1、BRITISH STANDARDBS ISO 16564:2004Incorporating Amendment No. 1Rubber, raw natural Determination of average molecular mass and molecular-mass distribution by size exclusion chromatography (SEC)ICS 83.040.10g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49
2、g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS ISO 16564:2004This British Standard was published under the authority of the Standards Policy and Strategy Committee on 1 November 2004 BSI 2006ISBN 0 580 44709 XNational forewordThis British
3、Standard reproduces verbatim ISO 16564:2004, including amendment May 2006, and implements it as the UK national standard.The start and finish of text introduced or altered by amendment is indicated in the text by tags !“. Tags indicating changes to ISO text carry the number of the amendment. For exa
4、mple, text altered by ISO amendment 1 is indicated in the text by !“.The UK participation in its preparation was entrusted to Technical Committee PRI/23, Test methods for rubber and non-black compounding ingredients, which has the responsibility to: aid enquirers to understand the text; present to t
5、he responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK.A list of organizations represented on this committee can be obtaine
6、d on request to its secretary.Cross-referencesThe British Standards which implement international publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electr
7、onic Catalogue or of British Standards Online.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations.Summary of pagesThis
8、document comprises a front cover, an inside front cover, the ISO title page, pages ii to iv, pages 1 to 8, and inside back cover and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments issued since publicationAmd. No. Date Comments1
9、6463 30 June 2006 See national forewordReference numberISO 16564:2004(E)INTERNATIONAL STANDARD ISO16564First edition2004-10-01Rubber, raw natural Determination of average molecular mass and molecular-mass distribution by size exclusion chromatography (SEC) Caoutchouc naturel brut Dtermination de la
10、masse molculaire moyenne et de la rpartition des masses molculaires par chromatographie dexclusion strique (SEC) BS ISO 16564:2004ii iiiContents Page Foreword iv 1 Scope 1 2 Principle . 1 3 Materials. 1 4 Apparatus 2 5 Procedure 2 6 Calibration . 3 7 Expression of results 5 8 Precision 5 9 Test repo
11、rt . 6 Annex A (informative) SEC analysis of natural rubber using cyclohexane as solvent and polyisoprene standards 7 Bibliography . 8 BS ISO 16564:2004iv 1Rubber, raw natural Determination of average molecular mass and molecular-mass distribution by size exclusion chromatography (SEC) WARNING Perso
12、ns using this International Standard should be familiar with normal laboratory practice. This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure comp
13、liance with any national regulatory limitations prior to use. 1 Scope This International Standard specifies a method of determining the average molecular mass and the molecular-mass distribution of raw natural rubber dissolved in tetrahydrofuran. A set of polystyrene standards is used for calibratio
14、n purposes (i.e. the method is a relative one). An alternative method, using cyclohexane as solvent and polyisoprene standards, is included in an informative annex. 2 Principle Dried natural rubber is dissolved in tetrahydrofuran at room temperature. The solution is filtered to remove “gel” (slightl
15、y crosslinked rubber) and other insoluble materials. The filtrate is used to determine the molecular mass by size exclusion chromatography1. From the chromatogram, the number-average molecular mass (Mn), the mass-average molecular mass (Mw) and a polydispersity value (Mw/Mn) are calculated. 3 Materi
16、als 3.1 The recommended solvent is HPLC-grade tetrahydrofuran (THF). If analytical-grade THF is used, 0,5 % of butylated hydroxytoluene (BHT) shall be added to the solvent as an antioxidant. The BHT is strongly retained and acts as a marker for the end of the chromatogram. It also allows minor varia
17、tions in run time to be corrected, as well as indicating when a serious change in the column conditions has occurred. 3.2 The solvent is filtered through a polytetrafluoroethylene (PTFE) membrane (4.2) before use. 3.3 A set of polystyrene standards with proper traceability shall be used, typically c
18、overing an Mwrange from 6,5 103to 1,06 107. An example of a suitable set of polystyrene standards is given in Table 1. Table 1 Polystyrene standards No. of standard MpMw/Mn1 3,55 1051,08 2 7,06 1051,12 3 2,89 1061,36 4 3,84 1061,30 5 4,48 1061,47 6 5,48 1061,40 7 6,77 1061,37 8 8,42 1061,33 BS ISO 1
19、6564:20042 4 Apparatus 4.1 Size exclusion chromatograph: Suitable high-performance liquid chromatography equipment consisting of the components specified in 4.1.1 to 4.1.7. 4.1.1 Solvent reservoir, large enough to hold an adequate quantity of solvent (see 3.1). About 3 l of solvent is recommended fo
20、r a complete analysis. Refilling can result in variations in dissolved air and the inclusion of impurities. When fresh solvent is added, it takes a long time to obtain a stable baseline. 4.1.2 Automatic on-line degassing system, to prevent the formation of bubbles in the mobile phase and to ensure a
21、 stabilized liquid feed flow. 4.1.3 Pump with temperature-control equipment, capable of delivering the solvent at a sufficient rate and a suitable temperature, for example 1,0 cm3/min at 35 C. 4.1.4 Injector. 4.1.5 Columns: Columns for the SEC of natural rubber are usually packed with rigid spheres
22、of crosslinked polystyrene gel (prepared from styrene-divinylbenzene copolymer) having a range of pore sizes. Molecular masses in the range 2 103to 1012can be measured, depending on the particular set of columns used. Other column packings can be used providing they are capable of separating the pol
23、ymers of different molecular mass in the range of interest and are not adversely affected by the solvent used. The columns shall be connected to the chromatograph in such a way that the sample is eluted through the column of greatest pore size first and the column of smallest pore size last. 4.1.6 R
24、efractive index detector (or other type of detector), capable of giving an adequate response to natural rubber at the concentrations used. 4.1.7 Data-processing unit, capable of calculating the required molecular masses and molecular-mass distributions. 4.2 PTFE membrane filters, with a porosity of
25、0,45 m to 1 m. The actual porosity used will depend on the columns being used and the laboratory set-up. 4.3 Syringes, capacity 100 mm3and 200 mm3(or larger). 4.4 Syringe, capacity 10 cm3. 4.5 Vortex shaker. 4.6 Auto-sampler, with vials (brown when available). NOTE An auto-sampler with associated so
26、ftware may be used for sample introduction. 5 Procedure 5.1 Preparation of solvent Filter the solvent through a PTFE filter (4.2) and degas under vacuum for 30 min. This shall be done 12 h prior to the analysis. 5.2 Setting up the SEC chromatograph Condition the chromatograph, fitted with a suitable
27、 set of columns, with the solvent flowing at 0,6 cm3/min until a stable baseline is obtained. The columns are normally kept at a constant temperature at least 10 C above room temperature, i.e. between 30 C and 45 C. Sufficient degassed solvent for the complete set of analyses shall be stored in the
28、solvent reservoir under an inert gas. BS ISO 16564:200435.3 Setting up the data-processing unit The data processor (4.1.7) converts the detector output into a digital signal, making measurements typically at a rate of 1 Hz to 5 Hz. If it is necessary to reduce noise, the measurements can be combined
29、 to give time slices with a width of 1 s to 2 s. 5.4 Preparation and injection of sample solution 5.2.1 Prepare a solution of the sample by placing 0,01 g or less of dried sample into a 20 cm3screw-cap vial (brown when available) containing 10 cm3of the previously prepared solvent (see 5.1). 5.2.2 S
30、tir the vial, using a vortex shaker (4.5), for 1 min at room temperature and then keep warm at 37 C for 24 h. Take care not to expose the sample solution to light, e.g. by shielding the vial from light during warming, or by using a brown vial. 5.2.3 Filter the solution through a PTFE membrane (4.2)
31、and introduce the filtrate into a syringe (4.3) or an auto-sampler vial. Record the mass of the dried PTFE membrane (4.2) before and after filtration and report the percentage “gel” content together with the porosity of the filter used. NOTE When the sample contains 30 % “gel”, a significant fractio
32、n of the rubber is not being measured. This seriously affects the use of the molecular-mass distribution data for the prediction of the behaviour of the rubber. 5.2.4 Inject the sample solution and carry out the sample run in the normal way. 6 Calibration 6.1 Polystyrene standards (see 3.3) dissolve
33、d in the prepared solvent (see 5.2) at a concentration of 0,1 g/l shall be used for calibration. The standards shall cover the entire molecular-mass range of the sample. Prepare the standards by swelling them in solvent overnight without shaking. Shaking will reduce the molecular mass and broaden th
34、e peak width. 6.2 Run three 100 mm3injections of each standard and read the retention times of the peaks. Take the average of the three retention times for each standard. 6.3 Plot the average retention time against the molecular mass. A typical molecular-mass calibration curve for a set of polystyre
35、ne standards is shown in Figure 1. 6.4 The calibration curve is used by the instrument software to calculate the sample parameters to be determined. BS ISO 16564:20044 Key X retention time, tR(min) Y logMwFigure 1 Typical calibration curve for a set of polystyrene standards Key X retention time, tR(
36、min) Y mass fraction Figure 2 Example of an SEC chromatogram BS ISO 16564:200457 Expression of results 7.1 For each sample solution, the following parameters are obtained automatically from the instrument: mass-average molecular mass (Mw); number-average molecular mass (Mn); z-average molecular mass
37、 (Mz); polydispersity (Mw/Mn); peak molecular mass (Mp). In addition, a graphical representation of molecular-mass fraction vs. retention time is obtained. 7.2 An example of a chromatogram is shown in Figure 2. BS ISO 16564:20048 Precision 8.1 An interlaboratory test programme (ITP) was carried out
38、using the guidelines established by ISO/TR 9272, Rubber and rubber products Determination of precision for test method standards, in the period June to July 2002. A type 1 precision was determined on the basis of two measurements of molecular mass Mwon each of two different days. Each measurement is
39、 designated as a test result. Homogenized natural rubber (NR) was used as the test material and each laboratory used a set of supplied polystyrene standards for calibration. Data on Mwobtained from four laboratories were used to calculate the repeatability and reproducibility. 8.2 The precision resu
40、lts as determined by this ITP may not be applied to acceptance or rejection testing for any group of materials or products without documentation that the results of this precision evaluation actually apply to the products or materials tested. 8.3 The precision results are given in Table 2. General s
41、tatements for the use of the precision results are cited below. These are given in terms of both the absolute precision, r and R, and the relative precision, (r) and (R). Repeatability: The repeatability, or local domain precision, of the test method has been established as the values given in Table
42、 2. Two individual test results (obtained by the proper use of this International Standard) that differ by more than the tabulated values of r, in measurement units, or (r), in percent, shall be considered as suspect, i.e. to have come from different populations. Such a decision suggests that some a
43、ppropriate investigative action be taken. Reproducibility: The reproducibility, or global domain precision, of the test method has been established as the values given in Table 2. Two individual test results obtained in different laboratories (by the proper use of this International Standard) that d
44、iffer by more than the tabulated values of R, in measurement units, and (R), in percent, shall be considered as suspect, i.e. to have come from different populations. Such a decision suggests that some appropriate investigative action be taken. Bias: Bias is the difference between a measured average
45、 test result and a reference or true value for the measurement in question. Reference values do not exist for this test method and therefore bias cannot be determined. “! BS ISO 16564:20046 9 Test report The test report shall include the following: a) a reference to this International Standard; b) a
46、ll details necessary to identify the sample analysed; c) the date and place of the analysis; d) a graphical representation of the molecular-mass distribution (see 7.1); e) the values obtained for the parameters Mn, Mwand Mw/Mn; f) the percentage “gel” content; g) the porosity of the filter used. ! T
47、able 2 Precision data for MwWithin laboratory Between laboratories Material Mean value of Mwsrr (r) sRR (R) NR 1 910 041 46 160 250 245 13 321 309 887 030 46 where sris the repeatability standard deviation; sRis the reproducibility standard deviation; r is the repeatability limit, in measurement uni
48、ts; R is the reproducibility limit, in measurement units; (r) is the repeatability (relative), in percent; (R) is the reproducibility (relative), in percent. “7 Annex A (informative) SEC analysis of natural rubber using cyclohexane as solvent and polyisoprene standards A.1 General This method is rec
49、ommended for laboratories which do not perform the test regularly and/or experience difficulties with moisture in the solvent when using THF. A.2 Procedure Dissolve the sample in cyclohexane (HPLC grade) and stabilize the solution with 2,6-di-tert-butyl-4-methylphenol (BHT), which also serves as an internal standard, in the proportion 120 mg BHT/30 cm3of solvent. Store the solutions at 30 C for 14 days, stirring gently for 1 h daily. A.3 Equipment The equipment consists of a conventional SEC system gas extractor,
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