Synthesis and Characterization of an Exact Polystyrene-graft

Mar 23, 2017 - Synthesis and Characterization of an Exact Polystyrene-graft-polyisoprene: A Failure of Size Exclusion Chromatography Analysis. Sanghoo...
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Synthesis and Characterization of an Exact Polystyrene-graf tpolyisoprene: A Failure of Size Exclusion Chromatography Analysis Sanghoon Lee,† Hyojoon Lee,† Taihyun Chang,*,† and Akira Hirao*,‡,§,∥ †

Division of Advanced Materials Science and Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang 37673, Korea ‡ Polymeric and Organic Materials Department, Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo 152-8550, Japan § Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan ∥ Department of Applied Chemistry, National Chiao Tung University, Hsinchu 30010, Taiwan ABSTRACT: An exact polystyrene-graf t-polyisoprene (PS-g-PI) synthesized by iterative anionic polymerization and graft reaction using diphenylethylene functional groups was characterized by liquid chromatography. As-prepared graft copolymer contains various byproducts other than the target PS-g-PI. After the fractionation by size exclusion chromatography (SEC), the PS-g-PI appears quite homogeneous by SEC analysis, but the temperature gradient interaction chromatography (TGIC) separation with respect to the PI branch number showed a significantly wide distribution in the number of PI grafts. The resolved TGIC peaks were fractionated, and the molecular weight of each block (backbone or branches) was estimated by liquid chromatography at critical condition (LCCC) analysis at the critical adsorption condition of the opposite block. The composition determined by LCCC analysis and 1H NMR analysis yielded a reasonably self-consistent result. Through this study, we demonstrated that SEC analysis of this type of branched copolymers can lead to erroneous result and needs to be done with proper caution.

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INTRODUCTION Graft copolymer is a polymer type having branch chains grafted to a backbone chain. If branch chains are chemically different from the backbone chain, they are called graft copolymers. Such graft copolymers exhibit phase behaviors different from linear block copolymers.1−4 Graft polymers are often prepared by separate anionic polymerizations of backbone and branch polymers and subsequent grafting reaction of them.5,6 An iterative synthetic method was proposed by Hirao and coworkers and applied to synthesize “exact” branched polymers having predetermined length and positions of branches.6,7 However, imperfect stoichiometry and side reactions in the grafting process commonly result various byproducts.5,6,8−11 Therefore, usually the final or intermediate products are subject to purification using fractional precipitation or preparative size exclusion chromatography (SEC). We have previously shown that such purification methods often fail to acquire pure target structures even though they show a narrow and unimodal peak in SEC analysis.8,9,11 SEC analysis is commonly adopted for polymer characterization, but SEC is not an ideal method to separate branched polymers according to molecular weight (MW). SEC is a method to separate polymer chains according to their hydrodynamic size and the chain size of branched polymers is not directly correlated with MW. Therefore, differently branched polymers may coelute in the SEC separation despite different MW of them. If SEC is the only available separation tool, SEC/multiple detection including light scattering © XXXX American Chemical Society

detection can be an efficient method to detect the presence of byproducts.8,9,11 Application of other chromatographic techniques than SEC has been increasing rapidly in recent years in response to the growing need of more precise characterization of complex polymers.12−17 Interaction chromatography (IC) and liquid chromatography at critical condition (LCCC) are the representative techniques to complement SEC. In principle, SEC, IC, and LCCC are not entirely different techniques, but they are three separation modes for polymer characterization that can be established with the same liquid chromatographic (LC) system by changing either mobile phase or column temperature.18−21 These separation modes have their own merits and limitations. Among the three LC separation modes of polymers, LCCC has been successfully employed for the chromatographic separation of polymer mixtures,22,23 cyclic polymers from linear precursors,24 and according to the functionality in polymers.19,25,26 LCCC has also been used widely for the analysis of individual blocks of block copolymers. The chromatographic critical condition for a homopolymer is defined as the condition where the size exclusion effect is precisely compensated by the interaction effect.27−29 At this condition, the retention of a homopolymer becomes independent of MW. The LCCC analysis of block copolymers Received: January 2, 2017 Revised: February 9, 2017

A

DOI: 10.1021/acs.macromol.6b02811 Macromolecules XXXX, XXX, XXX−XXX

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functionalized PS anion with SiOP and DPE moieties at the initiating and terminating chain-ends was separately prepared by the SiOPLiinitiated polymerization of styrene and the subsequent addition of 1,4bis(phenylethenyl)benzene (PEB). The functionalized PS anion thus prepared was then reacted in situ with the above 3-propyl bromidefunctionalized PS to result in a two segments PS with chain-end SiOP and in-chain DPE moieties. The target MW of a PS chain unit was 10K. By repeating the same reaction sequence three more times according to Scheme 1, the PS backbone in-chain-functionalized with four DPE moieties was prepared. Finally, a 2-fold excess of living polyisoprenyl anions (target MW was 13K) toward each DPE moiety was reacted with the DPE-functionalized PS backbone in order to introduce four polyisoprene branches. After the grafting reaction, the excess unreacted PI was removed from the as-synthesized PS-g-PI by the fractional precipitation using a mixed solvent of heptane and methanol. If the synthesis is performed as shown in Scheme 1, the resulting graft copolymer is exactly controlled in MW of the backbone chain and each graft chain and the distance between the graft chains. SEC and TGIC Analysis. All polymers used in this study including homopolymer standards and the PS-g-PI were characterized by SEC/ multiple detection. The specific refractive index increment (dn/dc) of PS and PI was measured in THF by using standard polymers as 0.185 and 0.120 mL/g, respectively. Three mixed bed SEC columns (Agilent Polypore 300 × 7.5 mm, Waters Styragel HR4 300 × 7.8 mm, and Jordi mixed bed 300 × 8.0 mm) were used at a column temperature of 40 °C. SEC chromatograms were recorded with a triple detector (Malvern, TDA 305). The solvent was THF (Samchun, HPLC grade) at a flow rate of 0.7 mL/min. Polymer samples for the SEC analysis were dissolved in THF at a concentration of ∼1 mg/mL, and the injection volume was 100 μL. For TGIC analyses, a C18 bonded silica column (Nucleosil C18, Macherey-Nagel, 250 × 4.6 mm, 500 Å pore, 7 μm particle) was used. 1,4-Dioxane (Samchun, HPLC grade) was the eluent at a flow rate of 0.5 mL/min using a Bischoff HPLC compact pump. Temperature of the column was controlled by circulating water from a programmable bath/circulator (Julabo, F25-HL) through a homemade column jacket. The TGIC column temperature was maintained at 12.5 °C for 15 min and then varied from 12.5 to 42.5 °C for 100 min at linear gradient. Sample solutions (∼3 mg/mL) were prepared by dissolving the polymers in a small volume of the corresponding eluent, and the injection volume was 100 μL. The chromatograms were recorded with a dual wavelength UV detector (Younglin, UV730). LCCC Analysis. Two C18 bonded silica columns (Nucleosil C18, Macherey-Nagel, 250 × 4.6 mm, 500 Å pore, 7 μm particle and 250 × 4.6 mm, 300 Å pore, 5 μm particle) and 1,4-dioxane (Samchun, HPLC grade) were used for the LCCC analysis of PS at the critical condition of PI. The column temperature was 47.4 °C. Two bare silica columns (Nucleosil, 250 × 4.6 mm, Macherey-Nagel, 500 Å pore, 5 μm particle and 250 × 4.6 mm, 300 Å pore, 5 μm particle) and a mixture solvent (THF/n-hexane, 45/55) were used for the LCCC analysis of PI at the critical condition of PS. The column temperature was 15 °C. Injection samples (∼1 mg/mL) were prepared by dissolving the polymers in a small volume of the corresponding eluent, and the injection volume was 100 μL. The chromatograms were recorded by a UV absorption detector (Younglin, UV730) operating at dual wavelengths of 230 and 260 nm. The PS (homemade) and PI standard samples (Polymer Source) were used as calibration standards for the MW analysis of individual blocks. Two-Dimensional (2D)-LC (TGIC x LCCC) Analysis. Comprehensive 2D-LC analysis was carried out by combining a temperature gradient IC (TGIC) and an LCCC online to separate as-prepared PSg-PI4 according to the number of PI branch and to separate the effluents further according to the PS backbone size only, respectively. The same stationary (Nucleosil C18, Macherey-Nagel, 7 μm, 500 Å pore, 150 × 4.6 mm) and mobile phases (CH2Cl2/CH3CN, Samchun, HPLC grade, 78/22, v/v) were used for both TGIC and LCCC except for the column temperature. The TGIC column temperature was maintained at 5 °C for 62 min, linear gradient from 5 to 29 °C for 600 min. Flow rate in the first-D TGIC was set at 0.04 mL/min in order to

is based on the assumption that a block at the critical condition of the same homopolymer is chromatographically “invisible”, and the retention of a block copolymer is governed solely by the other blocks in the block copolymer.12,18,30 Although both experimental and simulation studies showed that the block under the critical condition is not fully invisible, LCCC analysis can provide a good MW estimate of individual blocks.31−36 In this study, an exact polystyrene-graf t-polyisoprene (PS-gPI) was synthesized by the iterative synthetic method with a target structure of four equally spaced PI branches on a PS backbone by anionic polymerization of PS and PI blocks and subsequent linking reactions using 1,3-bis(1-phenylethenyl)benzene (PEB) moiety. As-prepared polymer contains various byproducts in addition to the target graft copolymers. Combining SEC, temperature gradient IC (TGIC), and LCCC, we were able to characterize the complex mixture of the graft copolymers in detail.

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EXPERIMENTAL SECTION

Synthesis of PS-g-PI. Except for the deprotection and transformation reactions, all of the polymerizations and linking and addition reactions were carried out under high-vacuum condition (10−6 Torr) in sealed glass reactors equipped with break-seals. All chemicals were from Aldrich. The reactors were always sealed off from the vacuum line and then prewashed with a red-colored 1,1-diphenylhexyllithium (ca. 0.05 M) in heptane solution prior to the polymerizations and reactions. All operations were performed according to the usual highvacuum technique reported.37 The PS-g-PI copolymer was synthesized by using PS backbone and PI branches as shown in Scheme 1. PS

Scheme 1. Synthetic Scheme of an Exact Polystyrene-graf tpolyisoprene

backbone was first synthesized stepwise by repeating the same reaction sequence involving the following three reaction steps: (1) a transformation reaction of 3-(tert-butyldimethylsilyloxy)propyl (SiOP) group to 3-bromopropyl function used as a new reaction site via deprotection of the silyl group, (2) a linking reaction to prepare the backbone chain with the incorporation of a DPE moiety, and (3) an addition reaction to introduce the backbone segment chain. Each of the reaction sequences involving steps 1−3 was carried out under the conditions similar to those previously reported.6 Styrene was first polymerized with 3-(tert-butyldimethylsilyloxy)-1-propyllithium (SiOPLi), followed by sequential treatments with (C4H9)4NF (deprotection) and CBr4/Ph3P (bromination) to afford a polystyrene functionalized with 3-propyl bromide at the initiating chain-end. A B

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Macromolecules synchronize with the second-D LCCC separations. The second-D flow rate was 0.8 mL/min at a column temperature of 46 °C, which is the critical condition of PI. The second-D LCCC run was repeated every 2.5 min while effluent of the first-D TGIC filled up a 100 μL storage loop. Two HPLC pumps (pump 1: Shimadzu, LC-20AD; pump 2: Bischoff compact pump 2250) and two UV detectors (Younglin, UV730) were used. The two LC systems were connected via electronically controlled 10-port 2-position switching valves (Alltech, SelectPro) equipped with two 100 μL storage loops. The 2D-LC system is similar to the one reported previously.38

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RESULTS AND DISCUSSION SEC Fractionation and TGIC Characterization. SEC analysis separates polymers according to the chain size in a good solvent. Polymer chain size depends on not only MW but also chemical composition as well as chain architecture. The relationship between chain size and chemical composition or chain architecture is not as simple as the chain size−MW relationship for linear homopolymers. Therefore, SEC analysis of copolymers or branched polymers is not straightforward and may lead to incorrect results. For an SEC analysis of polymers with chemical composition and/or architectural distribution, multiple detection is a powerful method to examine the homogeneity of the polymers. Since different detectors give signals proportional to different molecular characteristics of the polymers, nonoverlapping chromatograms obtained by different detectors provide good clues to notice the heterogeneity in the samples. The SEC chromatogram of the as-prepared PS-g-PI is shown in Figure 1a. In the SEC analysis, a refractive index (RI) detector (black line), a light scattering (LS) detector (red line), a dual wavelength UV detector (230 nm: blue line; 260 nm: green line) were used. Some amounts of both large (likely high MW) and small (likely low MW) species elute out before and after the main peak, respectively. When the four detector signals are normalized at the largest peak, the high-MW shoulder and low-MW peak show different intensities of the four detectors, indicating that they have different chemical compositions as well as MW. A peak eluting at tE ∼ 39 min is the solvent peak. The very large intensity of UV 230 nm of the solvent peak seems to be due to the remaining PEB in the sample coeluting with the injection solvent. Preparative SEC fractionation is often used in recent years instead of traditional fractional precipitation to purify such complex polymers.39−41 The as-prepared PS-g-PI was fractionated by collecting the portion eluting tE at 27.8−29.8 min (vertical bars shown in Figure 1a) to remove the high-MW and low-MW parts. The SEC chromatogram of the SECfractionated PS-g-PI (Figure 1b) appears to be highly homogeneous judging from that it shows a very narrow unimodal peak with decently matched signals of multiple detectors. The large solvent peak in Figure 1a also disappears. To examine the effectiveness of such an SEC fractionation, TGIC analysis was carried out. The TGIC separation condition is at the interaction mode of PI while at the SEC mode for PS, and it is supposed to separate the polymer species in the PS-g-PI samples according to the MW of PI mainly.42,43 TGIC chromatogram of as-prepared PSg-PI (Figure 2a) shows a multimodal distribution indicating that there are at least five major species differing in MW of PI. Multiple detection was also used in the TGIC analysis: UV 230 nm (blue solid line) and UV 260 nm (green dashed line) to estimate the composition of individual peaks. PS absorbs at both 230 and 260 nm while PI absorbs at 230 nm only, and the

Figure 1. SEC chromatograms of (a) the as-prepared PS-g-PI and (b) SEC fractionated PS-g-PI were recorded by a RI detector (black solid line), a LS (red dashed line) detector, and a UV detector at 230 nm (blue dashed line) and at 260 nm (green dashed line). Separation condition: three mixed bed columns (Agilent, Jordi, Waters). Eluent: THF at a flow rate of 0.7 mL/min; column temperature: 40 °C. A MW calibration curve made with PS standards (Mp: 1090K, 360K, 136K, 30.9K, 11.9K, and 3.8K) is also shown.

A260/A230 ratio is a good parameter to estimate the composition of PS-g-PI. The signal ratio A260/A230 decreases gradually as tE increases (as MW of PI increases) reflecting that the PS content decreases as MW of PI increases. It is consistent with the PS-gPI structure with increasing number of PI branches. However, a quantitative analysis is not possible since the PS-g-PI contains a PEB at each branching point that absorbs at 230 nm strongly. The SEC-fractionated PS-g-PI (Figure 2b) also shows a multimodal distribution not much improved in compositional homogeneity compared to the as-prepared sample (Figure 2a). Only noticeable changes in the TGIC chromatograms of the SEC-fractionated samples from the as-prepared PS-g-PI (Figure 2a) are the reduction of the late eluting shoulder (tE ∼ 70 min), relative intensity change of the two major peaks (tE ∼ 50 and 62 min), and the reduction of the second peak (tE ∼ 30 min) width. It is surprising to see that such a complex mixture of different PS-g-PI species can coelute in the SEC separation as a narrow peak without notable difference in the multiple detection signals (Figure 1b). They must have a similar chain size not to be separated by SEC and somehow hide their complex nature from the multiple detections. To figure out what the coexisting species are, the as-prepared PS-g-PI was fractionated into six fractions by TGIC as shown in Figure 2a and subjected to LCCC analysis to characterize individual PS and PI contents. (The chain structures illustrated on top of each peak are the structure of the corresponding PS-g-PI species as discussed later.) C

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Figure 3. LCCC chromatograms of TGIC-fractionated PS-g-PI and asprepared PS-g-PI at the critical condition of PI recorded by a UV detector at 260 nm (green solid line) and a light scattering (red dashed line) detector. Separation condition: two C18 columns. (Nucleosil C18, 250 × 4.6 mm, 500 Å, 7 μm and Nucleosil C18, 250 × 4.6 mm, 300 Å, 5 μm) Eluent: 1,4-dioxane at a flow rate of 0.3 mL/min. Column temperature was 47.4 °C. Linear PIs elute out at tE = 19.4 min near the solvent peak. A MW calibration curve of PS made with homoPS standards (Mp: 136K, 82K, 45K, 33K, and 11K) is also shown. Peak positions were labeled with the red arrows.

Figure 2. TGIC chromatograms of (a) the as-prepared PS-g-PI and (b) SEC fractionated PS-g-PI recorded by a UV detector at 230 nm (blue solid line) and at 260 nm (green dashed line). Separation condition: Nucleosil C18, 250 × 4.6 mm, 500 Å, 7 μm. Eluent: 1,4dioxane at a flow rate of 0.4 mL/min. Temperature program is also shown in plot. The chain structures illustrated on top of each peak will be discussed later.

Characterization of TGIC-Fractionated PS-g-PI. At the critical condition of a block (either PS or PI) in a diblock copolymer, the block under the critical condition is regarded not to contribute to the LC retention of the block copolymer (i.e., the block becomes chromatographically invisible), and the block copolymer elutes as if it consists of the other visible block only. Therefore, individual blocks of a block copolymer have been characterized by LCCC under the critical conditions of each block often.30 Rigorous experimental examination of the theoretical prediction revealed that the block under critical condition is not completely invisible, but LCCC allows reasonable estimation of the individual block length.31,33,35 Here we set the elution condition as SEC for the visible blocks and tried to estimate the size of the visible block using the calibration curve made with the corresponding homopolymer standards. Figures 3 and 4 show the LCCC chromatograms under the critical adsorption conditions of homo-PI and homoPS to estimate the MW of PS backbone and PI branches using the calibration curve of standard homo-PS and homo-PI, respectively. The LCCC separation does not have high resolution since the separation is done in the SEC mode with only two short columns, but it provides a reasonable landscape of the complex mixture. In the LCCC analysis of the TGIC fractions at the critical condition of PI, the fractions are separated in the SEC mode according to the PS size. The LCCC chromatograms in Figure 3 show that all TGIC fractions contain a common PS backbone eluting at tE ∼ 15 min. All chromatograms have solvent peak at tE ∼ 19.2 min. In addition, most of the fractions contain a longer PS backbone eluting at tE ∼ 14 min while F2 and F3

Figure 4. LCCC chromatograms of TGIC fractions and as-prepared PS-g-PI at the critical condition of PS recorded by a UV detector at 260 nm (green solid line) and a light scattering (red dashed line) detector. Separation condition: two bare silica column. (Nucleosil silica, 250 × 4.6 mm, 500 Å, 5 μm and Nucleosil silica, 250 × 4.6 mm, 100 Å, 5 μm) Eluent: THF/n-hexane (45/55, v/v) at a flow rate of 0.5 mL/min. Column temperature was 15 °C. Linear PSs elute out at tE = 13.2 min. A MW calibration curve of PI made with homo-PI standards (Mp: 149K, 80K, 43K, and 9.8K) is also shown. The peak positions are labeled with the red arrows.

have a shorter PS backbone (tE ∼ 18 min) as byproducts. Their MW can be estimated as 110K, 52K, and 12K using the calibration curve made by homo-PS standards as shown in the plot. On the other hand, LCCC analyses of the TGIC fractions at the critical condition of PS (Figure 4) show that major peaks of different fractions elute at different elution times. It indicates that all fractions have different size (number) of PI branches. The MW of PI increases with the fraction number in the LCCC analysis that is consistent with the TGIC separation scheme carried out at the PI interaction condition (Figure 2). F2 shows a bimodal peak, one of them eluting at the longer tE (low MW) is similar to F1, and the other eluting at the shorter tE (high MW) is similar to F3. F3 has a shoulder seemingly of the same D

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Macromolecules Table 1. Analysis Results of TGIC Fractions of PS-g-PI fraction

structure

Mp,PS-Mp,PIa (wt %)b

dn/dcc (mL/g)

PS (wt %)d (HPLC)

PS (wt %)e (NMR)

Mpf (MWLCCC)g

F1

PS-g-PI1

53.9K-14.4K

0.171

78.9

75.0

69.0K (68.3K)

F2

PS-g-PI2 PS1/5-g-PI1

53.6K-31.1K (32.5) 11.7K-16.5K (67.5)

0.161 0.147

48.6

50.5

85.9K (84.7K) 26.8K (28.2K)

F3

PS2-g-PI2 PS-g-PI2 PS1/5-g-PI1

107K-31.0K (3.4) 53.2K-31.0K (85.1) 12.4K-17.1K (11.5)

0.170 0.161 0.147

61.2

61.8

− 86.8K (84.2K) −

F4

PS2-g-PI3 PS-g-PI3

108K-47.9K (11.1) 52.4K-47.9K (88.9)

0.165 0.154

54.1

57.8

− 103K (100K)

F5

PS2-g-PIx≥4 PS-g-PI4

111K-93.6K (16) 51.7K-60.4K (84)

0.155 0.150

47.4

52.6

− 120K (112K)

F6

PSx≥2-g-PIx≥4 PS-g-PI4

100K-99.5K (50.2) 51.7K-67.8K (49.8)

0.152 0.148

46.7

48.2

− 124K (120K)

a

Peak MW of each block estimated from LCCC analysis at the critical condition of the other block. bRelative amount of each polymer species calculated from the area of RI signal in SEC analysis of fractionated samples taking into account of the composition dependence of dn/dc. cdn/dc in THF of the major species calculated using the chemical composition in the column 3 (a). (dn/dc)copolymer = (dn/dc)PS + WPS + (dn/dc)PI + WPI. d Average wt % of PS in the TGIC-fraction calculated from MW in the column 3 (a). ewt % of PS in the TGIC-fraction determined by 1H NMR f Peak MW determined by SEC-light scattering method (using dn/dc value in the column 4 (c)). gCalculated MW from LCCC analysis (a).

size PI as F1 or the low-MW peak of F2. It is apparent that F5 and F6 contain a high-MW fronting shoulder. By combining two LCCC results at the critical condition of each block and the consideration of the synthetic scheme, we can conclude that the major component in each fractions has an identical PS backbone but different number of PI branches. The MW of the PI branches at the major peaks is determined by the calibration curve shown in the plot, and the combined results are shown in Table 1. They are in good agreement with the synthetic scheme of PS-g-PI. The PS backbone MW of ∼52K is in good agreement with the target MW while the PI branch MW of ∼15K is somewhat larger than the target MW of ∼13K but in an acceptable error range considering the uncertainty in the MW determination by LCCC. The synthetic method appears to work reasonably well in synthesizing a PS backbone but failed to have all four PI branches attached to the backbone quantitatively. Furthermore, some longer PS backbones were also produced as byproducts. To complement the limitation of the LCCC analysis and to understand why the SEC fractionation failed to yield a fraction with a narrow distribution, SEC analysis of the TGIC fractions was carried out. In the SEC analysis (Figure 5a), the TGIC fractions elute at different elution times, indicating that size of the polymers are slightly different from one another. The SEC elution trend of the TGIC fractions is similar to the LCCC elution at the critical condition of PS (Figure 4). It is an expected result since the chain size should increase with the increase of the number of PI branches in the TGIC fractions. However, the increase of chain size with MW of branched polymers is not as large as linear polymers. Furthermore, the MW of the grafted branch (∼15K) is much smaller than the MW of the PS backbone (∼52K) that reduces the increment of the chain size with the addition of a branch to result a very small change in the SEC retention. Figure 5b shows the SEC chromatograms of as-prepared PS-g-PI (black solid line) and of the TGIC-fractions (various colors) together with the reconstructed chromatogram (red dashed line, weighted sum

Figure 5. (a) SEC chromatograms of TGIC-fractionated PS-g-PI recorded by a RI detector (black solid line), a LS detector (red dashed line), and a UV detector at 230 nm (blue dash-dotted line) and at 260 nm (green dash-dotted line). (b) Reconstruction of the SEC chromatogram of as-prepared PS-g-PI combining the SEC result of all fractions. Separation condition: three mixed bed columns (Agilent, Jordi, Waters), Eluent: THF at a flow rate of 0.7 mL/min. Column temperature: 40 °C.

of the fractions). The relative amounts of each fraction (according to A260) are also shown in the figure. The E

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The contrast between the A260 and A230 is less than expected as shown in Figure 2 since the PS backbone already contains four DPE units that absorb UV strongly at 230 nm. The combined effect of MW-dependent dn/dc and the DPE units in the PS backbone seems to be the reason for the surprisingly broad chemical composition distribution in the SEC fraction that appears to have a quite narrow distribution from the SEC/ multiple detection analysis. This example demonstrates that judicious precaution should be taken in judging the purity of this type of polymers with SEC analysis. We characterize the polymer species in F3, F4, F5, and F6 further by isolating the SEC peaks. The SEC chromatograms (RI signal) of three TGIC fractions are fitted with Gaussian shaped peaks as shown in Figure 6. We can predict the

reconstructed chromatogram reproduces the as-prepared PS-gPI well, indicating that the TGIC fractionation was done without noticeable loss. From the positions of the elution peaks of the fractions, it is understandable why the SEC fraction collected between tE = 27.8 and 29.8 mL shown in Figure 1b still contains the major species of all fractions just removing only parts of them. The chain size of PS-g-PI is mainly determined by the PS backbone and the different numbers of PI branches on a common PS backbone (the major elution peaks of F1−F6) has a small effect on the chain size. Therefore, the removed parts from the SEC fractionation are a part of F1, the shorter PS backbone portion in F2 eluting later at tE ∼ 32 min, and a significant portion of F6 containing a longer PS backbone to elute early at tE < 27.8 min. It is exactly what was observed from the comparison of the TGIC chromatograms of as-prepared and SEC-fractionated PS-g-PI shown in Figure 2a,b. We now try to identify the polymer species in each fraction combining the SEC and LCCC analyses. Since the TGIC separation of the as-prepared PS-g-PI could not resolve the individual polymer species completely as shown in Figure 2a, some overlap of the polymer species in the fractions is unavoidable. F1 has a common PS backbone (∼52K) from the LCCC results at the critical condition of PI (Figure 3) and a PI branch (∼15K) from the LCCC results at the critical condition of PS (Figure 4). Therefore, F1 can be assigned as PS-g-PI1. F2 shows two distinct peaks in all 3 LC analyses. It seems reasonable to speculate that there are two different polymer species. Figure 3 indicates that they are of two different PS backbones: a common one (∼52K) and a shorter one (∼12K). From the synthetic scheme, the target PS backbone has five PS chain units, and the ∼12K PS is likely to correspond to one PS chain unit. Figure 4 shows that F2 has two distinct PI also. One appears similar to PI in F3, and the other is similar to PI in F1. With the aid of the synthetic scheme of the graft copolymer, we may assign the two polymer species as PS1/5-g-PI1 and PS-g-PI2. F3 appears to contain essentially the same polymer species as in F2 judging from the elution peak positions in the three chromatograms. Only the relative amounts of the two polymer species are different. F2 contains more PS1/5-g-PI1 while F3 contains dominantly PS-g-PI2. It is not surprising from the TGIC fractionation range in Figure 2 that F2 and F3 share the same polymer species. F4 and F5 appear to contain PS-g-PI3 and PS-g-PI4 as the main species judging from the three chromatograms. Both of them contain weak fronting shoulders that contain a longer PS backbone (∼110K) from Figure 3 and a larger number of PI branches from Figure 4. In F6, presence of at least two different PS backbones is apparent (Figure 3) and larger number of PI branches (Figure 4). The major species appear to be PS-g-PI4 and PS2-g-PIx>4. We will discuss more about the high-MW byproducts later. The next question to be answered is why the SEC/multiple detection did not provide a good hint of this heterogeneity in the SEC fraction. The MW of the PS-g-PI fractions increases with the number of PI branches added. At the same time, dn/dc of PS-g-PI decreases as the number of PI branch added since dn/dc of PI (0.120 mL/g) is lower than PS (0.185 mL/g). Therefore, the LS signal, approximately proportional to (dn/ dc)2MW, changes less than similarly branched homopolymers due to the compensation of the MW increment with the dn/dc decrement by adding more number of PI branches. Therefore, the deviation of LS signal from the concentration detector signal is much less than the case of branched homopolymer.

Figure 6. Peak isolation of SEC chromatograms assuming Gaussian shape of the peaks. The experimental data of RI signal (black line) are fitted with Gaussian peaks (blue line) and the sum of Gaussian peaks (red line) are shown.

structures of corresponding isolated peaks from the synthetic scheme and MW obtained from the LCCC analysis as shown in Table 1: F3 contains PS2-g-PI2 (1) in addition to PS-g-PI2 (2) and PS1/5-g-PI (3). F4 contains PS2-g-PI3 (1) in addition to PSg-PI3 (2). F5 contains PS2-g-PIx≥4 (1) in addition to PS-g-PI4 (2) while F6 contains PSx≥2-g-PIx≥4 (1 + 2) and PS-g-PI4 (3). The chemical composition and the dn/dc values of the polymer species present in each fraction are calculated from the MW estimated by the LCCC analyses. For the calculation of dn/dc values of the polymer species (column 4), we used the weightaverage dn/dc of PS and PI in THF that works well for block copolymers.44 Using the dn/dc value of the polymer species and the RI signal intensity measured in Figure 6, the relative amount of polymer species in each fraction can be calculated from the formula ⎛ dn ⎞ I(Δn) = k ⎜ ⎟c ⎝ dc ⎠

(1)

where I(Δn) is the intensity of RI detector signal, k denotes the instrument constant, and c is the concentration of eluting polymer species. The weight fraction of PS in each TGIC-fraction (column 5) then can be calculated from the chemical composition and the relative amount of each species in the fraction. We also F

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Macromolecules measured the composition of the TGIC fractions by 1H NMR, and the results are shown in column 6 of Table 1. The PS wt % in each fraction calculated from the LCCC-estimated MW of each block shows the values close to the NMR analysis results, but they are not matched perfectly. The discrepancy would be mainly due to the limited accuracy in the MW measurement by LCCC. The MW determined for the “visible” block is slightly influenced by the length of the block under the critical condition, and the deviation becomes larger as the “invisible” block gets larger.31 In the last column of Table 1, the peak MW of the major species in each TGIC fraction determined by SEC/light scattering using the dn/dc value in column 4 are compared with the MW determined by LCCC analysis. They are in good agreement clearly showing that the LCCC analyses of this type of block copolymers can provide a good picture of the polymer species existing in the as-prepared PS-g-PI sample. The unexpectedly incomplete graft reaction indicates that the final linking reaction between the four DPE moieties in the PS backbone and living polyisoprenyl anions did not proceed well. The reaction of polyisoprenyl anion with DPE in a mixed solvent of heptane and THF (50:50 in volume) was observed to be much slower than polystyrenyl anion and a much longer reaction time was used for 12 h. Nevertheless, it was still too short as evidenced from the analysis results in this study. The DPE moieties introduced in the PS backbone correspond to the p-alkyl-substituted DPE derivatives in structure that could be less reactive than unsubstituted DPE since the electrondonating effect of their p-alkyl substituents (hyperconjugation) increases the electron densities on their CC bonds. The optimization should be needed to enhance the linking efficiency (for example, longer reaction time, higher temperature, polar solvent and addition of TMEDA or crown ether, etc.). The other side reaction of this synthesis is the formation of the higher MW backbone, PSx>1. The formation mechanism of the higher MW PS is not fully understood and deserves a further investigation. 2D-LC Separation of PS-g-PI4. 2D-LC separation is a combination of two independent LC separations that has a much larger peak capacity.45−47 It provides a visual overview of complex samples that can be appreciated more easily. Sometimes it can show features that cannot be found from two 1-D LC separations.33,38,48 The 2D-LC chromatogram shown in Figure 7 is a combination of a TGIC separation (Figure 2a) and an LCCC separation at the critical condition of PI (Figure 3). In this 2D-LC separation, a mixed solvent of CH2Cl2/CH3CN (78/22, v/v) instead of 1,4-dioxane in the previous separations was used since 1,4-dioxane is too viscous to carry out the second-D LCCC rapidly. Otherwise, the TGIC and LCCC separation of PS-g-PI is practically the same as before. Figure 7 shows a contour plot of the TGIC x LCCC 2D-LC chromatogram of the as-prepared PS-g-PI. The contour plots clearly display all the species in the PS-g-PI before (Figure 7a) and after (Figure 7b) the SEC fractionation and the major species in each peak are labeled with a schematic illustration of the polymer structures. The vertical dashed bars indicate the elution position of five segment PS backbone. The LCCC elution times of PSx>1-g-PIx>4 and PS1/5-g-PI1 are clearly distinguishable from the rest containing the five segment PS backbone. After the SEC fractionation they were removed largely but most of the major species with the five segment PS backbone remains.

Figure 7. TGIC x LCCC 2D-LC chromatograms of PS-g-PI before (a) and after (b) the SEC fractionation. The same stationary phase (Nucleosil C18, 500A, 7 μm, 500 Å pore, 150 × 4.6 mm) and mobile phases (CH2Cl2/CH3CN, 78/22, v/v) were used for both first-D TGIC and second-D LCCC. For the first-D TGIC, flow rate was 0.04 mL/min and the temperature was maintained at 5 °C for the first 62 min, then a linear gradient from 5 to 29 °C for 600 min. For the second-D LCCC, flow rate was 0.8 mL/min at a column temperature of 46 °C. The vertical dashed lines indicate the common elution time of five segment PS backbone.

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CONCLUSION In this study, a PS-g-PI was synthesized by iterative anionic polymerization methods and characterized by use of various HPLC methods. Combining TGIC, LCCC, and SEC/multiple detection analyses, most of the byproducts formed during the synthesis of the exact PS-g-PI were successfully isolated and identified. In so doing, we demonstrated that LCCC separation at the critical condition of each block can be used effectively for the characterization of this type of graft block copolymer. We also demonstrated that SEC/multiple detection method is not good enough to detect the heterogeneity in this type of graft copolymers and needs to be applied with proper caution.

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AUTHOR INFORMATION

Corresponding Authors

*(T.C.) Tel +82-54-279-2787; Fax +82-54-279-3399; e-mail [email protected]. *(A.H.) Tel +81-52-841-7579; Fax +81-52-841-7579; e-mail [email protected]. ORCID

Taihyun Chang: 0000-0003-2623-1803 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS T.C. acknowledges the support from NRF-Korea (2015R1A2A2A01004974). REFERENCES

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