NMR Study of Thermoresponsive Hyperbranched Polymer in Aqueous

Dec 3, 2013 - Department of Chemistry, School of Sciences, Tianjin University, and ... High-resolution 1H NMR has been used on the thermoresponsive ...
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NMR Study of Thermoresponsive Hyperbranched Polymer in Aqueous Solution with Implication on the Phase Transition Songzi Jiang,† Yefeng Yao,†,* Qun Chen,† and Yu Chen‡,* †

Department of Physics & Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, North Zhongshan Road 3663, 200062 Shanghai, P. R. China ‡ Department of Chemistry, School of Sciences, Tianjin University, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), 300072 Tianjin, P. R. China S Supporting Information *

ABSTRACT: High-resolution 1H NMR has been used on the thermoresponsive hyperbranched polyethylenimines (HPEIs) modified with isobutyramide (IBAm) groups (HPEI-IBAm), to study the structure and dynamics of the macromolecules in aqueous solution before and after the phase transition. It shows that the HPEI-IBAm macromolecule having a high IBAm substitution degree has a clear phase transition in aqueous solution, whereas the HPEI-IBAm macromolecule having a low IBAm substitution degree does not. The different phase transition behaviors have been attributed to the content as well as the distribution of the IBAm groups in the macromolecules. In order to deepen the understanding of the phase transition, the hydrophobic−hydrophobic interaction inside the HPEI-IBAm macromolecules was investigated by monitoring the 1H−1H NOEs between the different hydrophobic groups. An enhanced hydrophobic−hydrophobic interaction was observed in the HPEI-IBAm macromolecule having a high IBAm substitution degree after the phase transition, which provides a new perspective for our understanding of the phase transition of the macromolecules in aqueous solution. By using PFG diffusion NMR, the weight distributions of the moving particles in the solution were monitored. The β parameter used in the PFG diffusion NMR, which reflects the change of the weight distributions of the moving particles in solution, has proved to be a good way to monitor the aggregation process of the moving particles in the solution.

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INTRODUCTION During the past decade, polymers with stimuli-responsive properties, such as fast and reversible conformational or phase changes in response to variations in temperature and/or pH, have gained much interest in the field of biology and medicine.1,2 One of the most appealing stimuli-responsive species is the thermoresponsive hydrophilic polymer having the lower critical solution temperature (LCST) in aqueous solution, which means that above a specific temperature its solubility in water dramatically decreases. These thermoresponsive polymers show broad application potential in various interesting fields, such as protein chromatography,3 protein adsorption and tissue engineering,4 drug delivery, regenerative medicine,5,6 sensing devices, and so on.7 In 2004, Kono’s group reported their pioneering work on the thermoresponsive dendrimers whose LCST could be tuned over a broad range by varying the molecular weight.8 Following their pioneering work, additional thermoresponsive dendritic polymers, including dendrimers and hyperbranched polymers, were reported.9−16 Compared to the traditional linear thermoresponsive polymers that usually form a random-coil structure in solution,17,18 the dendrimeric/hyperbranched polymers have a compacted sphere-like structure. As a © 2013 American Chemical Society

consequence of such a structure, the thermoresponsive dendrimeric/hyperbranched polymers usually only have a minor conformation adjustment during the transition (i.e., the globule-to-globule transition), different from the coil-toglobule transition occurring during the transition of the traditional linear thermoresponsive polymers.19,20 Meanwhile, the thermoresponsive dendrimeric/hyperbranched polymers exhibit obvious differences in properties compared to the traditional thermoresponsive linear polymer.16,21−23 For instance, the phase transition temperature of thermoresponsive dendritic polymers is more sensitive to the addition of salts (including anions and cations) than those of thermoresponsive linear ones.16,22,23 A good understanding of the properties of the thermoresponsive dendrimeric/hyperbranched polymers requires a detailed knowledge of the structure and dynamics at a molecular level. However, to date, the study of thermoresponsive dendrimeric/hyperbranched polymers has mainly focused on the preparation of new polymers,8−16 the exploration of their Received: October 11, 2013 Revised: November 21, 2013 Published: December 3, 2013 9688

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potential application,24−26 and the measurement of their macroscopic phase transition behavior in solution using the traditional turbidity or calorimetric measurements.8−16,21−23 The detailed study of the phase transition behavior of the thermoresponsive dendrimeric/hyperbranched polymers at a molecular level has been very scarce. In the literature, Chai et al. studied the structure and conformation of DAB dendrimers in chloroform and benzene solutions via multidimensional NMR techniques.27 Wu et al employed FT-IR to investigate the detailed polar group interaction of thermoresponsive hyperbranched polymer during the heating−cooling process at the molecular level.28 Recently, Holycross and Chai reported a detailed NMR study on the structures of hyperbranched polyethylenimine (HPEI) in chloroform solution.29 NMR is a very powerful technique for the structure analysis of materials, because it supplies not only the information about the chemical nature of the molecules, but also some unique information, such as the spatial information on the groups, the inter/intramolecular interaction etc. NMR thus has been explored for studying the phase transition behavior of the traditional thermoresponsive linear polymers, and their interaction with various additives, and valuable information has been obtained.30−34 In this research, we applied a detailed NMR investigation on the thermoresponsive HPEI modified with isobutyramide (IBAm) groups (HPEI-IBAm). By combining different NMR techniques, we studied the content and distribution of the IBAm groups in the HPEI-IBAm macromolecules, which is considered to be very crucial for the phase transition of the samples. By monitoring nuclear Overhauser effects (NOEs), we studied the local dynamics of the different chemical groups in the HPEI-IBAm macromolecules. The inhomogeneity of the local dynamics in the HPEI-IBAm macromolecules was revealed. The hydrophobic− hydrophobic interaction inside the HPEI-IBAm macromolecule before and after the phase transition was studied. We monitored the weight distribution of the moving particles in the solutions before and after the phase transition by the PFG diffusion NMR. The molecular aggregation in the solutions was then discussed.

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were synthesized and these two samples have the substitution degrees of 15% (named as HPEI-IBAm-15) and 81% (named as HPEI-IBAm81), respectively. NMR Experiments. The NMR experiments were mainly performed on a Bruker DRX 500 MHz instrument equipped with 5 mm standard probe with a z-gradient. Some experiments were performed on a Varian 700 MHz instrument equipped with four RF channels, a z axis pulse field gradient (PFG) accessory and a 5 mm Varian 1H/13C/31P/2H four channel probe with a PFG coil. (a). 1D NMR Experiments. High resolution 1H NMR spectra were acquired on a 500 or 700 MHz instrument by using 5−7 kHz spectral width, 4 μs (45°) pulse width, a recycle delay of 4 s, and 32 scans. The data were processed with an exponentially decaying window function with a line broadening factor of 2 Hz. 13C NMR spectra were acquired on the 500 MHz instrument by using 38 kHz spectral width, 4 μs (45°) pulse width, a recycle delay of 2 s, and 1024 scans. The data were processed with Gaussian weighting function. All the experiments had TMS as the internal reference. (b). 2D NMR Experiments. 2D 1H−1H NOESY spectra were acquired at 500 MHz by using 2,500 Hz spectral windows in f1 and f 2, a recycle delay of 2 s and a 100 ms mixing time. Eight scans were averaged for each of 512 complex t1 increments. The data were processed with Gaussian weighting function in both dimensions and zero filling to a 1024 × 2048 data matrix before Fourier transformation. At 323 K, some 2D 1H−1H NOESY spectra were acquired on the Varian 700 MHz spectrometer by using 3,500 Hz spectral windows in f1 and f 2. The other parameters used on the 700 MHz instrument were same as those on the 500 MHz instrument. 2D 1H−13C HMBC spectra were acquired on a 700 MHz instrument by using 44 000 and 14 000 Hz spectral windows in the 13 C( f1) and 1H( f 2) chemical shift dimensions, respectively, and a 2 s recycle delay. 32 scans were averaged for each 1024 complex t1 increments. The data were processed with Gaussian weighting function in both dimensions and zero filling to a 2048 × 4096 data matrix before Fourier transformation. 2D 1H−1H COSY spectra were acquired on the 500 MHz instrument by using 3000 Hz spectral windows in f1 and f 2, and a 2 s recycle delay. Eight scans were averaged for each 512 complex t1 increments. The data were processed with Gaussian weighting functions in both dimensions and zero filling to a 1024 × 2048 data matrix before Fourier transformation. 2D 1H−13C HSQC spectra were acquired on the 500 MHz instrument by using 30 000 and 5000 Hz spectral windows in the 13 C( f1) and 1H( f 2) chemical shift dimensions, respectively, and a 2 s recycle delay. Four scans were averaged for each 512 complex t1 increments. The data were processed with Gaussian weighting function in both dimensions and zero filling to a 4096 × 2048 data matrix before Fourier transformation. (c). Diffusion NMR. 2D DOSY spectra were acquired on the 500 MHz instrument by using 10 kHz spectral width in f 2, and a recycle delay of 2 s. The pulse sequence used in the DOSY experiments is the Bruker pulse program “stebpgp1s”. The pulse gradient duration ranged from 2.0 to 4.5 ms in the different experiments, while the diffusion time (250 ms) was kept constant. The pulse gradients were incremented from 2% to 95% of the maximum gradient strength (50 G/cm) in a linear ramp (16 steps). Eight scans and 4 dummy scans were acquired on each sample. The data were processed with an exponentially decaying window function with a line broadening factor of 5 Hz and zero filling to 64k data points. Cloud-Point Measurement. The turbidity measurements were performed on a UV−vis spectrometer (Purkinje General T6) operated using a wavelength of 650 nm.

EXPERIMENTAL SECTION

Materials. Isobutyric anhydride (98%) was purchased from Alfa Aesar and used without further purification. Hyperbranched polyethylenimine (HPEI, Aldrich, Mn = 104g/mol, Mw/Mn = 2.5), was dried under vacuum prior to use. Benzoylated cellulose tubing (MWCO 1,200) was purchased from Sigma and used as received. Triethyl amine (A.R., TEA) was dried over CaH2 and distilled before use. Syntheses of HPEI-IBAm. The preparation procedure of HPEIIBAm is the same as that reported previously:16,23 Under nitrogen atmosphere, isobutyric anhydride was added dropwise to a mixture of HPEI and triethylamine in chloroform at 0 °C with vigorous stirring. Subsequently, the reaction mixture was kept at room temperature for 24 h. Finally, the reaction temperature was raised to 65 °C for 2 h to complete the reaction. After cooling to room temperature, the produced salt was filtered. Volatile materials in the filtrate were removed under vacuum and the residue was dissolved in 40 mL of methanol. About 1 g of potassium carbonate was added to the solution and the mixture was stirred at room temperature for 4 h. After filtration, the solution was concentrated to ca. 10 mL and then purified by dialysis against methanol using a benzoylated cellulose membrane (MWCO 1200 Da) for 2 days. Finally, the methanol solvent was removed under vacuum, and the product was dried in a vacuum pressure for 24 h. The IBAm substitution was controlled by adjusting the feed ratio of isobutyric anhydride to HPEI. Two HPEI-IBAms

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RESULTS AND DISCUSSION Turbidity Measurements. Four aqueous solutions of HPEI-IBAm-15 and HPEI-IBAm-81 were prepared for the turbidity measurement. The concentrations of the solutions are 1 and 20 mg/mL for each HPEI-IBAm sample. The light transmittances of the samples at different temperatures are 9689

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molecule is not thermoresponsive, but the HPEI-IBAm-81 macromolecule is thermoresponsive. Structural Analyses by NMR. Monitoring the Distribution of the IBAm Groups. Figure 2 exhibits the structure models and the 1H spectra of the two HPEI-IBAm samples. According to the literature and our results, we have assigned the signals in the spectra. A tentative signal assignment is shown in the spectra of the samples. For the details for the signal assignments, readers are referred to the 1H COSY, 13C DEPT-135, 1H−13C HSQC, and HMBC spectra in Supporting Information. For kinetics reasons, the isobutyric anhydride molecules are likely to first react with the amino groups on the periphery of the HPEI macromolecules. As the reaction proceeds, the isobutyric anhydride molecules may start to react with the amino groups inside the macromolecules. Therefore, it is anticipated that the IBAm groups in HPEI-IBAm-15 and HPEIIBAm-81 are different not only in the content, but also in the distribution. By combining the 1H COSY and NOESY experiments, we have distinguished the signals of the IBAm groups on the periphery and inside the macromolecules. Figure 3 shows the 1H COSY spectra of HPEI-IBAm-15 and HPEIIBAm-81 in aqueous solution. We paid close attention to the cross peaks between the CH3 signal (∼1.1 ppm) and the CH signals (2.5−3.0 ppm). In the 1H COSY spectrum of HPEIIBAm-15 (Figure 3a), only one set of cross peak between the CH3 signal and the CH signal at ∼2.5 ppm is observed, indicating that there is only one kind of CH group present in the macromolecules. In contrast, there are up to 4 sets of cross

shown in Figure 1. For the concentrated solution of HPEIIBAm-81 (20 mg/mL), a sharp transition appears at 25 °C. For

Figure 1. Light transmittance of the HPEI-IBAm-15 and HPEI-IBAm81 aqueous solutions at different temperatures. The solution concentrations are 1 and 20 mg/mL.

the dilute solution of HPEI-IBAm-81 (1 mg/mL), the transition temperature increases to ∼30 °C. Meanwhile, the transition progress is not as sharp as that in the concentrated solution. For HPEI-IBAm-15, no clear transition has been observed both in the concentrated solution (20 mg/mL) and in the dilute solution (1 mg/mL). From the macroscopic aspect, it can be therefore determined that the HPEI-IBAm-15 macro-

Figure 2. Structure models of (a) HPEI-IBAm-15 and (b) HPEI-IBAm-81 and the high resolution 1H NMR spectra of (c) HPEI-IBAm-15 and (d) HPEI-IBAm-81. The asterisk in part c indicates that there are some signals overlapping D. The assignment of the signal (∗) is discussed in the Supporting Information. These 1H NMR spectra were recorded at the 700 MHz instrument. The experimental temperature is 278 K. The solution concentration is 1 mg/mL for both samples. 9690

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Figure 3. 1H COSY spectra of (a) HPEI-IBAm-15 and (b) HPEI-IBAm-81. The two 1D spectra on the top of the 2D spectra are the 1D projections of the extracted areas marked by the gray dashlines in the 2D COSY spectra. These COSY spectra were recorded at the 500 MHz instrument. The experimental temperature is 278 K. The solution concentration is 1 mg/mL for both samples. The free software “Dmfit” was used for the signal decomposition.36

Figure 4. The 1H NOESY spectra of (a) HPEI-IBAm-15 and (b) HPEI-IBAm-81. These 1H NMR spectra were recorded at the 500 MHz instrument. The experimental temperature is 278 K. The solution concentration is 1 mg/mL for both samples.

peaks between the CH3 signal and the CH signals in the 1H COSY spectrum of HPEI-IBAm-81 (Figure 3b), indicating that there are multiple kinds of CH group present in HPEI-IBAm81. On the basis of the synthetic chemistry of the two HPEIIBAm samples, we assigned the CH signal at ∼2.5 ppm to the CH groups that are located on the periphery of the HPEI macromolecule, and assigned the three CH signals ranging from ∼2.7 ppm to ∼2.9 ppm to the CH groups located inside the HPEI macromolecule. The results from the 1H−13C HSQC and HMBC (see Supporting Information) are consistent with this assignment. On the basis of the above assignment, we thus

are able to calculate the ratio between the IBAm groups on the periphery and those inside the macromolecule. For this purpose, we extracted the areas marked in the 2D spectra and plotted the 1D projections of the extracted areas on the top of the 2D spectra. For HPEI-IBAm-15, only one peak is observed in the 1D projection spectrum, indicating that almost all of the IBAm groups are on the periphery of the macromolecule. For HPEI-IBAm-81, the 1D projection spectrum shows four well resolved peaks. Decomposition shows that the ratio of the IBAm groups on the periphery (I4) to those inside the macromolecule (I1 + I2+ I3) is about 100:31. 9691

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Figure 5. Temperature dependent high resolution 1H NMR spectra of (a) HPEI-IBAm-15 and (b) HPEI-IBAm-81. These spectra were recorded at the 500 MHz instrument. The solution concentration is 1 mg/mL for both samples.

Figure 6. 1H NOESY spectra of the HPEI-IBAm-15 dilute solution sample (1 mg/mL) acquired at (a) 278 and (b) 323 K. These spectra were recorded on the 500 MHz instrument. (c) 1H NOESY spectrum of the HPEI-IBAm-15 dilute solution sample (1 mg/mL) acquired in the 700 MHz instrument. The extracted 1D slices are normalized according the integral of the CH3 signals. The experimental temperature is 323 K.

Therefore, about 24% IBAm groups in a HPEI-IBAm-81 macromolecule are inside the macromolecule. The spatial locations of the IBAm groups in the HPEI macromolecules have been further studied by using the 1H NOESY experiment. Figure 4a shows the 1H NOESY spectrum of HPEI-IBAm-15. It is observed that the cross peaks are only present between A and B, and between C and D. From the chemical structure, it is known that A and B are from the IBAm group and, C and D are from the HPEI skeleton. The absence of the cross peaks between A/B and C/D indicates that in the HPEI-IBAm-15 macromolecules the IBAm groups are not in proximity to the HPEI skeleton. A possible distribution of IBAm groups in HPEI-IBAm-15 macromolecules thus is that the IBAm groups are located only on the periphery of the

HPEI-IBAm-15 macromolecules (see the cartoon picture in Figure 2a). Moreover, it is observed that the cross peaks between A and B in Figure 4a have very low signal intensities, although A (the CH3 group) and B (the CH group) are chemically bonded. These weak cross peaks indicate the high mobility of the IBAm groups. Considering the limited amount of the IBAm group in the HPEI-IBAm-15 macromolecule, the high mobility of the IBAm groups in fact indicates that the IBAm groups are sparsely distributed on the periphery of the HPEI-IBAm-15 macromolecule. Figure 4b shows the 1H NOESY spectrum of HPEI-IBAm81. Compared to Figure 4a, the cross peaks between A′ and B′ in Figure 4b show much stronger intensities, indicating a stronger NOE between A′ and B′ in HPEI-IBAm-81. This 9692

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Figure 7. 1H NOESY spectra of the HPEI-IBAm-81 dilute solution sample (1 mg/mL) acquired at (a) 278 and (b) 323 K at 500 MHz and at (c) 323 K at 700 MHz. The extracted 1D slices are normalized according the integral of the CH3 signals.

considered to have an intrinsic relation to their different phase transition behaviors. More details about the segmental dynamics of these two HPEI-IBAm macromolecules are revealed by following temperature dependent 1H NOESY spectra of the samples. Parts a and b of Figure 6 show the 1H NOESY spectra of the HPEI-IBAm-15 dilute solution (1 mg/mL) acquired at 278 and 323 K, respectively. These two experiments were performed on the 500 MHz instrument. In these spectra, we focus on the two sets of the cross peaks, that is, the cross peaks between A (the CH3 group, ∼1.1 ppm) and B (the CH group, ∼2.5 ppm), and the cross peaks between C and D (i.e., the two directly bonded CH2 groups). It is interesting to find that these cross peaks, which appear in the spectrum at 278 K (see Figure 6a), completely disappear in the spectrum acquired at 323 K (see Figure 6b). In the literature,27 it is known that the cross peaks in the 1H NOESY spectrum are from the 1H−1H NOE between the groups, which depends on the 1H Larmor frequency, ω, and the correlation time of the segmental motion (τc): When the segmental motion is in the slow regime, (ω2τc2 ≫ 1), positive cross peaks are anticipated; When the segmental motion is in the fast regime, (ω2τc2 ≪ 1), negative cross peaks are anticipated; When the segmental motion is in the intermediate regime, (ω2τc2 ∼ 1), the cross peak may disappear or show a dispersive line shape, if the signal is strongly modulated by J-coupling.35 Therefore, the disappearance of the cross peaks in the NOESY spectrum in Figure 6b indicates that, with increasing temperature, the dynamics of the CH2 groups of HPEI-IBAm-15 increases from the low frequency regime

stronger NOE between A′ and B′ in HPEI-IBAm-81 indicates the reduced segmental mobility of the IBAm groups that most likely can be attributed to the reduced free volume resulting from the increased number of the IBAm groups in the HPEIIBAm-81 macromolecule. Moreover, compared to HPEI-IBAm15, it is interesting to observe that the cross peaks between the CH3 groups (A′), and the CH2 groups ranging from ∼3.3 ppm to ∼3.6 ppm (C′), are present in the spectrum. Note that A′ (the CH3 group) belongs to the IBAm group and C′ (the CH2 group) belongs to the HPEI skeleton. Therefore, the presence of the cross peaks between A′ and C′ indicates that some IBAm groups in HPEI-IBAm-81 are in close proximity to the HPEI skeleton. In order to have such a spatial proximity, a possible structure is that some IBAm groups of HPEI-IBAm-81 are located inside the HPEI-IBAm-81 macromolecule (see the cartoon picture in Figure 2b). Segmental Dynamics of HPEI-IBAm Macromolecules. The segmental dynamics of the two HPEI-IBAm macromolecules was first studied by the temperature dependent 1H spectra in Figure 5. It is observed that the signals in the spectra of HPEI-IBAm-81 show clear line broadening with increasing temperature, whereas the signals in the spectra of HPEI-IBAm15 do not show clear changes in the linewidths and the signal intensities. These temperature dependent 1H spectra indicate that the segmental mobility of HPEI-IBAm-81 decreases with increasing temperature, whereas for HPEI-IBAm-15 the segmental mobility does not have such a temperature dependence. The different temperature dependences of the segmental mobility in HPEI-IBAm-15 and HPEI-IBAm-81 is 9693

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Figure 8. Natural logarithm of the normalized echo attenuation, I/I0, as a function of (γgδ)2*(Δ−δ/3) for (a) the dilute HPEI-IBAm-15 solution (1 mg/mL) and (b) the concentrated HPEI-IBAm-15 solution (20 mg/mL). The experimental temperatures are 278 and 323 K. The curve fitting was achieved by using eq 1. To demonstrate the influence of the polydispersity of the moving particles, the fitting curves using β = 1 (the dashed lines) are also plotted.

(ω2τc2 ≫1), to the intermediate regime (ω2τc2 ∼ 1). This point has been further proved by the 1H NOESY spectrum of the same sample at 323 K acquired on a 700 MHz instrument (see Figure 6c), where the cross peaks between D (∼2.7 ppm) and C (∼3.3 ppm) appear again at the same temperature. The reappearing of the cross peaks can be attributed to the Larmor frequency in the 700 MHz instrument that makes the local dynamics of the two CH2 groups fulfill ω2τc2 ≫ 1 again. It is interesting to find that the cross peaks between A and B show a dispersive line shape. The origin of this line shape can be attributed to the strong modulation of the J-coupling.35 Note that in the above discussion we have assumed that the cross peaks in the NOESY spectra in Figure 6 are mainly from the groups that are directly chemically bonded. In this context, therefore, it is the segmental dynamics, 1/τc, that dominates the NOEs between the groups. The variation in the spatial distance between the groups is considered to have a minor influence on the NOEs between the groups. Parts a and b of Figure 7 show the 1H NOESY spectrum of HPEI-IBAm-81 dilute solution (1 mg/mL) acquired at 278 and 323 K, respectively. These two experiments were performed on the 500 MHz instrument. In these spectra, similar to what is observed in the HPEI-IBAm-15 dilute solution, the cross peaks between C′ and D′ appear at 278 K (see Figure 7a), but completely disappear at 323 K (see Figure 7b), indicating that the dynamics of the CH2 groups of HPEI-IBAm-81 increases from the low frequency regime (ω2τc2 ≫1), to the intermediate regime (ω2τc2 ∼ 1), when the temperature increases from 278 to 323 K. Interestingly, accompanying with the disappearance of the cross peaks between C′ and D′, the cross peaks between A′ and C′ with positive intensities are still present in the spectrum at 323 K, indicating that the dynamics of the CH3 groups and the “interacted” CH2 groups is still in the low frequency regime (ω2τc2 ≫1). Moreover, comparison has shown that the intensities of the cross peaks between A′ and C′ increase when the temperature increases from 278 to 323 K. This indicates the NOEs between A′ and C′ become stronger when the temperature increases from 278 to 323 K. The above observations when combined reveal an interesting feature of the HPEI-IBAm-81 macromolecule in aqueous solution at 323 K, that is, the local dynamics in the skeleton structure of HPEI-IBAm-81 becomes inhomogeneous after the phase transition. At 323 K the CH2 groups that are in close

proximity to the IBAm groups have a segmental dynamics in the low frequency regime (ω2τc2 ≫1), whereas some CH2 groups still have a segmental dynamics in the intermediate regime (ω2τc2∼1). This inhomogeneous segmental dynamics reflects the inhomogeneity of the local packing of the CH2 groups in the skeleton structure of HPEI-IBAm-81, which likely results from the “equilibrium” between the hydrophobic− hydrophobic interaction (e.g., the interaction between the CH2 and IBAm groups) and the polar−polar interaction (e.g., the interaction between the amine groups). Note that the CH3 group, A′, and the CH2 group, C′, both are hydrophobic. Therefore, the increased NOEs between A′ and C′ actually reflects the increased hydrophobic−hydrophobic interaction in the skeleton structure. In the NOESY spectra in parts a and b of Figure 7, it is observed that the cross peaks between A′ and B′ keep the positive line shape when the temperature increases from 278 to 323 K. This indicates that the segmental dynamics of the IBAm groups on the periphery of the HPEI-IBAm-81 macromolecule are still in the low frequency regime (ω2τc2 ≫1) even at 323 K. This temperature dependence of the IBAm groups is different from that of the HPEI-IBAm-15 macromolecule where the mobility of the IBAm groups increases with increasing temperature. Figure 7c shows the 1H NOESY spectrum of HPEI-IBAm-81 acquired on the 700 MHz instrument at 323 K. In this spectrum, the cross peaks between D′ (∼2.7 ppm) and C′ (∼3.3 ppm) appear again. The reason for the reappearance of the cross peaks is as same as that of HPEI-IBAm-15. In summary, the 1H NOESY spectra in Figures 6 and 7 clearly demonstrated the different segmental dynamics in the HPEI-IBAm-15 and HPEI-IBAm-81 macromolecules. For the HPEI-IBAm-15 macromolecule, the segmental dynamics increases with increasing temperature. In contrast, the segmental dynamics in the HPEI-IBAm-81 macromolecule seems to be more complicated. The segmental dynamics of the skeleton structure of the HPEI-IBAm-81 macromolecule shows a clear inhomogeneity. This inhomogeneity can be attributed to the inhomogeneous packing of the CH2 groups in the skeleton structure. Meanwhile, it has been realized that the observed 1H NOEs actually reflect the hydrophobic−hydrophobic interactions in the macromolecules. It is also observed that the 9694

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Table 1. β and Diffusion Coefficients for the HPEI-IBAm-15 and HPEI-IBAm-81 Aqueous Solutions at 278 and 323 K

hydrophobic−hydrophobic interaction in the skeleton structure may increase after the phase transition. Molecular Aggregation of HPEI-IBAm before and after Phase Transition. The PFG NMR diffusion measurement has been used to monitor the aggregation process of the HPEI-IBAm macromolecules in the solution before and after the phase transition. In the literature,37,38 the signal decay in the PFG-NMR diffusion measurement can be described by eq 1 ln(I /I0) = [−Dg 2γ 2δ 2(Δ − δ /3)]β

sample

concentration (mg/mL)

temp (K)

β

diffusion coefficient (m2/s)

HPEI-IBAm15

1

278

0.82

2.56 × 10−11

323 278 323 278

0.82 0.82 0.82 0.95

1.05 2.75 1.02 2.52

323 278 323

0.80 0.95 0.74

1.23 × 10−10 2.50 × 10−11 1.17 × 10−10

20

(1)

HPEI-IBAm81

where I is the signal intensity measured under the gradient strength of g; I0 is the signal intensity without gradient; D is the diffusion coefficient; γ is the gyromagnetic ratio of the spin; δ is the duration of gradient pulse; Δ is the diffusion time; β is called the stretch parameter that will be the focus of the following study. In the literature, the β parameter has been used to quantify the influence of the polydispersity of the moving particles on the signal decay.38 For a polymer sample, β is equal to 1 for a monodisperse sample and, ranges from 0.5 to 1 for a polydisperse sample. Usually, the value of β decreases when the polydispersity increases. Figure 8a shows the plots of CH3 peak area versus [g2γ2δ2(Δ−δ/3)] of the dilute HPEI-IBAm-15 solution (1 mg/mL) at 278 and 323 K. The solution concentration (1 mg/ mL) was selected to ensure that the HPEI-IBAm-15 macromolecules do not aggregate in the solution. A clear bending-up curvature is observed in both of the decay curves in Figure 8a, indicating the presence of the polydispersity of the moving particles. Fitting these curves yields a β value of 0.82 and the diffusion coefficient of 2.56 × 10−11 m2 s−1 for the solution at 278 K and, a β value of 0.82 and the diffusion coefficient of 1.05 × 10−10 m2 s−1 for the solution at 323 K. The same β value at 278 and 323 K indicates that the polydispersity of the moving particles in solution does not change when the temperature increases. The unchanged polydispersity indicates that no aggregation process occurs in the dilute HPEI-IBAm-15 solution when the temperature increases from 278 to 323 K. The bending-up curvature of the curves thus can be simply attributed to the polydispersity of the HPEI-IBAm-15 macromolecules. Figure 8b shows the plots of CH3 peak area versus [g2γ2δ2(Δ−δ/3)] of the concentrated HPEI-IBAm-15 solution (20 mg/mL) at 278 and 323 K. Fitting the two curves yield the β value of 0.82 both at 278 K and at 323 K. This β value is the same as that in the dilute solution, indicating that no aggregation process occurs in the concentrated solution at both 278 and 323 K. The β values and the diffusion coefficients from the curve fitting are summarized in Table 1. Figure 9a shows the plots of CH3 peak area versus [g2γ2δ2(Δ−δ/3)] of the dilute HPEI-IBAm-81 solution at 278 and 323 K. It is clear that the decay curve at 323 K has a more clear curvature than that at 278 K. Fitting the curves yields a β value of 0.95 at 278 K and 0.80 at 323 K. The decreased β value at 323 K indicates the increase of the polydispersity of the moving particles, which most likely reflects the molecular aggregation in the solution. Figure 9b shows the plots of CH3 peak area versus [g2γ2δ2(Δ−δ/3)] of the concentrated HPEI-IBAm-81 solution at 278 and 323 K. The best fit of the curve at 278 K yields a β value of 0.95. This β value is the same as that of the dilute solution sample, indicating that at 278 K the HPEI-IBAm-81 macromolecules does not aggregate in this concentrated solution. For the curve obtained at 323 K, the best fit yields a β value of 0.74. This β

1

20

× × × ×

10−10 10−11 10−10 10−11

value is largely deviated from 1, indicating that the macromolecules strongly aggregate at this temperature. Table 1 summarizes the β values and the diffusion coefficients of the HPEI-IBAm-81 solutions obtained from the curve fitting. Implication to the Phase Transition. Because the HPEI macromolecules do not have the phase transition behavior, it is considered that the phase transition behavior of HPEI-IBAm macromolecule originates from the IBAm group substitution. For the HPEI-IBAm-15 and HPEI-IBAm-81 macromolecules, the different degrees of IBAm group substitution are expected because of their controlled chemical synthesis conditions. Reflecting the influence of the IBAm group substitution on the phase transition, the cloud point experiments have shown that the aqueous solutions of the HPEI-IBAm-81 macromolecule have a clear phase transition, whereas the aqueous solutions of the HPEI-IBAm-15 macromolecule do not. In this contribution, we find that the IBAm groups in the HPEI-IBAm-15 and HPEIIBAm-81 macromolecules differ not only in the substitution degree, but also in the location/distribution in the macromolecules. This opens a new perspective on the influence of the IBAm group on the phase transition of the HPEI-IBAm macromolecule, that is, the IBAm substitution degree and the location/distribution of the IBAm groups both may contribute to the phase transition behaviors of the HPEI-IBAm macromolecules. At this moment, however, we are not able to distinguish the influence from the substitution degree and the influence from the location/distribution of the IBAm groups on the phase transition behavior. Further study of the influences of the substitution degree and the location/distribution of the IBAm groups on the phase transition is the subject of ongoing studies in our laboratory. In the literature, it is considered that the phase transition behavior of HPEI-IBAm macromolecule is controlled by three factors, namely, the hydrophobic−hydrophobic interaction, the polar−polar interaction and the de/hydration of the hydrophobic and polar units. Among these factors, the hydrophobic− hydrophobic interaction is much less well observed compared to the others. In this contribution, it has been realized that the hydrophobic−hydrophobic interaction in the HPEI-IBAm-81 macromolecule is clearly reflected by the NOEs between the CH3 groups of the IBAm group and the CH2 groups of the HPEI skeleton structure. It is very intriguing to observe that the hydrophobic−hydrophobic interaction in the HPEI-IBAm-81 macromolecules increases after the phase transition (as indicated by the increased NOEs between the CH3 groups of the IBAm group and the CH2 groups of the HPEI skeleton structure at 323 K). The increased hydrophobic−hydrophobic interaction indicates that the hydrophobic units like the CH3 9695

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Figure 9. Natural logarithm of the normalized echo attenuation, I/I0, as a function of (γgδ)2*(Δ−δ/3) for (a) the dilute HPEI-IBAm-81 solution (1 mg/mL) and (b) the concentrated HPEI-IBAm-81 solution (20 mg/mL). The experimental temperatures are 278 and 323 K. The curve fitting was achieved by using eq 1. To demonstrate the influence of the polydispersity of the moving particles, the fitting curves using β = 1 (the dashlines) are also plotted.

and CH2 groups do rearrange themselves during the phase transition. Monitoring the NOEs between the hydrophobic units before and after the phase transition thus provides a feasible way to study the rearrangement of the hydrophobic units during the phase transition. Last but not least, from the NOESY spectra in Figure 6 and Figure 7, we realize that the hydrophobic−hydrophobic interaction is clearly present in the skeleton structure of the HPEI-IBAm-81 macromolecule, but not the HPEI-IBAm-15 macromolecule. From the chemical natures of the two macromolecules, the difference between the skeleton structures of the two HPEI-IBAm macromolecules is realized mainly in whether the IBAm groups are present or not. In this context, the hydrophobic−hydrophobic interaction in the skeleton structure of the HPEI-IBAm-81 macromolecule thus is attributed to the presence of the IBAm groups in the skeleton structure. This is possible because the IBAm groups can facilitate the formation of the hydrogen bonding network and consequently a dense packing arrangement of the hydrophobic units.28 Whereas, for the HPEI-IBAm-15 macromolecule the absence of the hydrophobic−hydrophobic interaction in the skeleton structure indicates that the CH2 groups in the HPEI skeleton structure themselves are not prone to form a dense packing. As a consequence, the segmental dynamics in the skeleton structure of the HPEI-IBAm-15 macromolecule only shows a normal temperature dependence. On the contrary, because of the presence of the hydrophobic−hydrophobic interaction, the segmental dynamics in the skeleton structure of the HPEI-IBAm-81 macromolecule shows a clear inhomogeneity after the phase transition, that is, some CH2 groups have intermediate segmental dynamics (ω2τc2 ∼ 1), whereas some CH2 groups that are in close proximity to the IBAm groups have a segmental dynamics still in the low frequency regime (ω2τc2 ≫1). On the basis of the above observations, we have made the cartoon pictures in Figure 10 to illustrate the structure and dynamics of the HPEI-IBAm-15 and HPEI-IBAm-81 macromolecules in aqueous solution at different temperatures. For the HPEI-IBAm-15 macromolecules, the IBAm groups are mainly on the periphery of the macromolecules (indicated by the cilium-like short lines around the spheres). At both low temperature (278 K) and elevated temperature (323 K), the

Figure 10. Cartoon pictures illustrating the structure and dynamics of the two HPEI-IBAm macromolecules in aqueous solution at different temperatures. In these pictures, the spheres represent the HPEI skeleton structure and the cilium-like short lines represent the IBAm groups. The ellipsoids represent the interaction between the IBAm groups inside the macromolecules and the CH2 groups of the HPEI skeleton structure. The red color and the blue color are used to indicate the relatively high and low mobility, respectively.

IBAm groups are well spaced from the HPEI skeleton structure. The segmental mobility in the HPEI-IBAm-15 macromolecules increases with increasing temperature. But the HPEI-IBAm-15 macromolecules do not aggregate at both 278 and 323 K. On the contrary, the IBAm groups in the HPEI-IBAm-81 macromolecules are not only on the periphery of the macromolecules, but also inside the macromolecules. Note that the IBAm groups inside the macromolecules may strongly interact with the CH2 groups of the HPEI skeleton structure at 278 K. This interaction (illustrated by the blue ellipsoids) is a hydrophobic−hydrophobic interaction and possibly is facilitated by the hydrogen bonding between the IBAm groups and the HPEI skeleton structure. This interaction enhanced at the elevated temperature (323 K), resulting in the inhomogeneous local dynamics in the skeleton structures. Accompanying with the changes in the structure and dynamics in the skeleton 9696

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structure at the elevated temperature, the HPEI-IBAm-81 macromolecules aggregate in the solutions.

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CONCLUSIONS To summarize, in this contribution we have demonstrated a detailed NMR investigation on two HPEI-IBAm macromolecules that are prepared from the same HPEI, but have different IBAm group substitution degrees. By combining different NMR techniques, we have shown that the IBAm groups of two HPEI-IBAm macromolecules differ not only in the substitution degree but also in the spatial distribution in the HPEI structure. This opens a new perspective as to how the IBAm groups influence the phase transition of the HPEI-IBAm macromolecules. By monitoring the NOEs between the hydrophobic groups in the HPEI-IBAm macromolecules before and after the phase transition, we demonstrate an unambiguous experimental proof that the hydrophobic−hydrophobic interaction in a HPEI-IBAm macromolecule increases after the phase transition. The results indicate that the IBAm groups inside the HPEI skeleton structure are very crucial to form the hydrophobic−hydrophobic interaction. Finally, we used the PFG diffusion NMR to study the aggregation process of the HPEI-IBAm macromolecules in aqueous solutions at different temperatures. By analyzing the β parameter in the diffusion NMR, we clearly demonstrate that the HPEI-IBAm macromolecules having a high IBAm substitution degree aggregate in both the dilute and the concentrated aqueous solution at 323 K, whereas the HPEI-IBAm macromolecules having a low IBAm substitution degree do not at the same temperature. This is consistent with the observations in the macroscopic turbidimetry measurements. The β parameter in the PFG diffusion NMR has been proved to be a good way to monitor the aggregation process of the moving particles in the solution.

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ASSOCIATED CONTENT

* Supporting Information S

Details for the signal assignments with the 1H COSY, 13C DEPT-135, 1 H− 13 C HSQC, and HMBC spectra and calculation of the IBAm group substitution.This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: (Y.Y.) [email protected]. *E-mail: (Y.C.) [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21174039, 21274106). REFERENCES

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