell mu
42、st 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 decomposition of ben
43、zoylperoxide, 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 (molarabsorptivity
44、 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 to do so may res
45、ult in erroneous MW-averages and adistorted MWD.7.8 Tubing and FittingsAll tubing between the sampleinjector and the detector shall be no greater than 0.25-mm0.010-in. internal diameter and of sufficient thickness for useat pressures up to 42 MPa. Connecting tubings shall be kept asshort as possible
46、 and all fittings and connectors must bedesigned 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 is recommended to plotthe chromatographic data. Choose a pen
47、response and signal-to-noise ratio so that the concentration signal is not appreciablyperturbed.7.10 Data Handling SystemsMeans must be provided fordetermining chromatographic peak heights or integrated areasegments at prescribed intervals under the HPSEC chromato-gram and for handling and reporting
48、 the data. This is bestaccomplished 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 differentmanufacturers now provide
49、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 performance require-ments of Section 12.
