1、Designation: D 7134 05Standard Test Method forMolecular Mass Averages and Molecular Mass Distributionof Atactic Polystyrene by Matrix Assisted Laser Desorption/Ionization (MALDI)-Time of Flight (TOF) Mass Spectrometry(MS)1This standard is issued under the fixed designation D 7134; the number immedia
2、tely following the designation indicates the 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 t
3、est method covers the determination of molecularmass (MM) averages and the distribution of molecular massesfor linear atactic polystyrene of narrow molecular mass distri-bution (MMD) ranging in molecular masses from 2000 g/molto 35 000 g/mol by matrix assisted laser desorption/ionizationtime-of-flig
4、ht mass spectrometry (MALDI-TOF-MS). This testmethod is not absolute and requires the use of biopolymers forthe calibration of the mass axis. The relative calibration of theintensity axis is assumed to be constant for a narrow MMD.Generally, this is viewed as correct if the measured polydis-persity
5、is less than 1.2 for the molecular mass range givenabove.1.2 The values stated in SI units are to be regarded as thestandard.1.3 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 app
6、ro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.NOTE 1There is no similar or equivalent ISO standard.2. Referenced Documents2.1 ASTM Standards:2D 883 Terminology Relating to PlasticsD 1600 Terminology for Abbreviated Terms Relating toPlas
7、ticsE 691 Practice for Conducting Interlaboratory Test Programto Determine the Precision of Test Methods3. Terminology3.1 DefinitionsFor definitions of technical terms pertain-ing to plastics used in this test method see Terminologies D 883and D 1600.4. Summary of Test Method4.1 The MALDI process in
8、volves the ablation and theionization of an analyte dispersed in an organic small moleculematrix, most commonly an organic acid. One way to cationizethe analyte is to add a metal salt. The process is as follows: Apolymer (biological or synthetic) is co-crystallized or co-mixedwith the matrix molecul
9、e in the solid phase and deposited onthe target often made of stainless steel (details of this processwill be described later). A short duration UV or IR laser pulseis used to ablate the matrix and the analyte mixture. Theablation process involves UV or IR absorption by the matrixmolecule. The laser
10、 energy excites the matrix molecule causingit to vaporize and decompose. Analyte and matrix leave thetarget surface in a plume. This ablation process involves thetransfer of energy from electronic or vibration modes intotranslational modes of the matrix. The MALDI-TOF-MSmethod described in this test
11、 method uses a UV nitrogen laseroperating at 337 nm. This laser has a pulse width of about 3 ns.4.2 In the test method described below, the polystyrenepolymer in the ablation plume gains an Ag cation and isaccelerated by a high voltage, often about 20 keV. Followingacceleration, the polymer species
12、drifts down the field freeflight tube and is detected at the end of the flight tube. Thetime-of-flight of the species is a measure of its mass. From thedistribution of arrival times and the calibration of the arrivaltimes with known mass standards, the mass distribution of thepolymer is determined.4
13、.3 This test method is valid only for polystyrene of narrowmolecular mass distribution (MMD) polymers, Mw/Mn 1.2with Mngreater than 3000 g/mol or less than 35 000 g/mol.1This test method is under the jurisdiction of ASTM Committee D20 on Plasticsand is the direct responsibility of Subcommittee D20.7
14、0 on Analytical Methods.Current edition approved April 1, 2005. Published June 2005.2For 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 p
15、age onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5. Significance and Use5.1 General UtilityThe molecular mass (MM) and mo-lecular mass distribution (MMD) are fundamental characteris-tics of a synthetic polymer t
16、hat result from the polymerizationprocess. The MM and MMD is useful for a wide variety ofcorrelations for fundamental studies, processing and productapplications. For example, it is possible to compare theobserved MMD to predictions from an assumed kinetic ormechanistic model for the polymerization
17、reaction. Differencesbetween the values will allow alteration of the model orexperimental design. Similarly, it is possible the strength, themelt flow rate, and other properties of a polymer are dependenton the MM and MMD. Determination of the MM and MMDare used for quality control of polymers and a
18、s specification inthe commerce of polymers.5.2 LimitationsIf the MMD is too wide, it is possible thatthe assumption of the constancy of the intensity scale calibra-tion is in serious error.6. Units and Symbols6.1 Units and symbols are given in Table 1.7. Apparatus7.1 A description of a typical MALDI
19、-TOF-MS instrumentfollows:7.1.1 Introduction to MALDI-TOF-MSMALDI-TOF-MSis a specific form of mass spectrometry. It is possible to viewmass spectrometry as comprised of three distinct processes:(1) The production of charged gas phase species from theoriginal analyte. This step involves a way to get
20、the analyte intothe gas phase and a way to ionize it. For MALDI these eventsoccur in the same process; for other MS techniques used onlower mass molecules, this is not necessarily the same process.(2) The separation of the analytes by mass or, morecorrectly, by m/z, the mass, m, divided by the charg
21、e, z.(3) The detection of the ions.7.2 We shall now consider here in detail the MALDI-TOF-MS (see Fig. 1 for the schematic of a linear MALDI-TOF-MS and Fig. 2 for the schematic of a reflectron MALDI-TOF-MS). The MALDI-TOF-MS is currently the type of massspectrometer most commonly used to analyze syn
22、thetic poly-mers.7.2.1 Essential ComponentsThe essential components ofthe MALDI-TOF-MS are: sample introduction chamber, alaser source, a flight tube with an acceleration region which isthe ion source, and an ion detector. It is possible that theinstruments will also have an ion deflector and an ion
23、 reflector.7.2.1.1 Sample Introduction ChamberA MALDI sampleconsists of a film of the analyte, matrix, and salt mixturedeposited onto a metal sample plate. The entire plate andMALDI sample is often referred to as a MALDI target. TheMALDI target is introduced into the spectrometer vacuumchamber by ei
24、ther a manual or an automatic operation. It ispossible that the MALDI target will contain many spots fordifferent samples that are accessible by the user through remotecontrol.7.2.1.2 Laser SourceThe laser system is comprised of apulsed nitrogen laser operating typically at a wavelength of 337nm and
25、 approximately a 3 ns pulse width, an attenuator whichallows for the adjustment of the laser power, beam splitters todirect a fraction of the light to a photodiode to start the timingfor the TOF measurement, and a lens and mirror system todirect the laser beam onto the MALDI target. The target ismov
26、eable, often by control of the operator through a mouse ona computer, so that the target can be moved around under thelaser beam.7.2.1.3 Flight TubeThe ion source consists of a positivelyor negatively charged electrode. The target is at a high voltageof 20 to 35 kV and just behind a grounded acceler
27、ation grid.The analyte/matrix/salt mixture is deposited on this electrodeand exposed to the pulsed laser beam. When the analyte/matrix/salt mixture is hit by the laser beam, gaseous analyteions are formed which are accelerated by the electric field, exitthe source and pass though into the flight tub
28、e, a field free driftregion.7.2.1.4 Ion detection in a TOF mass analyzer is based on thefast measurement of the electrode voltage resulting from an ionimpact. A detector in which the signal is proportional to thenumber of ions hitting the detector.7.2.1.5 RecorderMultichannel recorder with time step
29、sizes of 4 ns or less is acceptable7.2.1.6 Data HandlingUse any computer for data analy-sis. The computer and software must be able to read the outputof the recorder, store and analyze the data. Software must beavailable to determine a baseline, convert the data from time tomass though a calibration
30、 curve and obtain the moments of theMMD described below.8. Reagents and Materials8.1 MatricesAll-trans retinoic acid is the recommendedmatrix for this test method, but dithranol is also acceptable.Allof these materials must be at least 97 % pure. Store retinoicacid in a freezer and warm it to room t
31、emperature just beforeuse, as it degrades at room temperature. Also prevent lightexposure of retinoic acid to reduce degradation.8.2 Recommended solvent is tetrahydrofuran (THF) with orwithout antioxidant, but toluene is also a suitable solvent. Highpurity solvents are recommended. It is recommended
32、 to useTHF with an antioxidant like 0.025 to 0.1 % w/v butylatedhydroxy toluene and store it in an amber container. If THFwithout an antioxidant is used, store it in an amber containerunder an inert gas. Otherwise it will react with oxygen to formperoxides, which are hazardous upon evaporative conce
33、ntra-tion.8.3 SaltsSilver salts, silver triflouroacetate (AgTFA), inparticular, are recommended since they are soluble in THF andtoluene. The silver salt AgNO3dissolved in ethanol (EtOH) issuitable for use with the polymer and matrix in THF. The saltsTABLE 1 Units and Symbols Related to FunctionFunc
34、tion Common Unit SI UnitBasic PropertyDefinitionMolecular Mass(often calledMolecular Weight)g mol1Sample Mass mg gConcentration mg/mLAASame as common unit.D7134052must be soluble in the solvent chosen for the polymer and thematrix. (See 9.3 for a discussion of hazards ofAg compounds.)8.4 Biopolymer
35、Mass StandardsOne way of conductingthe calibration of the TOF MS mass axis is by using biopoly-mers in the range of the expected MM of the synthetic polymer.Suggested biopolymer and their masses are given in Table 2.9. Hazards9.1 Solvents used in this test method are likely to be toxicand highly fla
36、mmable, or both. Avoid direct contact with skinand inhalation of solvents. The user is advised to consult theliterature and follow recommended procedures pertaining tosafe handling of the solvent.FIG. 1 Linear MALDI-TOF MSFIG. 2 Reflectron MALDI-TOF MSTABLE 2 Molecular Mass Calibrants, Molecular Mas
37、s, g/molMolecular Mass Calibrants Average Molecular Mass, u Monoisotopic Mass, u Average Molecular Mass MH+ Monoisotopic Molecular Mass MH+DHB 154.12 154.03 155.03Sinnapinac Acid 224.21 224.07 225.08Angiotension II human 1046.2 1045.5 1047.2 1046.5ACTH(18-39) clip human 2465.3 2464.2 2466.7 2465.2In
38、sulin bovine 5733.5 5734.6Ubiquitin 8564.8 8565.8Cytochrome c-equine 12360.1 12361.1Myoglobin equine 17568Myoglobin apo-myoglobin 16951.5 16952.5Trypsin bovine 23311.5 23312.5BSA 66430 66431D71340539.2 Handle matrices and biological standards with care.Avoid direct contact with skin. The user is adv
39、ised to consultthe literature and follow recommended procedures pertainingto safe handling of these materials.9.3 AgNO3is light sensitive and is a strong oxidizing agent.All Ag compounds are poisonous. There is a danger ofpermanent blue-gray staining of eyes, mouth, throat and skin,as well as eye da
40、mage following long-term exposure to Agcompounds. There is a danger of deposition of black silverstains on the skin following short contact with Ag compounds.Ag compounds have the potential to be very destructive ofmucous membranes. The user is advised to consult the litera-ture and follow recommend
41、ed procedures pertaining to safehandling of these materials10. Preparation of Apparatus10.1 Preparation of ApparatusTypically on a TOF MSthe vacuum systems, high voltage power supplies and comput-ers and other parts of the data collection system are left on atall times. For some systems, the laser i
42、s not started until used.Allow the laser to warm up for times as prescribed in themanufacturers manual. If no times are prescribed, experienceshows a 30 min warm-up time is acceptable.11. Sample Preparation on the Sample Plate11.1 Recipes for Polymer/Matrix/Salt Solutions11.1.1 Recipe AThe following
43、 recipe has been found towork successfully on many instruments for polystyrene. This isthe preferred recipe:5 mg/mL of PS in THF75 mg/mL retinoic acid in THF5 mg/mL AgTFA in THFMix solutions by volume 1:10:1 of PS : retinoic acid : AgTFA.Use the solutions within 48 h after they are made. Use either
44、the method forsample plate preparation in 11.2.1 or the one in 11.2.2.11.1.2 Recipe BThe following other recipe has beenfound to work on many instruments for polystyrene:5 mg/mL of PS in THF75 mg/mL retinoic acid in THF5 mg/mL AgNO3in EtOHMix solutions by volume 1:10:1 of PS : retinoic acid : AgNO3i
45、n EtOH.It is critical to use the solutions soon after preparation. Use the method in11.2.2 for sample plate preparation.11.1.3 Recipe CThe following other recipe has beenfound to work on many instruments for polystyrene:5 mg/mL of PS in THF45 mg/mL dithranol in THF5 mg/mL AgTFA in THFMix solutions b
46、y volume 1:10:1 of PS : dithranol : AgTFA.The solutions can be kept in the dark in a refrigerator for as long as 48 h. Usethe method in 11.2.2 for sample plate preparation.11.2 Method to Deposit the Sample Solutions onto SamplePlateSample preparation is critical to the quality of theMALDI-TOF-MS dat
47、a obtained. The presumption is that thepolymer and the salt in the MALDI sample must be welldispersed in the final matrix mixture to achieve a one-to-onerepresentation of the polymer MMD in the solution to thepolymer MMD in the gas phase. Yet, the matrix is commonlycrystalline and the polymer atacti
48、c PS is glassy. Kineticprocesses occurring during the loss of solvent from the solutionof the mixture of matrix, salt and polymer must occur either toco-crystallize the polymer with the matrix and salt or to embedthe polymer in the defect structure of the organic matrix. Toobtain the correct represe
49、ntation of the MMD in the MALDIspectra, each n-mer in the MMD must occur in the MALDIspectra in proportion to its appearance in the original MMD.Thus a variety of methods have been developed to deposit thesample solutions onto the sample plate surface to obtain gooddispersion of the polymer and salt in the matrix. These aregiven in following sections.11.2.1 HandspottingThe solutions described in 11.1 arehand spotted from a L pipette onto a target plate; (0.5 to 2) Lof solution are used to deposit polymer, matrix, and saltmixtures onto the plate. The solvent is al
