Article pubs.acs.org/Macromolecules
SEC Gradients: An Alternative Approach to Polymer Gradient Chromatography. Separation of Poly(methyl methacrylate-statmethacrylic acid) by Chemical Composition Helena Maier,† Frank Malz,† Günter Reinhold,‡ and Wolfgang Radke*,† †
Fraunhofer Institute for Structural Durability and System Reliability LBF, Schlossgartenstrasse 6, D-64289 Darmstadt, Germany PSS Polymer Standards Service GmbH, P.O. Box 3368, D-55023 Mainz, Germany
‡
ABSTRACT: The development of a chromatographic method capable to separate poly(methyl methacrylate-stat-methacrylic acid) samples with methacrylic acid contents of up to 50% by chemical composition is described. For this purpose a gradient ranging from chloroform to dimethylacetamide on a PSS PROTEEMA column was applied. The application of a conventional gradient resulted in severe breakthrough peaks. Therefore, the recently developed concept of SEC gradients was used. No breakthrough peaks were observed, and the peaks corresponding to samples of different content of methacrylic acid were well resolved. A nearly linear relationship between elution volume and methacrylic acid content was observed. The developed method allows determination of the chemical composition distribution of the abovementioned class of polymers.
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composition,6,10−14 which allows obtaining the chemical composition distributions of such polymers. In gradient chromatography the sample is usually dissolved and injected in a solvent (preferably the eluent at the start of the gradient), which allows for adsorption of the sample components. The fraction of the desorption promoting liquid (desorli) is enhanced during the course of the chromatographic experiment, resulting in desorption and elution of the different sample components. Since the amount of desorli required to desorb a particular macromolecular entity depends on the chemical structure of the polymer, a separation as achieved. However, due to the solubility limitations frequently encountered for polymers, it is often not feasible to dissolve the polymer in the eluent at the beginning of the gradient. If the eluent strength of the solvent is higher than that of the initial eluent, parts of the sample or the complete sample are not adsorbed to the stationary phase but migrate through the column without retention, eluting close to the solvent peak. This results in so-called breakthrough peaks.15,16 These breakthrough peaks severely complicate method development and quantitation of the different polymeric components. Recently, an alternative concept called SEC gradients has been introduced to polymer chromatography. 17,18 This approach has high potential to overcome the problem of breakthrough peaks. Contrasting to conventional gradient chromatography, the sample is dissolved in a strong eluent and injected not at the start but into the end of the gradient, i.e., under desorbing conditions. As the polymer is injected into a nonadsorbing eluent, it experiences SEC conditions and
INTRODUCTION Interaction chromatography of polymers has become increasingly popular over the past years, as it allows polymer separations according to other molecular parameters than molar mass. In general, three different molar mass dependences of elution volume at isocratic conditions can be distinguished in polymer chromatography of nonfunctionalized homopolymers. The different molar mass dependences result from the magnitude of the dimensionless interaction parameter, c. This parameter characterizes the interaction of the repeating unit with the stationary phase and can for a given stationary phase be altered by variation of the mobile phase composition or temperature. If c < 0, the macromolecules elute in order of decreasing molar mass before the solvent peak. Such a behavior is termed SEC mode of polymer chromatography. If the interaction parameter c = 0, the macromolecules are neither adsorbed nor excluded from the pores of the stationary phase and elute together with the solvent peak irrespective of their molar mass. At c > 0, the repeating units adsorb to the stationary phase, resulting in a nearly exponential increase of retention time with molar mass and consequently elution after the solvent peak. Because of the strong increase in retention volume with molar mass, even a very weak adsorptive interaction of the repeating units with the stationary phase might result in incomplete polymer elution from the stationary phase for moderate to high molar mass samples, even for narrowly samples narrowly distributed in molar mass. Therefore, gradient methods are applied frequently, if samples containing molecules of different chemical composition need to be separated. Gradient chromatography has been applied to separate blends,1−4 graft,4−6 or block4,7−9 copolymers into the individual components. For statistical copolymers gradients were applied to separate the chains of different chemical © XXXX American Chemical Society
Received: November 15, 2012 Revised: December 21, 2012
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dx.doi.org/10.1021/ma3023553 | Macromolecules XXXX, XXX, XXX−XXX
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Poly(methyl methacrylate-stat-methacrylic acid) samples with different amounts of methacrylic acid were prepared by free radical polymerization in bulk, using 0.1 mol % AIBN as initiator. The polymerizations were performed for 5 h at 65 °C. After the polymerization reaction the samples were dissolved in THF and precipitated from heptane. Characterization details of the samples are given in Table 1.
consequently migrates faster than the surrounding solvent, thereby passing through eluent compositions of decreasing (increasing) amounts of the desorli (adsorli). This overtaking of different eluent compositions continues until the polymer reaches an eluent composition which will result in adsorption to the stationary phase (adsorption threshold). Since the polymer cannot overcome the adsorption threshold, it will continue traveling through the column with the same velocity as the solvent and will finally elute from the column at the composition of the adsorption threshold. The eluent composition of the adsorption threshold depends on the chemical composition of the polymer. Consequently, macromolecules differing in chemical structure elute from an SEC gradient at different retention times; i.e., they are separated by chemical composition. The above explanation of the principle implies that the polymer migrates with a faster velocity than the surrounding solvent due to the exclusion of the polymer from the pores. Consequently, the approach is the more effective, the stronger the polymer is excluded from the pores of the stationary phase. Therefore, columns having small pores but a large pore volume should favorably be applied. To distinguish SEC gradients from conventional gradient chromatography, we will once more clarify and summarize the differences between both modes of polymer gradient chromatography. 1. In conventional gradients the sample is dissolved and injected at adsorbing conditions at the start of the gradient. The sample components elute after the solvent peak. In SEC gradients the sample is dissolved and injected at desorbing conditions into the end of the gradient. The sample components elute before the injected solvent. 2. In conventional gradients the macromolecule remains adsorbed onto the stationary phase until an eluent of sufficient strength desorbs it. In SEC gradients the macromolecule remains desorbed and migrates through eluent layers of different compositions, until it reached the adsorption threshold. Therefore, the macromolecule it is never completely adsorbed. 3. In conventional gradient chromatography the separation can be enhanced by lowering the gradient slope. In SEC gradients the separation range is limited by the exclusion limit and the separation limit of the column. The concept of SEC gradients has been successfully applied to the separation of homopolymer blends. The present paper will extend the application area of SEC gradients to separations of copolymers by chemical composition. Copolymers of (meth)acrylates containing ionic groups are applied as tablet coating (EUDRAGIT), in paint formulations, as adhesives or additives for surface modifications. No separations according to the amount of ionic groups have been reported so far, to the best of our knowledge. Therefore, we will report on the development of a chromatographic method capable to separate poly(methyl methacrylate-stat-methacrylic acid) samples with methacrylic acid contents ranging up to 50% according to the amount of acid groups.
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Table 1. Characterization Details of Samples Used in the Investigation sample
Mn (g/mol)
Mw (g/mol)
methacrylic acid (wt %)
1 2 3 4 5
67 000 62 000 49 000 75 000 22 000
161 000 176 000 164 000 172 000 97 000
9.12 25.12 31.31 41.75 48.23
SEC. For SEC analysis an Agilent Series 1100 (Agilent Technologies, Santa Clara, CA) chromatography system consisting of a Degasser (G1379A), a quaternary pump (G1311A), an autosampler (G1313A), RI detector (G1362A), and a column thermostat Jetstream 2Plus (Duratec Analysentechnik GmbH, Hockenheim, Germany) operated at 50 °C was used. SEC separations were performed in DMAc with 50 mmol/L LiCl and 50 mmol/L acetic acid using a GRAM linear XL column (5 μm particles, 300 mm length and 8 mm i.d.),19 produced by PSS Polymer Standards Service GmbH, Mainz, Germany. The sample concentration and injection volume were 2 g/L and 50 μL, respectively. Calibration was performed using PMMA standards in the molar mass range M = 1020−1.2 × 106 g/mol (PSS Polymer Standards Service GmbH, Mainz, Germany). HPLC. HPLC measurements were performed using degasser Uniflow Degasys (DG-2410), Agilent Series 1200 binary pump (G1312A), an Agilent Series 1200 autosampler (G1329A) (both Agilent Technologies, Santa Clara, CA), and a column thermostat K4 (TECHLAB, Erkerode, Germany) operated at 60 °C. For detection an evaporative light scattering detector (ELSD) ELS 1000, (Polymer Laboratories, Church Stretton, England) was used at the following operating parameters: gas flow = 1.5 SL/min, nebulizer temperature = 100 °C, evaporator temperature = 200 °C. Data acquisition and evaluation were performed using PSS WINGPC Unity Software (PSS Polymer Standards Service GmbH, Mainz, Germany). Separations were carried out at 1 mL/min using a Proteema SEC-column (3 μm particles, pore size 100 Å, 300 mm length, and 8 mm i.d.), produced by PSS Polymer Standards Service GmbH, Mainz, Germany. Samples were dissolved at a concentration of 1 g/L in the respective solvent. The injection volume was typically 100 μL. Since the HPLC system does not allow starting the gradient prior to sample injection, the gradient was started by a blank run, and sample introduction was performed by a separate injection. Furthermore, flushing and equilibration steps needed to be implemented as part of the gradient program. Since the gradient is set to the initial conditions during the sample pretreatment phase of the autosampler, the time between the end of the blank run and the sample injection was optimized using the “overlap injection cycle” option. Alternatively, an isocratic gradient step at the same composition as at the time of injection can be added at the beginning of the gradient program (e.g., gradient given in Figure 3). Determination of Recoveries. For the determination of the recoveries defined amounts of the samples were injected and run isocratically. After the column a well-defined volume of the effluent covering the complete elution range of the sample was collected. Using the injected mass and the collected volume, the theoretical sample concentration was calculated. The true sample concentration was determined from a response calibration curve established by injecting well-defined amounts of the respective samples. The recoveries were taken as the ratios of the experimentally determined concentrations to the theoretical ones.
EXPERIMENTAL SECTION
Samples and Solvents. Chloroform (CHCl3) and N,Ndimethylacetamide (DMAc) were obtained from Merck Schuchardt OHG (Hohenbrunn, Germany) and used as received. Technical tetrahydrofuran (THF, BASF Ludwigshafen, Germany) was dried over calcium hydride and distilled. B
dx.doi.org/10.1021/ma3023553 | Macromolecules XXXX, XXX, XXX−XXX
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1 H NMR Spectroscopy. The compositions of the samples were determined by 1H NMR spectroscopy in deuterated dimethyl sulfoxide (DMSO-d6) using the intensity ratio (signal area) of the resonances of the methoxy protons at 3.8−3.4 ppm to the methyl resonances at 1.5− 0.5 ppm. The 1H NMR spectra were acquired on a 400 MHz (9.4 T) Mercury-VX (Varian Inc., Sao Palo, CA) spectrometer equipped with a 5 mm inverse probe. 1H measurements were executed using a 90° pulse, 2.6 s acquisition time (32K data points, 16 ppm spectral width), 10 s relaxation delay, 128 accumulated scans, simple zero addition, and exponential multiplication with lb = 0.3 Hz, respectively. The 1H NMR spectra were recorded at 30 °C and processed and evaluated using MestReNova 7 software (Mestrelab Research, Santiago de Compostela, Spain).
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RESULTS AND DISCUSSION
In order to establish a separation by chemical composition, a suitable adsorli and a suitable desorli needed to be identified. The Proteema column was had been used in a previous investigation and had been identified to adsorb PMMA from chloroform as the eluent, while THF had been used to desorb PMMA. However, isocratic runs of the poly(methyl methacrylate-stat-methacrylic acid) samples using THF as eluent revealed complete adsorption for the samples of high acrylic acid content. Thus, a solvent of higher eluent strength was required. Therefore, DMAc was applied, which in isocratic experiments resulted in elution of all samples at an elution volume of ∼6.1 mL despite the significant lower molar mass of sample 5 as compared to the others. This indicates that the pore size of the column is too low to allow for an effective separation by size for the samples under investigation. Small pore size relative to the size of the molecules is a prerequisite for an effective separation by chemical composition using SEC gradients, in order to minimize molar mass effects on the elution volume. Since the sample of highest acid content was not soluble in pure CHCl3, the samples were dissolved in DMAc and a first attempt was undertaken using a fast but conventional gradient from CHCl3 to DMAc. These experiments resulted in bimodal peaks for all samples. The intensity of the first peak, which eluted close to the injected solvent, increased with deceasing amount of methacrylic acid, while a second peak eluted within the gradient Thus, the first peak showed the typical behavior of a breakthrough peak. Thus, either optimization of the sample solvent or the application of a SEC gradient was required to overcome the problem. The next attempt was undertaken using a SEC gradient from 100% CHCl3 to 100% DMAc within 6 min. Figure 1 shows the corresponding chromatograms. All samples clearly elute at retention volumes higher than 7 mL. Since the elution time of all samples in pure DMAc was found to be ∼6.1 mL, this indicates that all samples have reached their adsorption thresholds within the column and were retarded. With increasing methacrylic acid content the samples elute at higher elution volume. This is due to the fact that these samples already adsorb at quite low CHCl3 amounts. Because the CHCl3 amount increases toward the lower elution volumes, the samples of higher acrylic acid content are more strongly retarded than those of lower content. The samples containing more than 20% acrylic acid are already nicely separated from each other, while no separation is observed for the samples containing 10 and 20% acrylic acid. The maximum separation range in SEC gradients is limited by the exclusion and separation limits of the column. Since the separation limit of the applied Proteema column was estimated
Figure 1. Comparison of the normalized chromatograms in a linear SEC gradient ranging from 100% CHCl3 to 100% DMAc. Samples dissolved in DMAc. Gradient: 0−5 min: 100% DMAc; 5−20 min: 100% CHCl3; 20−26 min linear increase from 100% CHCl3 to 100% DMAc. Injection time 26 min. Sample 1 (); sample 2 (· · ·); sample 3 (− −); sample 4 (···); sample 5 (− - −).
to be ∼12 mL, using low molar mass standards, attempts were undertaken to further optimize the separation, by stronger retarding the late eluting peaks; i.e., the adsorption thresholds have to be adjusted closer to the injection solvent. In other words, the DMAc content at the time of injection should be lowered. Therefore, an experiment was run in which the final DMAc content was restricted to 50%. The corresponding chromatograms are shown in Figure 2. Clearly the separation
Figure 2. Comparison of the chromatograms in a linear SEC gradient ranging from 100% CHCl3 to 50% DMAc. Samples dissolved in DMAc. Gradient: 0−5 min: 100% DMAc; 5−20 min: 100% CHCl3; 20−26 min linear increase from 100%CHCl3 to 50% DMAc. Injection time 26 min. Sample 1 (); sample 2 (· · ·); sample 3 (− −); sample 4 (···); sample 5 (− - −).
range has been expanded. While in Figure 1 all peaks eluted within an elution volume range of 1.5 mL, the separation range in Figure 2 now spans 3.5 mL. This is mainly due to the fact that the samples of higher acrylic acid content are more retarded. In addition, the peaks of lower acrylic acid content are also slightly stronger retarded, which results now in a beginning separation of the formerly nonresolved peaks for the samples of 10 and 20% acrylic acid. C
dx.doi.org/10.1021/ma3023553 | Macromolecules XXXX, XXX, XXX−XXX
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It is interesting to note the continuous decrease in the peak intensities with increasing retention time (acid content), which brings up the question of sample recovery. Therefore, recoveries were estimated for the two samples of highest amounts of methacrylic acid determined in 50:50 mixtures of CHCl3 and DMAc. The determined recoveries amounted to 80 and 85%. Therefore, the decrease of peak intensities cannot be explained by decreasing sample recoveries. It seems more plausible that the continuously decreasing intensities are due to an eluent depending response. To further optimize the separation of the early eluting peaks, the gradient was started at a slightly higher DMAc content (5%). This was expected to slightly move the peaks to lower elution volume. The resulting chromatograms are depicted in Figure 3. A clear separation of all five peaks is observed. Figure 4. Dependence of elution volume on composition of poly(methyl methacrylate-stat-methacrylic acid) for a linear SECgradient ranging from 95% CHCl3 to 50% DMAc.
the respective chromatograms allows determination of the chemical composition distributions, which are represented in Figure 5. For the calculation of the chemical composition
Figure 3. Comparison of the normalized chromatograms in a linear SEC gradient ranging from 95% CHCl3 to 50% DMAc. The straight line represents the eluent composition at the detector. Samples dissolved in DMAc. Gradient: 0−3 min; 50% DMAc; 3−8 min: 100% DMAc; 8−23 min: 100% CHCl3; 23−26 min: 5% DMAc; 26−32 min: linear increase from 5% to 50% DMAc. Injection time 32 min. Sample 1 (); sample 2 (· · ·); sample 3 (− −); sample 4 (···); sample 5 (− - −).
At this point it should be repeated that the effectivity of the SEC gradient approach crucially depends on an effective exclusion of the analyte molecules from pores of the stationary phase. Samples of lower molar mass might not be sufficiently excluded to catch up with the solvent front corresponding to the adsorption threshold. As a consequence, the elution volume for a particular sample in a SEC gradient is limited by the sample’s elution volume at pure SEC conditions on the one hand and the column’s separation limit on the other. In the present investigation the SEC elution volumes for all samples, even of samples 5 having the lowest molar mass, nearly coincide at ∼6.1 mL. Therefore, the separation should not be affected by the molar masses and molar mass distributions of the samples. We noticed from preliminary solubility experiments that ∼12% of DMAc in CHCl3 is required to dissolve the sample of highest methacrylic acid content. Since Figure 3 shows that only the sample with the lowest methacrylic acid content elutes below this critical value, the separation must be based on adsorption rather than on precipitation. In Figure 4, the elution volume is plotted versus the composition of the samples. A nearly linear dependence is observed. The use of this calibration curve in conjunction with
Figure 5. Chemical composition distributions of poly(methyl methacrylate-stat-methacrylic acid)s containing 10−50% methacrylic acid. Sample 1 (); sample 2 (· · ·); sample 3 (− −); sample 4 (···); sample 5 (− · −).
distributions the effect of the variation of the detector response with sample composition was neglected; i.e., it was assumed that the response across a single peak stays constant. The chemical composition distribution was calculated according to CCD(w) = C −1S(V )/σ C=
∫ S(V )/σ dV
(1)
where CCD(w) is the value of the chemical composition distribution at a weight fraction w of methacrylic acid, S(V) is the detector signal at the elution volume, and σ = dw/dV is the slope of the dependence of polymer composition on elution volume, which in the present case is assumed to be constant (see Figure 4). C is the normalization constant. D
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(7) Park, S.; Park, I.; Chang, T.; Ryu, C. Y. J. Am. Chem. Soc. 2004, 126 (29), 8906−8907. (8) Fandrich, N.; Falkenhagen, J.; Weidner, S. M.; Staal, B.; Thuenemann, A. F.; Laschewsky, A. Macromol. Chem. Phys. 2010, 211 (15), 1678−1688. (9) Trathnigg, B.; Malik, M. I.; Pircher, N.; Hayden, S. J. Sep. Sci. 2010, 33, 2052−2059. (10) Krämer, I.; Hiller, W.; Pasch, H. Macromol. Chem. Phys. 2000, 201, 1662−1666. (11) Braun, D.; Kramer, I.; Pasch, H. Macromol. Chem. Phys. 2000, 201 (10), 1048−1057. (12) Albrecht, A.; Brüll, R.; Macko, T.; Pasch, H. Macromolecules 2007, 5545−5551. (13) Brun, Y. J. Liq. Chromatogr. Relat. Technol. 1999, 22, 3067− 3090. (14) Striegel, A. M. J. Chromatogr., A 2002, 971, 151−158. (15) Jiang, X.; van der Horst, A.; Schoenmakers, P. J. J. Chromatogr., A 2002, 982 (1), 55−68. (16) Reingruber, E.; Bedani, F.; Buchberger, W.; Schoenmakers, P. J. Chromatogr., A 2010, 1217, 6595−6598. (17) Schollenberger, M.; Radke, W. J. Chromatogr., A 2011, 1218 (43), 7828−7831. (18) Schollenberger, M.; Radke, W. Polymer 2011, 52, 3259−3262. (19) Adler, M.; Pasch, H.; Meier, C.; Senger, R.; Koban, H.-G.; Augenstein, M.; Reinhold, G. e-Polym. 2004, 55.
Apparently, there is a distinct overlap of neighboring peaks indicating a substantial chemical heterogeneity of the samples. For example, the sample having an average methacrylic acid content of 30 wt % contains chains with methacrylic acid contents ranging from 25% to 40%. The derived chemical heterogeneity distributions are rather symmetrical, which probably is a consequence of very similar reactivities of the monomers, resulting in a low compositional drift throughout the polymerization process. It can also be seen that the chemical composition distributions become broader with increasing amount of the methacrylic acid. This increase in heterogeneity with increasing methacrylic acid content can be explained simply, assuming similar reactivities of both monomers at the polymerization conditions. Since a pure PMMA as well as a pure poly(methacrylic acid) are chemically homogeneous, while the copolymerization will in any case result in some heterogeneity due to the statistics involved in any polymerization process, the dependence of heterogeneity on composition has to be a maximum function. The exact position of the maximum will depend on the reactivity parameters and the polymerization conditions. The chemical heterogeneity might severely affect the application properties of these materials. For example, the time for dissolution and the solvent composition at which dissolution occurs clearly depend on the chemical composition. Thus, at a given average composition, a sharper transition for dissolution is expected for samples of lower chemical heterogeneity, while a broad chemical composition distribution will result in a less defined transition. Thus, the developed method might be helpful to understand the application properties of such kinds of materials and their dependences on polymerization conditions.
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CONCLUSIONS
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AUTHOR INFORMATION
The recently developed concept of SEC gradients was applied to set up a method to separate poly(methyl methacrylate-statmethacrylic acid) samples according to the amount of methacrylic acid. Suitable adjustments of the eluent compositions at the beginning and end of the gradient allowed optimizing the separation of the peaks. High recoveries were obtained. The developed method allows determination of the chemical composition distribution of poly(methyl methacrylate-stat-methacrylic acid)s. A clear dependence on chemical heterogeneity on average chemical composition could be verified.
Corresponding Author
*E-mail
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
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