Utility of Chromatographic and Spectroscopic Techniques for a

Laboratory for Polymer Chemistry and Technology, National Institute of Chemistry, Hajdrihova 19, SI-1000 ... Publication Date (Web): September 4, 2012...
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Utility of Chromatographic and Spectroscopic Techniques for a Detailed Characterization of Poly(styrene‑b‑isoprene) Miktoarm Star Copolymers with Complex Architecture Tina Šmigovec Ljubič,† Katja Rebolj,† David Pahovnik,† Nikos Hadjichristidis,§ Majda Ž igon,†,‡ and Ema Ž agar*,†,‡ †

Laboratory for Polymer Chemistry and Technology, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia Chemical and Life Sciences & Engineering Division, KAUST Catalysis Center (KCC), King Abdullah University of Science and Technology (KAUST), Bldg Ibn Sina (#3) West, Thuwal 23955-6900, Kingdom of Saudi Arabia ‡ Centre of Excellence for Polymer Materials and Technologies, Tehnološki Park 24, SI-1000 Ljubljana, Slovenia §

S Supporting Information *

ABSTRACT: We analyzed various miktoarm star copolymers of the PS(PI)x type (x = 2, 3, 5, 7), which consist of one long polystyrene (PS) arm (82 or 105 kDa) and various numbers of short polyisoprene (PI) arms (from 11.3 to 39.7 kDa), prepared by anionic polymerization and selective chlorosilane chemistry. The length of the PI arm in stars decreases with the number of arms, so that the chemical compositions of all PS(PI)x samples were comparable. Our aim was to determine the purity of samples and to identify exactly the constituents of individual samples. For this purpose we used a variety of separation techniques (size-exclusion chromatography (SEC), reversed-phase liquid-adsorption chromatography (RP-LAC), and two-dimensional liquid chromatography (2D-LC)) and characterization techniques (UVMALS-RI multidetection SEC system, NMR, and MALDI-TOF MS). The best separation and identification of the samples’ constituents were achieved by RP-LAC, which separates macromolecules according to their chemical composition, and a subsequent analysis of the off-line collected fractions from the RP-C18 column by SEC/UV-MALS-RI multidetection system. The results showed that all PS(PI)x samples contained the homo-PS and homo-PI in minor amounts and the high-molar-mass (PS)y(PI)z (y > 1) species, the content of which is higher in the samples PS(PI)5 and PS(PI)7 than in the samples PS(PI)2 and PS(PI)3. The major constituent of the PS(PI)2 sample was the one with the predicted structure. On the other hand, the major components of the PS(PI)x (x = 3, 5, and 7) samples were the stars consisting of a smaller number of PI arms than predicted from the functionalities of chlorosilane coupling agents. These results are in agreement with the average chemical composition of samples determined by proton NMR spectroscopy and characterization of the constituents by MALDI-TOF MS.

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INTRODUCTION Anionic polymerization using the high-vacuum technique has been proven to be a very powerful tool for the synthesis of welldefined macromolecules with complex macromolecular architectures.1−6 Using this technique, a variety of well-defined block copolymers with star structure (miktoarm star copolymers) and with narrow molar mass and compositional dispersity have been synthesized in order to establish the relationship between the structure and properties (e.g., self-assembly in selective solvents and in bulk), which is essential for designing polymeric materials with predetermined properties and high-tech applications (nanolithography, drug delivery, etc.).7−14 Ideally, miktoarm star copolymers, prepared by anionic polymerization high-vacuum techniques, should be with a defined number of chemically different arms and with a narrow molar mass distribution of arms’ lengths.1−4 In practice, however, even this methodology results in a small amounts of byproducts, even if the reaction mixture is purified by fractional precipitation. © 2012 American Chemical Society

Thus, miktoarm star copolymers can show multiple distributions in terms of various molecular properties, such as the molar mass, the chain architecture, and the chemical composition, as well as the presence of residual homopolymers. The structural uniformity of miktoarm stars is usually examined by conventional size-exclusion chromatography (SEC), membrane osmometry (MO), light scattering (LS), viscometry, and NMR. However, these characterization techniques usually misleadingly indicate a high degree of structural homogeneity of the miktoarm star copolymers due to their low resolution power, small differences in hydrodynamic volume of the miktoarm star with changing star functionality, and/or small amounts of byproducts and/or homopolymers being present. A much more efficient method for the Received: June 18, 2012 Revised: August 24, 2012 Published: September 4, 2012 7574

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spectroscopy and SEC/UV-MALS-RI to determine the chemical composition and the molar mass, respectively. Size-Exclusion Chromatography Coupled to a Multidetection System (SEC/UV-MALS-RI). Theoretical background of SEC/UVMALS-RI is in the Supporting Information. The separations of original samples and fractions by SEC were carried out in THF using an Agilent 1260 HPLC chromatograph, a ResiPore analytical column (7.5 mm × 300 mm) with a precolumn (Polymer Laboratories, UK). The ResiPore column has a wide pore size distribution, which covers the molar masses from 200 to 400 000 Da. For the detection we used successively connected detectors online: an ultraviolet (UV) detector, operating at a wavelength of 260 nm (Agilent 1260 VWD); a multiangle lightscattering (MALS with 18 angles) detector (DAWN-HELEOS, Wyatt Technology Corp.); and a differential refractive index (RI) detector (Optilab rEX, Wyatt Technology Corp.). The nominal eluent (THF) flow rate was 1 mL/min, the injection volume was typically 100 μL, and the mass of the samples injected onto the column was typically 150 μg. Reversed-Phase Liquid-Adsorption Chromatography (RPLAC). For the gradient LC experiments a Nucleosil C18 reversedphase column was used (250 mm × 4 mm i.d.; pore size: 120 Å; particle size: 5 μm; Macherey-Nagel, Germany), and the acetonitrile (ACN gradient grade; Sigma-Aldrich, Germany) and tetrahydrofuran (THF p.a.; Merck, Germany) as the solvents. The miktoarm star copolymers were dissolved in THF/ACN = 80/20, v/v, at a solution concentration of 5 mg/mL. The gradient that was used ran from 60 to 80% v/v of THF in ACN in 15 min. The flow rate for the one-dimensional gradient LC was 1 mL/min, and the injected volume was 20 μL. For the detection an LC-235 DAD (PerkinElmer, operating at 254 nm) and an evaporative light-scattering detector ELS 1000 (Polymer Laboratories, UK) connected in series were used. The retention behavior of the PS and PI homopolymers on reversedphase HPLC column was checked using standards with narrow molar mass distributions (PDI < 1.10), i.e., PS: 97.2, 196.0, and 390.0 kDa and PI: 7.57, 23.5, and 110.0 kDa. The solution concentration of the PS and PI standards in THF was 1 mg/mL. Two-Dimensional Liquid Chromatography (RP-LAC × SEC 2D-LC). The flow rate in the first dimension (RP-LAC) was 0.1 mL/ min, and the injected volume was 50 μL. For the first dimension the same column, solvents, and gradient were used as for the RP-LAC. For the second dimension (SEC) a linear porosity column PSS SDV-M, HighSpeed (50 mm × 20 mm i.d., particle size: 5 μm, Polymer Standards Service, PSS GmbH, Germany) was used, and the THF flow rate was set to 5 mL/min. The high-speed SDV column exhibits a wide pore-size distribution, covering the molar masses from 102 to 106 Da. The fractions were transferred from the first to the second dimension every 2 min. On average, 70 fractions were transferred in one run. The SEC column was calibrated using PS standards of a narrow molar mass distribution, which were dissolved in THF at a concentration of 1 mg/ mL. For the detection in 2D-LC the same detectors were used as in RPLAC. For the data acquisition and evaluation a WinGPC v.7 (Polymer Standards Service GmbH, Germany) was utilized. Fractionation Collection Conditions for Miktoarm Star Copolymers. The samples’ solutions were prepared by the same procedure as described in the chapter on RP-LAC analysis. The samples were fractionated according to their chemical composition by a Nucleosil C18 reversed-phase column (Macherey-Nagel). The HPLC system was equipped with an UV detector (operating at 260 nm) and an online fraction collector 1260 Infinity (all from Agilent Technologies). The same solvents and the same mobile-phase flow rate were used as for the one-dimensional RP-LAC analysis. NMR. The 1H NMR spectra of the PS(PI)x miktoarm star copolymers and their fractions were recorded using a 300 MHz Agilent Technologies DD2 spectrometer in CDCl3-d1. Mass Spectrometry. For the preparation of samples, trans-2-[3-(4tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB, Sigma-Aldrich) and silver trifluoroacetate (AgTFA, Sigma-Aldrich) were used as a matrix and a cationizer, respectively. The mass spectra were acquired with a Bruker UltrafleXtreme MALDI-TOF-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany).

characterization of copolymers with complex architectures is interaction liquid chromatography, e.g., a temperature-gradient interaction chromatography (TGIC)15−17 and a liquid chromatography at the critical point of adsorption (LC-CC).18−21 Both techniques separate the copolymers according to the enthalpic interactions of the solute macromolecules with the stationary phase. The former technique is sensitive to the length of the stronger adsorbing block in the block copolymer, whereas the latter technique provides the selective information about the block length of the particular block of the block copolymer, since the other block is chromatographically “invisible” under its critical conditions and does not contribute to retention. The TGIC alone or in combination with SEC, i.e., twodimensional liquid chromatography (TGIC × SEC 2D-LC), has proven to give valuable composition and structural information about the PS-b-PI-b-PS linear triblock copolymer17 and the PS(PI)3 miktoarm star copolymer15,17 as well as for other, more complex, branched polymers.22,23 The TGIC technique is extremely successful in the separation of individual structures of complex polymers since it has a superior resolution compared to SEC. In addition to TGIC, the LC-CC technique also gives an efficient separation of the blends of the PS and the PI homopolymers,20 the linear PS-b-PI copolymers,18 and the miktoarm star copolymer, consisting of one PS arm and three PI arms [PS(PI)3].19 The objective of the present work is to demonstrate the necessity of using a combination of different characterization techniques for the determination of the chemical and structural dispersity of four miktoarm star copolymers of the PS(PI)x type (x = 2, 3, 5, and 7), prepared by anionic polymerization highvacuum techniques and chlorosilane chemistry. For this purpose we used a variety of separation techniques (size-exclusion chromatography (SEC), reversed-phase liquid-adsorption chromatography (RP-LAC), and characterization techniques (UVMALS-RI multidetection SEC system, NMR and MALDI-TOF MS). The RP-LAC was also combined with SEC into a twodimensional liquid chromatographic system (RP-LC × SEC 2DLC) to simultaneously determining the variation in composition and molar mass. The average chemical composition of PS(PI)x samples and the molar masses of their constituents were determined by proton NMR spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), respectively. Finally, the miktoarm star copolymers were off-line fractionated using RP-LAC and the fractions, with a narrow chemical-composition distribution, were analyzed by NMR and SEC/UV-MALS-RI techniques.

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EXPERIMENTAL SECTION

Materials. A series of miktoarm star copolymers (PS(PI)x; x = 2, 3, 5, and 7) were synthesized by anionic polymerization high-vacuum techniques and selective chlorosilane chemistry.2−4,15 The PS(PI)x copolymers theoretically consisted of one long PS arm and various numbers and lengths of PI arms connected to one central junction point, so that all the samples should have approximately the same overall molar mass and the chemical composition, but different architectures, i.e., PS(PI)2: 82 kDa PS arm and two 39.7 kDa PI arms; PS(PI)3: 82 kDa PS arm and three 26.0 kDa PI arms; PS(PI)5: 105 kDa PS arm and five 15.5 kDa PI arms; and PS(PI)7: 105 kDa PS arm and seven 11.3 kDa PI arms. The dispersity values (Mw/Mn) of all precursor homopolymers were below 1.06 as determined by low-angle light scattering (LALS, Mw) and osmometry (Mn). Each PS(PI)x sample was also fractionated by RP-LAC into several fractions with a narrower chemical composition distribution than that of the original sample. The fractions were characterized by proton NMR 7575

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RESULTS AND DISCUSSION The chromatographic behavior of four PS(PI)x (x = 2, 3, 5, and 7) miktoarm star copolymers with theoretically comparable chemical compositions, but different architectures were studied. The samples consisted of one long PS arm and various numbers of PI arms, the length of which decreases with the number of arms in the copolymer (see Materials section). Size-Exclusion Chromatography Coupled to a Multidetection System (SEC/UV-MALS-RI). The SEC curves recorded by the RI, UV, and LS detectors show narrow molar mass distributions of the samples (Figure S1, Supporting Information), which is also reflected in their low molar mass dispersity (DM: 1.06 for PS(PI)2, 1.14 for PS(PI)3, 1.15 for PS(PI)5, and 1.10 for PS(PI)7). All the samples exhibit shoulders at the high-molar-mass end of the distribution curves, as indicated by all three detectors (Figure S1). The intensity of the LS detector’s response in this molar mass region is the most pronounced, since it is the most sensitive to high-molar-mass species (Figure S1, bottom picture). The fraction of high-molarmass species grows with increasing number of PI arms in the copolymer. At larger elution volumes, the SEC chromatograms recorded by the RI detector show the peaks of very low intensities (Figure S1, upper picture), which were not detected by the UV detector at 260 nm (Figure S1, middle picture). The apex of this peak shifts toward the larger elution volume with the number of arms in the copolymer (7.40, 7.70, 8.02, and 8.40 mL for PS(PI)2, PS(PI)3, PS(PI)5, and PS(PI)7, respectively). The intensity of this peak decreases in the order PS(PI)3 > PS(PI)2 ∼ PS(PI)5 > PS(PI)7. Since the PI does not absorb the UV light at 260 nm, whereas the RI detector is sensitive to both the PS and the PI, these results reveal the presence of excess homo-PI in the samples. SEC combined with a multidetection system (UV, MALS, and RI) enables a precise determination of the molar masses and the chemical compositions of individual constituents of the complex, multicomponent macromolecules if the two components give a different ratio of the UV to RI signals.24−28 In our system, only the PS component was UV active at 260 nm, whereas the PI component was invisible at this wavelength. Based on the known: (i) dn/dc values of both the homopolymers, (ii) specific extinction coefficient of the PS, and (iii) lengths of the PS and the PI arms, the molar masses of individual components (PS and PI) of the constituting species of star copolymers and, consequently, the copolymer’s chemical composition as well as the structure can be determined, provided that the separation is efficient. However, the separation of constituting components of the miktoarm star copolymers by SEC was insufficient, as can be seen from the curves representing the molar mass as a function of elution volume (Figure S2), which show different slopes over the copolymers’ peaks. Namely, only the horizontal line represents macromolecules with a uniform molar mass distribution. Therefore, the exact identification of individual components of the miktoarm stars and the structural purity of PS(PI)x samples was not successful using this technique. The inefficiency of SEC method for the separation of various byproducts in miktoarm star copolymers, especially the stars containing different numbers of arms, has already been observed and is due to a small change in hydrodynamic volume of the stars with an alteration of the number of star’s arms.15 Reversed-Phase Liquid-Adsorption Chromatography (RP-LAC). Interaction chromatography (IC), either the TGIC15−17 or the LC-CC,21 has proved to be an efficient

separation method for miktoarm star copolymers. We applied another type of interaction chromatography for separation of the constituents according to chemical composition, i.e., a reversedphase liquid-adsorption chromatography (RP-LAC). As in the case of TGIC, the separation in RP-LAC is also directed by the enthalpic interactions of polymeric chains with stationary phase; only that in RP-LAC the interaction strength is controlled by changing the solvent composition, whereas in TGIC by changing the temperature. We carried out the separations on a reversedphase C18 bonded silica using a linear gradient from 60 to 80% v/v of THF in ACN in 15 min. In the first set of experiments the retention behaviors of the PS and the PI homopolymers of different molar masses and narrow molar mass distributions were studied. Under the chosen chromatographic conditions, the PS homopolymers hardly interact with the stationary phase and are eluted in SEC mode (from higher to lower molar masses), whereas the PI homopolymers elute in the IC regime (from lower to higher molar masses) (Figure S3). Such retention behavior of the PS and PI homopolymers on the reversed-phase C18 bonded silica is similar to that obtained by TGIC separation technique.15 The RP-LAC traces of miktoarm star copolymers exhibit a much larger number of peaks than were found in their SEC traces, demonstrating the presence of various constituents in the PS(PI)x samples and, thus, the higher resolution power of RPLAC as compared to SEC (Figure 1). The peaks are located

Figure 1. RP-LAC chromatograms of the miktoarm star copolymers recorded by the ELS-detector. The RP-LAC chromatograms between the elution volumes 0−4 mL are magnified. Stationary phase: C18 bonded silica column; mobile phase: THF/ACN gradient.

mainly between the elution volumes 1−3 mL and above ∼8 mL. Above the elution volume of ∼8 mL, the number of peaks with high intensity depends on the type of miktoarm star. The PS(PI)2 star shows one peak, the PS(PI)3 two baseline separated peaks, the PS(PI)5 two partially overlapping peaks, and the PS(PI)7 again only one peak. All samples show, above and below these intense peaks, the peaks of significantly lower intensities, visible only after magnification of the chromatograms. On the basis of elution behavior of the PS and PI homopolymers in the RP-LAC under the same chromatographic conditions, we expected that the constituents of the miktoarm star copolymers would elute in a sequence of increasing content of the PI. This was proved by determination of the chemical 7576

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and PI (5.22−5.02 and 4.80−4.62 ppm for the protons of double bonds of the 1,4- and 3,4-isoprene repeat unit, respectively) in the proton NMR spectra of samples, the molar ratios between the PS and PI repeat units in fractions were determined (Table 1).29 As we expected, the PS/PI molar ratio decreases when going to fraction with larger number, which is retained in the RP column for a longer time. The last eluting fractions consist only of PI (the F6 of PS(PI)3 and the F8 of PS(PI)7) or contain much less PS than PI (the F4 of PS(PI)2 and the F6 of PS(PI)5), as also revealed a comparison of HPLC chromatograms of PS(PI)x samples recorded by the ELS (visible PI) and UV (invisible PI) detectors. Two-Dimensional Liquid Chromatography (RP-LAC × SEC 2D-LC). In order to exactly identify the peaks in the RPLAC chromatograms of PS(PI)x miktoarm star copolymers, the RP-LAC was combined with the SEC into an online twodimensional chromatographic system (2D RP-LAC × SEC). Using the 2D RP-LAC × SEC, it is possible to correlate a certain chemical composition with the molar mass. Figure 2 displays the RP-LAC × SEC 2D-LC contour plots of the PS(PI)x star copolymers recorded by the ELS detector. The y-axis demonstrates the separation according to chemical composition obtained by the 1st-D RP-LAC, and the x-axis represents the separation according to size (2nd-D SEC) of the species coming from the RP-LAC. The constituents of miktoarm

composition of eluting species from the C18 column by proton NMR spectroscopy (Table 1). The regions where the fractions Table 1. Molar Ratios between the PS and the PI Repeat Units As Determined by 1H NMR Spectroscopy for PS(PI)x (x = 2, 3, 5, and 7) Miktoarm Star Copolymers and Their Fractions molar ratios between the PS and the PI repeat units as determined by 1H NMR fraction

PS(PI)2

PS(PI)3

PS(PI)5

PS(PI)7

F1 F2 F3 F4 F5 F6 F7 F8 original PSPIx sample

1:0 1:0.89 1:1.41 1:2.10

1:0 a 1:0.53 1:0.92 1:1.39 0:1

1:0 a 1:0.49 1:0.74 1:1.01 1:2.44

1:1.53

1:1.14

1:1.00

1:0 a 1:0.34 1:0.80 1:0.90 1:0.95 1:0.99 0:1 1:0.98

a

Fraction F2 of samples PS(PI)x (x = 3, 5, and 7) is the breakthrough peak.

were collected are indicated by vertical lines in the RP-LAC chromatograms of samples (Figure 1). From the characteristic signals of PS (7.24−6.28 ppm for the protons of phenyl group)

Figure 2. 2D RP-LAC × SEC contour plots of the PS(PI)x (x = 2, 3, 5, and 7) miktoarm star copolymers recorded by the ELS detector. The 1st-D RPLAC: a C18 bonded silica column, THF/ACN gradient; the 2nd-D SEC: an SDV linear-M high-speed column, THF eluent. 7577

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Figure 3. Enlarged RI chromatograms of the fractions of the PS(PI)x (x = 2, 3, 5, and 7) star copolymers together with the molar masses of the PS and the PI components vs elution volume. The crosses represent the PI component and the pluses the PS component of the stars. The following fractions of the samples are presented: PS(PI)2: F3 (black), PS(PI)3: F4 (blue) and F5 (red), PS(PI)5: F3 (red), F4 (blue), F5 (magenta), and F6 (green), PS(PI)7: F3 (magenta), F4 (black), F5 (red), F6 (green), and F7 (purple).

earlier eluting spots (1−3 mL) are identified as the PS precursors and/or the (PS)2 species, as indicated by the relative molar masses of these species, which were determined in the 2nd-D SEC using a calibration based on PS standards (PS(PI)2: Mn(PS2) = 182 kDa, ĐM = 1.08); PS(PI)3: Mn(PS) = 78 kDa, ĐM = 1.06) and Mn(PS2) = 164, ĐM = 1.05); PS(PI)5: Mn(PS) = 105

star copolymers elute in the 1st-D RP-LAC in a sequence of increasing PI content, whereas in the 2nd-D SEC they elute from larger to smaller size. The contour plots of samples indicate a different number of spots, which are in the 1st-D RP-LAC located between the elution volumes 1−3 mL and above ∼8 mL (Figure 2). The 7578

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Table 2. Results of Characterization of the Individual Fractions of PS(PI)x (x = 2, 3, 5, and 7) Miktoarm Star Copolymers Obtained by SEC/UV-MALS-RIa fraction

PS(PI)2

PS(PI)3

PS(PI)5

PS(PI)7

F1 F2 F3 F4 F5 F6 F7 F8

(PS)2 PSPI (PS)2(PI)2b PS(PI)2 PS(PI)2 + PS(PI)3 + PI

PS + (PS)2 c PSPI (PS)2(PI)2b PS(PI)2 (PS)2(PI)3b PS(PI)3 + (PS)2(PI)4b PI4

PS c PS(PI)2 (PS)2(PI)3b PS(PI)3 (PS)2(PI)4b PS(PI)4 ((PS)2(PI)7 + (PS)3(PI)8 + PI2)b PS(PI)5 ((PS)2(PI)8 + PI6)b

PS c (PS)2(PI)5 PS(PI)4b PS(PI)5 (PS)3(PI)11b PS(PI)5.6 (PS)2(PI)12b PS(PI)6 (PS)2(PI)12b PS(PI)7 (PS)2(PI)12b PI7

a

The most abundant fractions in samples are underlined. bThe component represents small portion in fraction. cFraction F2 of samples PS(PI)x (x = 3, 5, and 7) is the breakthrough peak.

Table 3. Calculated Molar Ratios of the Most Abundant Components in Each Individual Fraction of PS(PI)x (x = 2, 3, 5, and 7) Miktoarm Star Copolymers, As Determined by SEC/UV-MALS-RI and the Known Molar Masses of the PS and the PI Armsa calculated molar ratios between PS and PI repeat units fraction F1 F2 F3 F4 F5 F6 F7 F8 a

PS(PI)2 1:0 1:0.74 1:1.48 1:2.96

PS(PI)3 (PS)2 PSPI PS(PI)2 PS(PI)3

1:0 1:0.48 1:0.97 1:1.45 0:1

PS(PI)5

PS + (PS)2 b PSPI PS(PI)2 PS(PI)3 PI4

1:0 1:0.45 1:0.68 1:0.90 1:1.13

PS(PI)7 PS b PS(PI)2 PS(PI)3 PS(PI)4 PS(PI)5

1:0 1:0.41 1:0.82 1:0.92 1:0.99 1:1.15 0:1

PS b (PS)2(PI)5 PS(PI)5 PS(PI)5.6 PS(PI)6 PS(PI)7 PI7

The most abundant fractions in the samples are underlined. bFraction F2 of samples PS(PI)x (x = 3, 5, and 7) is the breakthrough peak.

kDa, ĐM = 1.04); PS(PI)7: Mn(PS) = 107 kDa, ĐM = 1.05) and/ or the chemical composition of the fractions determined by proton NMR spectroscopy (Table 1). The number and shape of the latter eluting spots (above ∼8 mL) depend on the type of miktoarm star. The PS(PI)2 star shows one high-intensity spot, centered at the elution volume of 11.5 mL (Figure 2). This spot is narrow along the y-axis, indicating the uniform chemical composition of the component. Below and above this spot, some other spots of very low intensity can be seen. In contrast to the PS(PI)2, the PS(PI)3 star exhibits, in this region, two wellresolved narrow spots instead of one, which somewhat differ in their average molar mass (Figure 2). The upper spot closely coincides with that of the PS(PI)2 (∼11.5 mL), whereas the second one elutes earlier in the 1st-D RP-LAC. These two spots represent two main constituents of the PS(PI)3 star, which differ in PI arm number, as indicated by the difference in their molar masses. In contrast to the PS(PI)3, in this region the PS(PI)5 and the PS(PI)7 samples exhibit only one intense spot, which are asymmetrical and very broad along y-axis and x-axis, suggesting the presence of several constituents in the samples (Figure 2). The RP-LAC × SEC 2D-LC gave a somewhat better insight into the complexity of samples’ composition; however, an exact identification of individual constituents was not possible using only the ELS or the UV concentration detectors and the calibration of SEC column with PS standards. The RP-LAC and RP-LAC × SEC 2D-LC results of the PS(PI)3 sample are very similar to those reported by Chang et al. on the characterization of the PS(PI)3 sample with longer PS and PI arms (200 and 50 kDa, respectively), synthesized by a similar synthetic procedure to that used for our PS(PI)3 sample, using the RP-TGIC15 and the RP-TGIC × SEC 2D-LC techniques.17 A comparison of the results obtained by RP-LAC and RP-TGIC reveals the comparable resolution power of both separation

techniques for separation of the constituents of the PS(PI)3 star copolymer according to chemical composition. Fractionation of PS(PI)x Star Copolymers by RP-LAC and a Subsequent Analysis of the Fractions by SEC/UVMALS-RI. In order to unambiguously identify the individual components of the miktoarm stars, we collected fractions at the outlet of C18 column and analyzed them with SEC/multidetection system for the molar mass, the chemical composition, and the structure (Figure 3 and Table 2). Figure 3 shows the enlarged RI chromatograms of the fractions of PS(PI)x star copolymers together with the molar masses of the PS and the PI components as a function of elution volume. The fractions that are present in samples in trace amounts are not shown. For the PS(PI)2 sample the calculated molar masses of the PS and PI components are comparable, which is in agreement with the molar masses of individual arms and the predicted structure of this star copolymer: PS: 82 kDa and PI: 2 × 39.7 = 79.4 kDa (Figure 3). The chromatograms of the fractions of PS(PI)x (x = 3, 5, and 7) samples show a slight shift of the peak toward a lower elution volume with an increasing fraction number (Figure 3). In each PS(PI)x sample the molar mass of the PS component is constant, regardless of the fraction number, whereas the molar mass of the PI component increases with the fraction number due to the increasing number of PI arms in the star structure. The chromatogram of the fraction F6 of the PS(PI)5 sample also shows the presence of the high-molar-mass homo-PI, which elutes at the low-molar-mass end of the PS(PI)5 distribution curve. The homo-PS arm also elutes at the low-molar-mass end of the distribution curves (not shown). On the other hand, the chromatogram of the fraction F3 of the PS(PI)7 sample shows the presence of the (PS)2(PI)5 as a byproduct. The presence of high-molar-mass (PS)y(PI)z (y = 2 and 3) species in the miktoarm star copolymers, especially in the samples PS(PI)5 and 7579

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Mass Spectrometry. The number of peaks in the mass spectra of PS(PI)x miktoarm star copolymers grows with the number of PI arms in copolymer, indicating an increasing number of sample constituents, which is in agreement with the previous analysis (Figure 4). In the case of the PS(PI)2 and

PS(PI)7, is evidenced from the shoulders at the high-molar-mass end of samples’ distribution curves (Figure 3), indicating that in RP-LAC the (PS)y(PI)z (y = 2 and 3) species coelute with the PS(PI)x species that have a similar PS/PI molar ratio. On the basis of the results obtained on fractions using SEC/ multidetection system and the known lengths of the PS and the PI arms, we also determined the most probable structure of samples’ components (Table 2). Each PS(PI)x sample consists of several constituents, i.e., the PS and PI homopolymers in trace amounts and the miktoarm stars of different number of arms, which are the major constituents of star copolymers. The PS(PI)2 sample consists primarily of the star with one PS and two PI arms, which is the targeted product. The PS(PI)3 sample consists of the stars with one PS and two or three PI arms in a weight ratio of about 2:1. In the case of the PS(PI)5 and the PS(PI)7 samples the desired products, i.e., PS(PI)5 and PS(PI)7, represent a smaller proportion of the samples. The main fractions of the PS(PI)5 sample are the stars with one PS and four or three PI arms, whereas in the PS(PI)7 sample the major fractions are the stars with one PS arm and six or five PI arms. The fact that the PS(PI)x (x = 3, 5, and 7) samples consist mainly of species with a smaller number of PI arms than predicted from the chlorosilane functionality is also evidenced from a comparison of the calculated molar ratios of the PS/PI repeat units for the most abundant constituent in individual fractions (Table 3) with those experimentally determined by NMR spectroscopy (Table 1). From the chemical composition determined by NMR spectroscopy and the known lengths of the PS and the PI arms, we calculated the average number of PI arms in the samples, i.e., 2.0 for PS(PI)2, 2.3 for PS(PI)3, 4.0 for PS(PI)5, and 5.6 for PS(PI)7. These results agree very well with the results obtained for the fractions using SEC/multidetection system. In addition to the homo-PS and homo-PI as well as the stars with smaller number of PI arms than predicted (byproducts), the investigated PS(PI)x samples also contain the high-molar-mass (PS)y(PI)z (y = 2 and 3) constituents, particularly the samples PS(PI)5 and PS(PI)7. The (PS)y(PI)x‑y+1 (y = 2 and 3) species result from a double or triple linking of PSLi to chlorosilane coupling agents, which is a consequence of a local excess of living PSLi over the chlorosilanes, even though it was added dropwise. The reason for much higher content of (PS)y(PI)x−y+1 (y = 2 and 3) in the PS(PI)5 and PS(PI)7 samples than in the PS(PI)2 and PS(PI)3 samples is that in the former case the living PSLi was added to the chlorosilane linking agent in stoichiometric amounts, whereas in the latter case it was added in a huge excess. Thus, the formation of these species is more likely for the PS(PI)5 and PS(PI)7 samples. In addition, the formation of high-molar-mass (PS)y(PI)z (y = 2 and 3) species most probably also took place through the coupling of the species with not completely reacted silane groups into the siloxanes (Si−O−Si).16 Such species contain more isoprene arms than it is possible for a single coupling agent. The calculated molar ratios of the PS and PI repeat units of the main constituent in fractions (Table 3), which were identified by SEC/UV-MALS-RI, fit very well with the fraction’s chemical composition determined by proton NMR (Table 1). The slight deviation between the experimentally obtained PS/PI molar ratios of the fractions by NMR spectroscopy and those calculated for the most abundant component in the fraction, as determined by SEC/UV-MALS-RI, was observed only in cases when fractions contained larger amounts of other species, i.e., the neighboring species, homo-PI and/or (PS)y(PI)z (y = 2 and 3).

Figure 4. MALDI-TOF mass spectra of PS(PI)x (x = 2, 3, 5, and 7) miktoarm star copolymers.

PS(PI)3 samples, the peak for the mass ion of homo-PS coincides with that of the double-charged ions of the PS(PI)2 and PS(PI)3 constituents. The intensity of the peak due to homo-PS is very high in all the mass spectra since the homo-PS ionized much more easily in a mixture of different species than the homo-PI and the copolymers of the PS and PI under the experimental conditions used. Besides the homo-PS, the mass spectrum of the PS(PI)3 sample shows a high-intensity peak due to the mass ion of the major sample’s constituent with one PS and two PI arms and a very-low-intensity peak for a constituent with one PS and one PI arm, which is present only in trace amounts. The mass spectrum of the PS(PI)3 sample indicates the peaks for the homo-PS and the stars with one PS arm and with two and three PI arms. The mass spectra of the PS(PI)5 and PS(PI)7 samples are very complex due to the large number of constituting species and due to the presence of double-charged ions. However, both mass spectra show, besides the homo-PS, the presence of major components, i.e., the stars with a smaller number of PI arms than predicted from theoretical structure. The results obtained by MALDI-TOF mass spectrometry are thus in good agreement with those obtained by RP-LAC fractionation and the subsequent analysis of fractions using SEC/multidetection system. 7580

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Article

CONCLUSIONS We demonstrated that the combination of different characterization techniques is necessary for a thorough determination of the molar mass, the chain architecture, and the chemical composition of miktoarm star copolymers of the PS(PI)x type, where x is 2, 3, 5, and 7. The SEC with UV-MALS-RI multidetection system indicates the presence of high-molar-mass (PS)y(PI)z (y = 2 and 3) species and the homo-PI in original PS(PI)x samples. Because of insufficient separation of the stars with different numbers of PI arms by SEC, an exact identification of the samples’ constituents was not possible, even using a UVMALS-RI multidetection SEC system. Better separation of the samples’ constituents was achieved by RP-LAC, which separates the macromolecules according to chemical composition. The efficiency of the RP-LAC separation decreases with decreasing length of the PI arm due to a too small difference in chemical composition between the PS(PI)x and the PS(PI)x−1. Additionally, in RP-LAC the (PS)y(PI)z (y = 2 and 3) species and the PS(PI)x species with comparable PS/PI molar ratios coelute, as indicated by subsequent SEC/UV-MALS-RI analysis of fractions collected at the outlet of RP-C18 column. A somewhat better insight into the complexity of miktoarm star copolymer composition is given by RP-LAC × SEC 2D-LC analysis; however, an exact identification of individual constituents was not possible using only ELS or RI concentration detectors and conventional SEC with PS calibration. Unambiguous information about the molar mass, the chemical composition, and the structure of the constituents of PS(PI)x miktoarm star copolymers was obtained by fractionation of samples by RPLAC into the fractions with narrow chemical composition distribution and their subsequent characterization by SEC/UVMALS-RI, which efficiently separates the high-molar-mass (PS)y(PI)z (y = 2 and 3) species from the PS(PI)x species and allows a simultaneous determination of the molar mass, chemical composition, and structure of individual constituents. The results show that all PS(PI)x samples contain byproducts, i.e., the homo-PS and homo-PI in minor amounts and the highmolar-mass (PS)y(PI)z (y = 2 and 3) species, the content of which is higher in star copolymers with larger numbers of PI arms (PS(PI)5 and PS(PI)7). The major constituent of the PS(PI)2 sample is the one with the predicted structure, whereas, the major components of other PS(PI)x (x = 3, 5, and 7) samples are the stars, containing smaller number of PI arms than predicted from the functionalities of chlorosilane coupling agents. These results are in agreement with the average chemical compositions of samples determined by proton NMR spectroscopy and the determination of individual constituents by mass spectrometry. The results of this study show that by conventional characterization techniques the detailed differentiation and evaluation of the various types of byproducts in the polymers with complex architecture are not feasible. A comprehensive study, as presented in our work, is necessary to improve and optimize the synthesis by suppressing the formation of byproducts, which are in the case of PSPIx miktoarm star copolymers related to the chlorosilane coupling reaction.

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theoretical background of SEC/UV-MALS-RI. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support of the Ministry of Higher Education, Science and Technology of the Republic of Slovenia through (a) the Slovenian Research Agency (Program P2-0145) and (b) the Centre of Excellence - Polymer Materials and Technologies for MALDI-TOF MS analysis.

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ASSOCIATED CONTENT

S Supporting Information *

Details regarding the synthetic procedures for preparation of the PS(PI)x miktoarm star copolymers, the experimental conditions for the characterization methods we applied, Figures S1−S3, and 7581

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