Comprehensive Two-Dimensional Liquid Chromatography of

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Comprehensive Two-Dimensional Liquid Chromatography of Stereoregular Poly(Methyl Methacrylates) for Tacticity and Molar Mass Analysis Khumo Maiko,† Mathias Hehn,‡ Wolf Hiller,‡ and Harald Pasch*,† †

Department of Chemistry and Polymer Science, University of Stellenbosch, Private Bag X1, Matieland, 7602 Stellenbosch, South Africa ‡ Technical University of Dortmund, Faculty of Chemistry and Chemical Biology, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany ABSTRACT: For the first time, stereoregular poly(methyl methacrylates) (PMMAs) were separated according to tacticity on a carbon-based stationary phase using solvent gradient interaction chromatography (SGIC). This stationary phase provides superior separation capabilities, enabling the baseline separation of highly isotactic and syndiotactic PMMAs of different molar masses. Twodimensional liquid chromatography was performed with the SGIC method separating, according to tacticity in the first dimension coupled to size-exclusion chromatography separating according to molar mass in the second dimension, thus providing comprehensive information on both microstructure and molar mass.

S

Cho et al. used temperature gradient interaction chromatography (TGIC) with MALDI-TOF MS detection for the analysis of stereoregular PEMAs and studied the tacticity effect on the SEC behavior of stereoregular PEMA.21,22 They showed that TGIC on its own is not sufficient to characterize both tacticity and molar mass. Carbon-based stationary phases have numerous advantages over silica-based materials in that they are very stable in aggressive solvents, there is no shrinkage or swelling, and column bleeding is significantly reduced. They can be used with reversed phase and normal phase eluents. They can be utilized over the whole pH range [0−14] as well as at very high temperatures (∼200 °C). These materials are also believed to have increased selectivity regarding closely related compounds.23 They have been extensively utilized for the separation of carbohydrates,24 peptides,25 natural products,26 and pharmaceuticals.27 In polymer science, the vast majority of separations utilizing carbon-based stationary phases so far were high temperature applications for the analysis of polyolefins. Macko and Pasch separated polypropylene with respect to tacticity using hightemperature adsorption liquid chromatography.28,29 Ginzburg et al. carried out high temperature 2D chromatography, separating polypropylene with respect to tacticity and molar mass.30

ynthetic polymers can be heterogeneous with respect to molar mass, chemical composition, chain architecture, functionality, and tacticity. It has been shown that the tacticity influences the physical properties of poly(methyl methacrylate) (PMMA).1−7 Various techniques have been used to study stereoregular PMMAs.8−15 Stereoregular PMMAs have also been explored as stationary phases in chromatography.16 The molecular structure of isotactic (mmm), syndiotactic (rrr), and atactic (mrr) PMMA are presented in Figure 1. The first liquid chromatographic separation of stereoregular PMMAs was presented by Inagaki et al. using thin layer chromatography, the separation being influenced both by tacticity and molar mass.17 Berek et al. attempted to separate PMMA with regard to tacticity using liquid chromatography at limiting conditions on bare silica stationary phases. Although there were slight differences in the curves of different stereoregular PMMAs, the differences were not sufficient for a proper separation.18 Later, Berek et al. used liquid chromatography at the critical adsorption point (LC-CAP) on a Nucleosil silica stationary phase using various binary solvents to separate stereoregular PMMA with respect to tacticity without the influence of molar mass. However, the disadvantage of this method was the total adsorption of high molar mass PMMAs.19 Following this work, Janco et al. separated poly(ethyl methacrylate) (PEMA) by online twodimensional liquid chromatography (2D-LC) with SEC as the first dimension separating with regard to molar mass on a poly(styrene-divinylbenzene) stationary phase followed by LCCAP as the second dimension separating with regard to tacticity on a NH2-silica stationary phase.20 © 2013 American Chemical Society

Received: July 18, 2013 Accepted: September 20, 2013 Published: September 20, 2013 9793

dx.doi.org/10.1021/ac402203c | Anal. Chem. 2013, 85, 9793−9798

Analytical Chemistry

Article

Figure 1. Structure of isotactic, syndiotactic, and atactic poly(methyl methacrylate).

Table 1. Chemical Structure, Molar Masses, and Tacticities of Stereoregular PMMAs

molar massa (g/mol)

a

percentage tacticityb

manufacturer

label

Mn

Mw

Mp

mm (iso)

mr (hetero)

rr (syndio)

PSS PSS PSS PSS PL PL

it-PMMA 4890 it-PMMA 12000 st-PMMA 7870 st-PMMA 24100 st-PMMA 4900 st-PMMA 60150

3750 11500 7310 23200 4530 57450

4660 12400 7660 24400 4960 58900

4890 12000 7870 24100 4900 60150

86.5 92 1.5 1.1 4.4 1.0

3.1 3.0 24.5 21.3 38 19.1

9.4 5.0 74 77.6 57.2 79.9

Molar masses as given by the manufacturer. bPercentage triad tacticity as determined by 1H NMR.

2.2. Instrumentation. All experiments were carried out on an Agilent 1200 series HPLC system (Agilent Technologies, Böblingen, Germany). This system consisted of a degasser, quaternary pump, auto sampler, thermostatted column compartment, isocratic pump, variable wavelength UV detector, instant palm pilot, and an Agilent 1260 evaporative lightscattering detector (ELSD). The recording and processing was carried out with WinGPC software (version 8.0) (Polymer Standards Service GmbH, Mainz, Germany). All the 1H NMR spectra were acquired on a Varian Unity 400 MHz spectrometer (Agilent, Santa Clara). All samples were dissolved in CDCl3. 2.3. One-Dimensional SGIC and SEC Experiments. The development of the solvent gradient method for the separation of the stereoregular PMMA was carried out on a Hypercarb column (Thermo Scientific, Dreieich, Germany), 150 × 4.6 mm i.d., packed with porous graphite particles with an average particle diameter of 5 μm and a pore size of 250 Å. For the SGIC, dichloromethane (DCM),and acetone were used as solvents at a flow rate of 0.5 mL/min. The solvent gradient program was as follows: time/DCM/acetone (min/vol%/vol %): 0/53.5/46.5, 5/53.5/46.5, 10/100/0, 12/100/0, 14/53.5/ 46.5, and 20/53.5/46.5. The column oven was maintained at a temperature of 30 °C. For the SEC experiments, THF was used

To the best of our knowledge, 2D-LC of stereoregular poly(methyl methacrylates) with regard to tacticity and molar mass on carbon-based stationary phases has never been reported. In this study, solvent-gradient interaction chromatography (SGIC) shall be used to separate stereoregular PMMAs with regard to tacticity. This separation is coupled online to SEC for the separation of the PMMA fractions with respect to molar mass.

2. EXPERIMENTAL SECTION 2.1. Materials. Highly isotactic PMMA (it-PMMA) and syndiotactic PMMA (st-PMMA) were products of Polymer Laboratories (PL)/Varian (Church Stretton, England) and Polymer Standards Service GmbH (PSS) (Mainz, Germany). All samples had the same end groups, as shown in Table 1. HPLC-grade solvents were purchased from Sigma Aldrich (Steinheim, Germany): dichloromethane (>99.8% purity) with amylene as a stabilizer and acetone (>99.8% purity). The tacticities and molar masses of the samples are summarized in Table 1. The stereoregular PMMAs were synthesized via anionic polymerization with 1,1-diphenylhexyl lithium (DPHLi) as an initiator, as described by Allen et al.31 9794



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as a solvent at a flow rate of 1 mL/min. The sample concentration was 1 mg/mL with the sample dissolved in the initial composition of the gradient solvent. To adjust the critical conditions for st-PMMA, as shown in Figure 2a, only approximate concentrations were used. The injection volume was 20 μL. The ELSD was set at a gas pressure of 3.0 bar, an evaporating temperature of 90 °C and a gain of 3.

valve EC8 W (VICI VALCO instruments, Texas) equipped with two 100 μL loops. After the initial injection (50 μL), fractions from the first dimension were transferred into the second dimension every 8 min in order to inject 100 μL of eluate from the first dimension into the second dimension.

3. RESULTS AND DISCUSSION 3.1. SGIC and SEC. In a first attempt to separate PMMA according to tacticity, the critical point of adsorption for stPMMA was determined. LC-CAP is known to be a very sensitive method for the separation of complex polymers regarding chemical composition or microstructure irrespective of molar mass. For the determination of the critical point, a series of st-PMMA with different molar masses were analyzed in DCM-acetone as the mobile phase. The composition of the mobile phase varied from an exclusion-promoting mobile phase (80% DCM) to an adsorption-promoting mobile phase (50% DCM). As can be seen, the different modes of liquid chromatography of polymers, SEC, LC-CAP, and LAC, were obtained in different mobile phase compositions (see Figure 2a). Under the LC-CAP conditions, all st-PMMA samples elute at the same elution volume irrespective of their molar masses (see Figure 2b). Unfortunately, it was found that under the LCCAP conditions for st-PMMA, all it-PMMA samples irreversibly adsorbed on the stationary phase (see Figure 2b for itPMMA 12000). Therefore, LC-CAP conditions were found not to be suitable for the tacticity separation. The different peak intensities for st-PMMA were caused by the fact that only approximate concentrations were used and that the ELSD signal response is molar mass dependent. As separation under isocratic conditions was not successful, the following experiments were conducted under solvent gradient conditions (SGIC). Starting from a mobile phase with a low content of DCM, the DCM content in the mobile phase was increased with increasing elution volume. Similar to the previous experiments, for SGIC, a mixture of DCM and acetone was found to be suitable as a mobile phase. Figure 3 (panels a and b) show the overlaid chromatograms of individual injections of isotactic and syndiotactic PMMAs of different molar masses from both manufacturers. The volume percentage DCM was increased linearly from 53.5 vol % to 100 vol %, as indicated by the dashed line. Under these conditions, the entire sample is eluted from the column with full recovery. The SGIC chromatograms show the baseline separation of the isotactic and syndiotactic PMMAs, with the order of elution in agreement with that observed by Cho et al. on a C-18-bonded silica stationary phase with it-PEMA more strongly retained than st-PMMA.21 The late elution of it-PMMA in the present case indicates that the separation is based mainly on full adsorption−desorption. Preliminary gradient experiments with an initial composition of DCM of 40 vol % showed a molar mass dependency for the syndiotactic PMMAs, which were eluting in liquid adsorption (LAC) mode. To avoid increased adsorption with increasing molar mass, LC-CAP conditions for st-PMMA at a composition of DCM-acetone 53.5:46.5 vol % were used as the initial eluent of the SGIC separations. The percentage triad tacticity of st-PMMA 4900 g/mol, as determined from the bulk 1H NMR, was as follows: mm/mr/rr (4.4/37.5/57.2). Since the st-PMMA 4900 g/mol (and also the st-PMMA 60150) eluted in two peaks (see Figure 3a), we fractionated this sample and analyzed the two resulting fractions with 1H NMR. The first fraction had the following

Figure 2. Calibration curves of st-PMMA. (a) Various molar masses at various solvent compositions showing SEC, LC-CAP, and LAC modes and an overlay of st-PMMA and it-PMMA at a mobile phase composition of DCM-acetone 53.5:46.5% by volume. (b) Stationary phase: hypercarb, mobile phase: DCM−acetone 80:20 (black), 53.5:46.5 (red), 50:50 (green) % by volume.

2.4. Two-Dimensional Liquid Chromatography. The sample concentration for the 2D-LC experiments was approximately 10 mg/mL with an injection volume of 50 μL. The first dimension separation with respect to tacticity was carried out on the Hypercarb column with the specifications mentioned above at a flow rate of 0.0125 mL/min. The second dimension separation with respect to molar mass was carried out on a PL gel Mixed E column (PL/Varian, Church Stretton, England), 300 × 7.5 mm i.d., packed with styrenedivinylbenzene copolymer with a particle diameter of 3 μm. This column was kept at room temperature (∼ 20−23 °C) and operated at a flow rate of 1 mL/min. The coupling of SGIC and SEC was achieved by an electronically controlled eight port 9795



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higher than the triad percentage in the first fraction. This PMMA fraction was labeled as “unidentified” tacticity PMMA (ut-PMMA). Figure 3c shows the overlaid SEC chromatograms of the individual injections of all stereoregular PMMAs. All samples had a unimodal molar mass distribution, which was expected as these samples were synthesized via anionic polymerization. itPMMA 4890 and st-PMMA 4900 eluted at the same elution volume, thus showing the inability of SEC alone to separate polymers with respect to tacticity. 3.2. Two-Dimensional Liquid Chromatography. After having obtained a baseline separation of st-PMMA and itPMMA by SGIC, the molar masses of the samples and their respective fractions were to be analyzed by SEC. For a complete correlation of the tacticity and the molar mass separations, comprehensive two-dimensional liquid chromatography (2D-LC) was used. The separation efficiency of the 2DLC method was tested by injecting a binary mixture of itPMMA and st-PMMA of similar molar masses. The 2D-LC contour plot based on ELSD detection is shown in Figure 4, showing the corresponding reconstructed SGIC chromatogram on the y axis, as the first dimension and the SEC chromatogram on the x axis as the second dimension. This 2D-LC contour plot shows the superiority of this separation method, where the two components of blend 1 with similar molar masses are baseline separated in the first dimension with respect to tacticity. The st-PMMA has a unimodal distribution in the tacticity dimension due to the incorporated LC-CAP conditions before the start of the gradient, while the it-PMMA component seems to indicate a molar mass separation as shown by the multimodal peak observed in the tacticity dimension. The 2D-LC method was also tested using a quaternary mixture, as shown in Figure 5. The SGIC projection on the y axis showed two distinct regions of elution, the first region had a unimodal distribution due to the two st-PMMAs, eluting in the LC-CAP mode. The second region had a multimodal distribution, the first peak belonging to the low molar mass utPMMA of the st-PMMAs. This peak was more visible in the quaternary blend as compared to the binary blend. The remaining peaks belonged to the two it-PMMAs, eluting in the LAC mode. Due to the inability to obtain it-PMMAs with a molar mass higher than 12000 g/mol, a more detailed study on resolution could not be conducted. The resolution in the SEC dimension for the it-PMMAs was not as good as observed for the st-PMMAs, which had a greater variety of molar masses. Further optimization of this separation is part of future investigations.

4. CONCLUSION Blends of stereoregular poly(methyl methacrylates) with different isotactic and syndiotactic sequence distributions were successfully separated using SGIC at ambient temperature. Highly syndiotactic PMMAs were retained less strongly than the highly isotactic PMMAs on a carbon-based stationary phase with a gradient of DCM and acetone. There were two distinct regions of elution, the first region due to the st-PMMAs and the second region due to the it-PMMAs. The SGIC method utilizes only 10 mL of mobile phase for the total analysis which is less than any other HPLC method developed for the separation of polymethacrylates with respect to tacticity. The SGIC method is superior in that it separates with respect to tacticity more or less irrespective of molar mass.

Figure 3. Overlays of SGIC chromatograms of syndiotactic and isotactic PMMAs (a) (st-PMMA 4900 + st-PMMA 60150, it-PMMA 4890 + it-PMMA 12000) and (b) (st-PMMA 24000 + it-PMMA 12000). (c) Overlay of SEC chromatograms (st-PMMA 4900 + stPMMA 7870 + st-PMMA 24000 + st-PMMA 60150, it-PMMA 4890 + it-PMMA 12000). All molar masses are in g/mol.

percentage triad tacticity: mm/mr/rr (7.7/35.7/56.8), being more or less the same composition of mm, mr, and rr triads, as observed for the bulk 1H NMR analysis. The second fraction contained an impurity which could not be identified and which overlapped with the mm and rr triad signals of the PMMA present in this fraction. We could only accurately determine the mr triad percentage in the second fraction, which was 10% 9796



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Figure 4. Online 2D-LC contour plot of binary blend 1 (st-PMMA 4900 + it-PMMA 4890) showing the SGIC and SEC projections. All molar masses are in g/mol.

Figure 5. Online 2D-LC contour plot of quaternary blend 2 (it-PMMA 4890 + it-PMMA 12000 + st-PMMA 7870 + st-PMMA 24000) showing the SGIC and SEC projections. All molar masses are in g/mol.

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ACKNOWLEDGMENTS The authors would like to thank the National Research Foundation and Stellenbosch University, both South Africa, for funding and PSS GmbH for the stereoregular poly(methyl methacrylate) samples. The support of Thermo Scientific by providing the Hypercarb column is greatly appreciated.

The 2D-LC separation was carried out by combining SGIC (separating PMMA by tacticity) and SEC (separating PMMA by hydrodynamic size). The sensitivity of the Hypercarb stationary phase to the tacticity of the polymer allowed for the successful characterization of the stereoregular PMMAs with very high selectivity.

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AUTHOR INFORMATION

Corresponding Author

REFERENCES

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*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 9797



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Comprehensive Two-Dimensional Liquid Chromatography of

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