Dual Detection High-Performance Size-Exclusion Chromatography

Chapter 24

Dual Detection High-Performance Size-Exclusion Chromatography System as an Aid in Copolymer Characterization Downloaded by UNIV OF MINNESOTA on October 14, 2014 | http://pubs.acs.org Publication Date: September 29, 2005 | doi: 10.1021/bk-2005-0916.ch024

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Tatjana Tomić , Marko Rogošić , Zvonimir Matusinović , and Nikola Šegudović 1

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Strategic Development, Research and Development Sector, INA Industrija nafte d.d., Lovinčićeva bb, HR-10000 Zagreb, Croatia Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev Trg 19, HR-10000 Zagreb, Croatia 2

Compositional heterogeneity is one of the essential factors controlling the physical and physico-chemical properties of copolymers; its determination is in most cases a rather difficult and challenging scientific task. The overall composition of a copolymer comprising two kinds of repeating units can be determined as a function of molecular mass by the dual detector high performance size exclusion chromatography (HPSEC) technique. The common combination oftherefractive index (RI) detector with the UV spectrophotometer can be applied if at least one of the monomer units absorbs at a suitable wavelength and if the UV-spectra of both components are sufficiently different. In this paper, synthesized random copolymers of styrene and methyl methacrylate were dissolved in tetrahydrofuran and characterized by the RI-UV HPSEC technique, at the UV wavelength of 254 nm. Home made software and a corresponding hardware interface were used for the equipment control, data acquisition, data processing and 325 In New Polymeric Materials; Korugic-Karasz, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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Downloaded by UNIV OF MINNESOTA on October 14, 2014 | http://pubs.acs.org Publication Date: September 29, 2005 | doi: 10.1021/bk-2005-0916.ch024

calculation. The obtained results show that the technique is capable for fast, routine determination of average copolymer composition. There is no significant variation of chemical composition with molecular mass for investigated copolymers with lower polystyrene content. For copolymers rich in polystyrene, chemical composition varies with molecular mass. In this case, calculated apparent mass fractions of styrene may exceed 100%, and this is explained by the presence of small quantities of low-molecular-mass styrene homopolymer.

Introduction The properties of a copolymer are affected not only by its average composition and molecular mass, but they also depend on the sequential arrangement of monomeric units along the chain, i.e. on the chain architecture. From the viewpoint of the chain architecture, copolymers composed of chemically différait monomers can be classified into four main types, i.e. alternating, statistical, block and graft copolymers [1]. The composition of graft copolymers generally correlates with the molecular mass, but this may not be true for statistical and block copolymers [2]. In particular, compositional heterogeneity may be the essential factor controlling the physical and physicochemical properties of statistical copolymers. In the last twenty years, size exclusion chromatography (SEC, former GPC, gel permeation chromatography) became the most common method for the determination of molecular mass and molecular mass distribution (MMD) of polymers [3]. SEC separates the solutes in solutions based on the size and hydrodynamic volume of molecules. This kind of sorting takes place by passing the solution through a column containing a separation packing. For simple homopolymers, the apparatus can be calibrated to relate size (as measured by the volume of mobile phase eluted between injection and elution of the molecule) to molecular mass using simple concentration detector, such as refractive index detector (RI). For homopolymers, the size of a molecule (as well as other molecular properties) depends not only on its molecular mass but on other variables as well, such as the degree of branching. For copolymers, even more structural properties (chemical composition distribution, functionality distribution, details

In New Polymeric Materials; Korugic-Karasz, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.


327 of molecular architecture, etc.) may affect the size of a molecule in solution, although elution volumes are often given only as functions of molecular mass [4]. By taking into account above mentioned effects, other analyses, like those of composition and configuration of copolymer molecules are also possible by using modified SEC. In other high performance liquid chromatography (HPLC) techniques, common in analysis of small molecules, like normal (NP) and reverse (RP) phase chromatography it is common to extend separation and identification by coupling different detectors [5-7] to chromatographic columns. Moreover, multidimensional HPLC coupled with a range of detectors may be a method of choice for performing difficult separations and analyses of complex petrochemical samples according to the size, volatility and polarity of constituent molecules [6, 8]. Using various detection systems with SEC, one may characterize copolymers according to different properties, such as chemical composition distribution, grafting efficiency, graft ratio, graftfrequency,etc. In addition, the conversion of monomers during copolymerization may be monitored on-line using SEC [9-11]. When spectroscopic detectors (UV-Vis, DAD, 3R) are used, it is expected that at least one component of a copolymer is an U V or IR absorbing medium, and that its spectrum differs as much as possible from that of another component. Wavelengths to be monitored should be chosen to maximize the difference of molar absorbance of the parts. At the same time, corresponding mobile phase (solvent) must possess low UV cut-off properties. The aim of this paper was to describe the simple case of copolymer compositional heterogeneity characterization, using SEC dual detection system equiped with UV spectrophotometer at 254 nm as a selective detector and RI detector as a concentration detector. The model copolymers to be studied were statistically prepared styrene - methyl methacrylate copolymers. Home made software and a corresponding hardware interface were used for data acquisition, processing and manipulation.

Experimental

Sample preparation Statistical copolymers of styrene and methyl methacrylate were prepared by the batch radical solution copolymerization at 60°C in toluene, under nitrogen


328 atmosphere. Total monomer concentrations were kept approximately constant (3 mol dm" ), but monomer mole fractions as well as initiator (azobisisobutyronitrile, ΑΠ3Ν) concentrations and reaction times wore varied in order to produce copolymers having different average compositions and différait molecular mass distributions. Yet, overall monomer conversions were kept under 10% by mass to prevent the copolymer composition shift. Synthesized copolymers were purified by the three times repeated dissolution/precipitation process, using toluene as a solvent and cold methanol as a non-solvent. Copolymer compositions were determined by the elemental analysis using evaporative combustion/GC coupled CHNS analysis system (Perkin Elmer 2400, ser. 2). The resulting C/H/O mass fractions were converted to styrene/methyl methacrylate molar fractions using simple stoichiometric relations. The data related to the sample preparation and elemental analysis are given in Table I.


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Table I. Homo and copolymerization conditions Sample ID fMJ / F X m / PS 100 9.4 100 3.00 190 1.0 ίοSMMA 0.2x2 78.6 3.00 240 4.1 84.2 2.5 10" 80.1 SMMA 0.2x4 3.00 480 4.7 84.2 6.3 ΙΟ" 46.5 SMMA 0.5x2 3.00 2.9 45.3 2.5 10 160 50.0 SMMA 0.5x4 45.3 3.00 390 3.3 6.3 ΙΟ SMMA 0.8x2 3.00 11.3 17.5 240 6.9 2.5 10" 17.7 SMMA 0.8x4 3.00 4.5 6.3 ΙΟ" 360 11.3 PMMA 0 0 2.58 34.8 1.0 10 210 [M] total monomer concentration, mol dm"; [I] initiatior concentration, mol dm"; t reaction time, min; X total monomer conversion, wt%; / styrene molefractionin feed, mol%; F styrene molefractionin copolymer as determined by elemental analysis, mol% 2

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SEC measurements All measurements were performed on a modular SEC system comprising the Waters 6000A high pressure pump, Waters U6K manual injector capable for injection of up to 2.0 ml of sample solution, three PL-gel mixed Β columns (30.0 χ 0.76 cm, 10 μτη), Waters 440 UV fixed wavelength detector (at 254 nm) and Waters 401 RI detector. A l l sample solutions were prepared in concentrations of 0.52±0.02 g / 100 mL, by dissolving the samples in freshly distilled tetrahydrofuran (THF). Sample solutions were left to rest overnight to allow the dissolution process to reach its equilibrium. Before injection, all


329 solutions were filtered through 0.5 μιη membrane filters (Millipore MX) and 50μ£ of solutions were injected. Flow rates were kept constant, at the value of 1.0 mL min" . The separations were performed at ambient temperature. For the calibration of both detectors, narrow molecular mass polystyrene standards (molecular mass ranging from 1.3xl0 to 2.9xl0 were used. This procedure is rather common, but the calculated molecular masses of SMMA copolymers may be considered only as relative values. The time lag between detectors was 9 seconds (150 (iL). Equipment control, data acquisition, reduction, processing and reporting were performed using home made software and a corresponding hardware interface. The interface is able to monitor both the injection event and the evolution of chromatogram. Analog signals from both detectors are amplified and forwarded to AD connector (16 channels and 12 bit resolution) to obtain digital values. The software is divided into three main parts: 1) on-line data acquisition and plotting of the chromatogram, 2) calibration part and 3) data reduction procedures and post-run manipulation. In the acquisition and plotting part, a sequence of digitalized raw data is plotted against time as a chromatogram, starting from the injection event and ending at any appropriate time chosen by the operator. In the calibration part, chromatogram peaks are recognized and converted to corresponding retention times. By the input of corresponding molecular mass values, individual calibration data are then converted to calibration curves, described by 1 or 3 order polynomials. The data reduction part enables post-run calibration and manipulation of the acquired data. The following features are included: Φ datafilteringusing Savitzky-Golay filters • automatic recognition of onsets and offsets of chromatogram peaks • baseline deduction • calculation of differential and integral molecular mass distributions using previously determined or externally supplied calibration curves • calculation of average molecular mass values (M„, M , M ) • fitting of experimental distribution curves data with some theoretical functions (Flory, Schulz, Tung, Wesslau) • generation of final reports 1


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Results and discussion Before running of SEC analyses of copolymers, UV-spectra of corresponding homopolymers (polystyrene, PS and polymethyl methacrylate, PMMA) were recorded (Fig 1). The results showed the wavelength of 254 nm to be appropriate for subsequent measurements, because at that wavelength the



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Wavelength (nm) Figure 1. UV spectra ofPS and PMMA


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Figure 2 UV chromatograms ofPS andPMMA

absorbance of PMMA could be neglected compared to the absorbance of PS, and UV détecta* could be taken as a selective detector. The UV cut-off of THF was below 254 nm. SEC-UV chromatograms of the same samples at equal concentration are shown in Fig 2 , confirming that the UV signal of PMMA can be neglected compared to the PS signal. The chromatograms clearly indicate that the almost complete UV signal in copolymers belongs to PS. Figs 3 and 4 present chromatograms of PS with différait injection volumes, obtained with I R and UV detector, respectively. 25, 50 and 75 JIL of the stock solution in THF were injected in the system to obtain response factors for both detectors. The R I response factor for PMMA was determined in very much the same way. The elemental analysis of prepared copolymers has shown that three different copolymer compositions were obtained (molar fraction of M M A component of approximately 0.2, 0.5 and 0.8). Taking every composition, there were two samples with the expected difference in molecular mass, based on the variations in the initiator concentration. Fig 5 shows UV chromatograms of the three samples (differing in composition) that are prepared at a higher initiator concentration. Chromatograms of the samples prepared at a lower initiator concentration look very much the same (Fig 6). Corresponding molecular mass averages (M„ and M ) determined by R I and UV specific calibration curves are presented in Table II. w


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Table Π. Molecular mass averages and average sample composition determined by SEC Sample ID M„ (Rl) M„(UV) M„(RI) M„(UV) F(UV)/v»t% PS 24500 43400 84500 78.3 SMMA 0.2x2 38700 43800 85600 139600 78.8 SMMA 0.2x4 71400 43900 142800 103700 51.3 94400 SMMA 0.5x2 53300 40400 142500 51.0 SMMA 0.5x4 78400 146500 50800 20.0 SMMA 0.8x2 144000 139300 78300 58800 20.0 SMMA 0.8x4 169500 87200 67500 172000 PMMA 55100 91300 M„ and M number and mass average molecular mass, respectively, as determined by UVOT RI detector response, F styrene mole fraction in copolymer as determined by UV response at a peak W

Upon the comparison of the raw U V chromatograms of copolymer and homopolymer (PS) samples, it becomes obvious that the height of the chromatogram is related to the overall content of PS component. From this observation, taking the height of the PS homopolymer chromatogram as a fixed 100% (by mass) point, mass fractions of PS in copolymer samples were calculated. The results are included in Table II as the sixth column; calculated average compositions correspond well with the results obtained by the different method (Table I, column 7). Γ

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Figure 3. RI chromatograms ofPS with different concentrations



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Figure 4. UV chromatograms ofPS with different concentrations 35

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Figure 5. UV chromatograms of copolymer samples with different compositions (higher initiator concentration)



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Figure 6. UV chromatograms ofcopolymer samples with different compositions (lower initiator concentration)


335 There is significant doubt in the application of the height of chromatogram in HPLC, when the chromatogram is not a narrow one [12]. In the first instance, die height may be used for symmetric (that is, relatively narrow) peaks at M and only if characteristic detector values (refractive index increment and molecular absorptivity, respectively) ar not A/ -dependent However, it seems that the usage of the height as a representative of chromatogram has given qualitatively correct results in the determination of average copolymer composition. The observed quantitative deviations deserve further discussion. Molecular masses for copolymer samples, calculated from the RI and UV detector responses using corresponding specific polystyrene calibration curves, agree well in the higher molecular mass range, i.e. in A/ -values. The discrepancy is found primarily in the lower molecular mass range, i.e. in M„-values, because the tailing parts of the U V chromatogram are more pronounced. Even more, in the case of copolymer samples with high styrene contents (SMMA 0.5 and particularly SMMA 0.2) there is some indication of a bimodal distribution, the second peak appearing in an oligomer molecular mass range. The indices of homogeneity (MJM„-vahies), calculated according to the RI response are approximately 2.0, but those calculated according to the U V response are much higher, approaching the value of 3.0, depending on the average composition of copolymer. A possible explanation for the low molecular weight peak may be found in the secondary polymerization reaction, that takes place either simultaneously or prior to the main polymerization reaction. The relative height of the second peak seems to increase with the content of styrene cornonomer units; hence, one immediately suspects the styrene homopolymerization to be the secondary reaction. It is well known that styrene is prone to thermally initiated polymerization (see e.g. [13]). This may occur during the warm-up period of the reactor, i.e., before the injection of the initiator solution, but also during the storage of styrene monomer after the removal of inhibitor. The second peak is missing in RI detector responses and it is probably unimportant in determining the overall copolymer proparties, except Mn (see Fig 6 for the three samples prepared at the higher initiator concentration). U V detector is much more sensitive than RI detector in the oligomer molecular mass range, due to the high molecular absorptivity. In dual detection SEC systems, chemical composition along the peak is calculated from the ratio of detector signal intensities (X\ /X , in this case UV response / RI response [9]). The results of the calculations of chemical composition distribution (CCD) data are shown in Fig. 7, together with the corresponding RI detector responses. The data are given for the three samples prepared at the higher initiator concentration, but the results for the set of samples prepared at the Iowa* initiator concentration look very similar. The data indicate that there is no variation in composition across die copolymer elution volume range in case of the copolymer with lower styrene content. w


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Figure 7 UV chromatograms ofcopolymer samples with different compositions (curved lines) and corresponding apparent styrene mass fractions (line connected symbols)

However, there is a strong variation in cases of the copolymers with higher styrene content; in some cases apparent massfractionsexceed the value of 1.0. The possible explanation for the observed, obviously unrealistic styrene massfractionvalues may be given as follows. In this case, investigated samples (especially at higher styrene content) may be regarded as polymer blends, consisting of a major component, SMMA copolymer and a minor component, styrene homopolymer. The chemical composition distributions of the main sample polymer, i.e. SMMA copolymer have to be considered as uniform, since overall monomer conversions were kept below 10% by mass to prevent the copolymer composition shift. Thus, it may be suspected that a stronger U V signal, obtained at higher elution volumes (lower molecular mass range), may be a consequence of the minor polystyrene homopolymer quantities, present in the samples due to the thermal homopolymerization of styrene. Upon decreasing of the molecular mass (i.e. increasing of the elution volume), the samples are gradually enriched with the P§ homopolymer component. Therefore, the discrepancies of the average and die fractional styrene content increases in the same direction, thus producing styrene mass fraction values above 1.0, as a consequence of the applied calculation procedure. As mentioned before, the quantities of styrene homopolymer in investigated copolymer samples remain rather low, thus not affecting the average sample properties.



337 The effect of variation of the apparent composition distribution in both the high and low molecular mass ranges was observed earlier [9], and two reasons were suggested for the explanation [14]: in dual detection SEC systems the chemical composition data will be influenced by the peak dispersion between the detectors as well as by the incorrect interdetector volume (time lag). The obtained results have also pointed out the possible role of the more sensitive UV detector, not only in the copolymer but also in the homopolymer (PS) characterization. Due to the high molecular absorptivity, U V detector is more sensitive in the low molecular mass range and is therefore capable to detect low molecular mass tails or secondary peaks in SEC chromatrograms, not observable by the RI detector. This feature may partially explain the big discrepancies in MMD (especially in A/„-values) amongst different laboratories, obtained previously on selected test samples [15].

Conclusion Dual detection SEC technique comprising RI and U V detection in the characterization of statistical styrene - methyl methacrylate copolymers was described. The obtained results show that the technique is capable for the fast, routine determination of average copolymer composition. The results are comparable to those obtained by other technique (elemental analysis). Calculated molecular mass distributions and averages vary according to the copolymerization conditions. However, the results are dependent on the type of the detector used; lower number average molecular mass values and broader distributions are obtained using the U V calibration curve. Preliminary results show that there is no significant variation of chemical composition with molecular mass for copolymers with lower PS content. For copolymers rich in styrene monomer units, chemical composition varies with molecular mass. In this case, calculated apparent mass fractions of styrene may exceed 100%.

References 1. Hamielec, A.E.: Pure Appl.Chem. 54(29) (1982) 293.



338 2. Inagaki, H; Tanaka, Τ: "Developments in Polymer Characterization" J. V Dawkins, Ed.; Applied Science: London 1982; Vol.3. 3. Yau, W.W.; Kirkland, J.J.; Bly, D.D.: Modern Size Exclusion Liquid Chromatography Wiley- Interscience: New York, 1979. 4. Meister, J.J.; Nicholson, J.C.; Patil, D.R.; Field, L.R.: Macromolecules 19 (1986) 803. 5. Pasadakis, N.; Gaganis, V.; Varotsis, N.: Fuel 80 (2001) 147. 6. Schoenmakers, P.; Blombery, J.; Keskuliet, S.: LC/GCIntern.11 (1996) 718. 7. Sarowha, S.L.S.; Sharma, B.K.; Sharma, C.D.; Bhagat, S.D.: Energy and Fuels 11 (1997) 566. 8. Gratzfeld-Huesgen, A.: HPLC Appl. (87-5) Hewlett Packard note 9. Mori, S.; Suzuki, T.: J. Liq. Chromatog. 4(10) (1981) 1685. 10. Fodor, Zs.; Fodor, Α.; Kennedy, J.P.: Polym. Bull. 29 (1992) 689. 11. Huang, N.J.; Sundberg, D.C.: Polymer 35(26) (1994) 5693. 12. Kipiniak, W.: J. Chromatog. Sci. 19 (1981) 332. 13. Hui, A.W.; Hamielec, A.E.: J. Appl. Polym. Sci. 16 (1972) 749. 14. Trathnigg, B.; Ferchtenhofer, S.; Kollroser, M . : J. Chromatogr. A. 786 (1997) 75. 15. Berek, D.: Results of the Round Robin test of IUPAC WP 1V.2.2 project No. 4., personal communication


Dual Detection High-Performance Size-Exclusion Chromatography

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