Article pubs.acs.org/Macromolecules
Dissecting the Mechanism of the Heat-Induced Phase Separation and Crystallization of Poly(2-isopropyl-2-oxazoline) in Water through Vibrational Spectroscopy and Molecular Orbital Calculations Yukiteru Katsumoto,*,† Aki Tsuchiizu,† XingPing Qiu,‡ and Françoise M. Winnik*,‡ †
Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima 739-8526, Japan Department of Chemistry and Faculty of Pharmacy, University of Montreal, CP 6128 Succursale Centre Ville, Montreal, QC H3C 3J7, Canada
‡
S Supporting Information *
ABSTRACT: Aqueous solutions of amphiphilic polymers often undergo a heat-induced phase separation, which is known as the lower critical solution temperature (LCST) phase transition. In the case of aqueous poly(2-isopropyl-2oxazoline) (PIPOZ) solutions, the phase separation is followed, upon prolonged heat treatment, by an irreversible crystallization of the polymer. Optical microscopy observation of a PIPOZ solution (60 g L−1) in water revealed that liquid− liquid phase separation of the aqueous PIPOZ solution occurs at the cloud point (Tc) and that PIPOZ crystallizes in the polymer-rich liquid phase upon prolonged heating of the mixture at a temperature T > Tc. Vibrational spectroscopy combined with molecular orbital (MO) calculations and spectral measurements with model compounds were employed to monitor water/ polymer interactions and changes in polymer conformation during the LCST-type phase separation. The thermally induced spectral variations suggest that the dehydration of the PIPOZ amide functions occurs gradually as the temperature is raised from 20 °C up to Tc. Upon prolonged heating of the phase-separated mixture at constant temperature (Tc + ∼2 °C), the infrared spectrum of the polymer undergoes further changes ascribed to conformational transitions of the polymer backbone. These changes, which are irreversible upon cooling the solution below Tc, lead to the conformation taken by the polymer in the crystalline phase. This situation facilitates crystallization of the polymer by a nucleation/growth mechanism in the polymer-rich phase, a process akin to the crystallization of proteins from solution.
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clusters,5 does not apply to the case of proteins in solutions due to the short range of their attractive interactions. Experimental evidence of liquid/liquid phase separation was reported in the case of lysozyme crystallization6 as well as in solutions of normal and sickle cell hemoglobin.7 The phase behavior of synthetic polymer/solvent mixtures, in which both crystallization and liquid/liquid phase separation may occur, has been a topic of heated discussions since the early 1940s when Richards envisaged this situation for the first time.8 Soon after, it was demonstrated experimentally with several polymer/solvent pairs for which the upper critical solution temperature (UCST) was located above the melting temperature.9 In these systems, the liquid/liquid phase can be identified by the apparition of turbidity upon cooling the solution below a given temperature, the cloud point (Tc). Crystallization occurs by nucleation and growth within the polymer-rich liquid phase and can be detected by light scattering between crossed polars.10 The phase diagram of a
INTRODUCTION Crystallization from solutions is a ubiquitous phenomenon that drives a variety of industrial manufacturing processes, geological events, laboratory manipulations, physiological pathways, and pathological situations. Under specified conditions, the process generates reproducibly particles with desired size, shape, crystal form, and purity. Yet, controlling the crystallization of molecular and nanoscale systems remains a challenge in physics, chemistry, and biology. For instance, although crystallization is central to protein characterization, there is still no set of rules for the rational in vitro production of protein crystals.1 The phase behavior and association dynamics of protein solutions, however, are reasonably well understood.2 It is generally assumed that protein crystallization proceeds via a two-step process involving, first, the formation of liquid droplets of high protein concentration and, second, the creation of ordered protein clusters within the dense liquid intermediate immediately prior to nucleation leading to protein crystals.3 The development of ordered clusters is the ratelimiting step in this two-step nucleation model.4 The classical nucleation model, which involves only one step, namely the generation of ordered crystalline nuclei from dense molecular © 2012 American Chemical Society
Received: February 5, 2012 Revised: March 25, 2012 Published: April 4, 2012 3531
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Given the nontoxicity of polyoxazolines,24 further studies of the temperature responsiveness of its aqueous solutions are easily justified, as they may lead the way to new biomedical applications of PIPOZ and its copolymers.25 Driven by these opportunities, we set about to gain a detailed insight into the overall phenomenon by gathering local nanoscopic information, in addition to the thermodynamic data and mesoscopic structural observations currently available. Various spectroscopic tools can be used to gain local information about polymer conformation, association, and dynamics in solution, including magnetic resonance spectroscopy,26 fluorescence spectroscopy,27 electron paramagnetic spectroscopy,28,18d and infrared spectroscopy (IR).29 The latter is particularly powerful to monitor local conformation changes of the polymer chain as well as changes in polymer/water interactions believed to play an important role in the LCST-type phase separation of aqueous polymer solutions. For instance, studies of the phase behavior of PNIPAM in water by IR spectroscopy gave direct evidence that heating the system brings about the dehydration of the polymer side chains,30 in agreement with the theoretical treatment of the PNIPAM phase separation in water introduced by Okada and Tanaka.31 In a previous report, we pointed out that conformational changes of the polymer also occur during the phase separation.32 Moreover, a detailed analysis of the IR spectra with the aid of molecular orbital (MO) calculations led to the conclusion that intramolecular interactions among the side chains of PNIPAM compete with the hydration/ dehydration process.33 In the first part of this article, we describe results of studies by optical microscopy, which allowed us to observe a liquid/ liquid phase separation in aqueous PIPOZ solutions heated through their Tc. Polymer-rich micrometer-sized droplets formed above Tc. The droplets grew in size with increasing temperature. At sufficiently high temperature, PIPOZ crystallization took place within the droplets. Next, we examine by vibrational spectroscopy the heat-induced liquid−liquid phase separation of aqueous PIPOZ solutions and compare them to the vibrational spectra of new model compounds analogous to the dimer of the PIPOZ repeat unit. Analysis of the spectra of the model compounds combined with molecular orbital (MO) calculations yields a precise molecular picture of the temperature-induced changes in polymer conformation and hydration throughout the liquid/liquid phase separation. Our study provides important mechanistic insights onto the evolution of the liquid/liquid phase separation that leads the way to polymer crystallization. Overall, the phenomena observed bear remarkable similarities to the two-step nucleation of protein crystals from aqueous solutions.
polymer/solvent system changes depending on the molecular weight of the polymer. A decrease in molecular weight lowers the UCST line but hardly affects the melting line. Hence, situations arise where the UCST lies below the melting temperature. Under equilibrium conditions, such a UCST line has no significance. However, under nonequilibrium conditions liquid/liquid separation may still take place if the energy barrier of crystallization is sufficiently high.11 This phase separation process underlies an important processing technique in material sciences, known as thermally induced phase separation (TIPS), a process using nonequilibrium thermal quenching to create intricate liquid/liquid morphologies that are “frozen” upon crystallization of the polymer-rich liquid phase.12 It is used in the fabrication of microcellular foams, microporous membranes,13 and, more recently, scaffold materials in tissue engineering.14 Poly(2-nonyl-2-oxazoline) and poly(2-isobutyl2-oxazoline) in mixed ethanol/water solutions also undergo crystallization upon annealing below their UCST, by a process involving intermediate liquid/liquid separation.15 Liquid/liquid phase separation can also occur upon heating a polymer solution that undergoes a lower critical solution temperature (LCST)-type phase transition. The phenomenon has been observed in aqueous solutions of amorphous polymers. For example, in the course of a study of the heatinduced phase separation of aqueous poly(vinyl methyl ether), Tanaka observed that a temperature jump above the LCST line triggers the formation of a polymer-rich phase dispersed in the lean phase in the form of micrometer-sized droplets that remain stable up to 2 days with no sign of coalescence although they undergo fast Brownian motion.16 Tanaka attributed this remarkable stability to a viscoelastic effect17 that prevents polymer chains from interdiffusing upon collision of two moving droplets. The same effect has also been put forward to account for the formation of stable mesoglobules upon heatinduced phase separation of aqueous poly(N-isopropylacrylamide) (PNIPAM) solutions,18 although in these solutions liquid/liquid phase separation, per se, has never been reported. In the case of the solutions of semicrystalline polymers that undergo a LCST-type phase behavior, liquid/liquid phase separation may be accompanied by simultaneous or subsequent polymer crystallization. This phenomenon is well-known in the case of blends of amorphous and crystalline polymers.19 As far as we are aware, there are few, if any, examples of polymer solutions where LCST-type liquid/liquid phase separation and crystallization compete or coexist. Aqueous solutions of poly(2isopropyl-2-oxazoline) (PIPOZ, Figure 1), a crystalline polymer
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EXPERIMENTAL SECTION
Materials. N,N,2-Trimethylpropionamide (TMPAm) was purchased from Wako Pure Chemical Ltd. PIPOZ was prepared as described previously.34 The number-average molecular weight Mn, the polydispersity Mw/Mn, and the cloud point Tc of the samples are listed in Table 1. The cloud point of this sample was measured by turbidimetry. The dimer model compound, N,N′-1,2-ethanediylbis(N,2-dimethylpropanamide) (EDMPAm), was prepared by reaction of isobutyryl chloride with N,N′-dimethylethylenediamine in dry dichloromethane at 0 °C in the presence of triethylamine.35 The crude amide was purified by fractional distillation at reduced pressure (bp 190−194 °C, 15 mmHg). The 1H NMR spectrum of EDMPAm was obtained using a JEOL JNM-LAMBDA spectrometer (500 MHz, Natural Science Center for Basic Research and Development (NBARD), Hiroshima University). 1H NMR (500 MHz, DMSO-d6 at
Figure 1. Chemical structures of TMPAm, EDMPAm, and PIPOZ.
with a melting temperature of ∼200 °C,20 present the necessary features, since they undergo an LCST-type phase separation around 40 °C,21 and PIPOZ crystallizes from the solutions when they are heated to temperatures in the vicinity of 65 °C.22,23 The possibility of a liquid/liquid phase separation prior to crystallization was invoked. However, direct evidence of the phenomenon was not provided. 3532
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ATR cell was controlled by a homemade thermoelectric device with an accuracy of ±0.1 °C. The cell was sealed by a toggle-clamp type cover to prevent solvent evaporation. Data processing, such as the background solvent spectrum subtraction, derivative calculation, and the nonlinear least-squares method, was performed by a software composed by one of the authors (Y.K.).36 The pretreatment procedure for ATR/IR spectra of polymers in solutions, including the subtraction of the solvent spectrum from a sample spectrum, was described previously.32 Raman Spectroscopy. Raman spectra were recorded on a JASCO NRS-2100 Raman spectrometer equipped with a CCD detector (Princeton Instrument LN/CCD-1100 PBUVAR). The excitation wavelength of Raman scattering was 514.5 nm from an Ar ion laser (Spectra-Physics 2016). The spectral resolution and laser power at the sample position were 4 cm−1 and 200 mW, respectively. Raman spectra were acquired for a 10 wt % solution of EDMPAm in D2O. MO Calculations. All MO calculations were performed using the B3LYP functional with a 6-31+G(d) basis set.37 For the calculation of solvent effects, the polarizable continuum model using the integral equation formalism variant (IEFPCM) was used. The computations were carried out on the GAUSSIAN 03 program.38 Optimized geometries and vibrational frequencies of the model compounds were calculated. The zero-point energy correction was carried out. The harmonic wavenumbers νharm obtained were scaled by the wavenumber linear scaling (WLS) method: νscale = νharm × (1.003 − 0.000021 × νharm).39 WLS parameters were chosen so as to reproduce the experimental IR spectra in the fingerprint region. IR and Raman spectra were simulated by assuming a Lorentzian band shape with a bandwidth of 7 cm−1.
Table 1. Characterization of PIPOZ Samples sample
Mn/g mol−1
Mw/Mn
Tc/°C
concn/wt %
solvent
PIPOZ-10K
10 400
1.06
5800
1.20
44.0a 38.3 41.4 40.4 38.6
0.1 2.0 2.0 2.0 6.0
H2O D2O H2O D2O D2O
PIPOZ-6K
a
Reference 34.
120 °C): δ 1.09 (d, 12H, −CH(CH3)2), 2.76 (m, 2H, −CH(CH3)2), 3.07 (m, 6H, −NCH3), 3.54 (s, 4H, −N(CH2)2N−). The chemical structures of PIPOZ, EDMPAm, and TMPAm are shown in Figure 1. Procedures for Direct Monitoring of the Liquid−Liquid Phase Separation and Crystallization of PIPOZ. Samples of a PIPOZ solution (6 wt %) in water were held between two microscope slides as follows: The solution (10 μL) to be monitored was placed on a glass slide; a smaller glass coverslip was placed on top of the drop. The edge of the coverslip was sealed with an epoxy glue. The liquid spread over a circular area ∼10 mm in diameter. The solution thickness, estimated from the solution volume and the area of the spread solution, was ∼100 μm. The slides were mounted on the temperature control stage (THMS 600 with TMS94, Linkam) of a microscope (Axioskop 2, Carl Zeiss). The heating rate was 10 °C min−1, unless otherwise specified. Turbidimetry. The temperature dependence of transmittance of 650 nm light was monitored using a homemade apparatus equipped with a Si photodiode detector (S2386-18K, Hamamatsu Photonics). The temperature was controlled by a homemade thermostat with an accuracy of 0.1 °C. Heating rate was less than 0.2 °C min−1. Tc was taken as the inflection point of the transmittance curves. IR Spectroscopy. IR spectra were measured at a resolution of 2 cm−1 with coaddition of 512 scans using a Nicolet 6700 Fouriertransform IR spectrometer equipped with a liquid-nitrogen-cooled MCT detector. The attenuated total reflection (ATR) technique was employed to obtain IR spectra of polymer solutions (2 wt %). An ATR cell made of a horizontal ZnSe crystal (RI = 2.403) with an incident angle of 45° was filled with a polymer solution. The temperature of the
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RESULTS AND DISCUSSION
Direct Observation of the Temperature-Induced Phase Transitions of an Aqueous PIPOZ Solution. The temperature of an aqueous solution of PIPOZ-6K (6 wt %) placed between two sealed microscope slides was raised from 20 to 70 °C while recording the changes in appearance of a specific area of the specimen, as displayed in Figure 2a. Within a few seconds of reaching 48 °C, droplets of 1−2 μm in size
Figure 2. Optical micrographs of a solution of PIPOZ-6K in water (6 wt %) undergoing liquid/liquid phase separation and crystallization (heating rate = 10 °C min−1): (A) 20 °C, (B) 48 °C, (C) 50 °C, (D) incubated for 2 h at 50 °C, (E) incubated for 2 h at 70 °C, (F) incubated for 30 h at 70 °C, (G) the transmittance image through crossed Nichols polarizers for (F). The image (H) is a composite of the gray-scaled (F) with the image (G) after applying the edge detection. The micrographs from (I) to (L) show the growth of droplets by heating at a rate of 0.3 °C min−1: (I) 39.8, (J) 40.1, (K) 40.9, and (L) 41.2 °C. 3533
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Figure 3. Temperature and time dependence of IR spectra of a solution of PIPOZ-10K in D2O (2 wt %). The spectra shown were obtained after subtraction of the IR spectrum of D2O from spectra measured under the following conditions. (a) 20 °C, (b) 40 °C, (c) 42 °C, (d) after incubation for 13 h at 42 °C, (e) after incubation for 20 h at 42 °C, (f) solution (e) cooled to 20 °C and kept at 20 °C for 12 h. The inset represents the temperature dependence of the νCO band in water after baseline correction.
concentration of the polymer-rich phase using various in situ methods, including FTIR and Raman imaging, but failed to obtain reliable data. Then, we sought means to trigger the macroscopic liquid/liquid phase transition in a vial and to separate the dense layer. Analysis of this layer by refractometry or densitometry would lead to its polymer concentration. We placed a 1 mL aliquot of the PIPOZ solution in a conical vial and heated it to 50 °C. The solution became turbid as the temperature reached 40 °C. The turbidity persisted upon prolonged incubation at 50 °C, yet the suspension showed no tendency at all to separate into two liquid layers, confirming the stability against coalescence of the polymer-rich liquid droplets. This point will be addressed in detail in a forthcoming publication.40 Next, we used optical microscopy to visualize the crystallization of PIPOZ from hot water. A fresh sample was incubated at 50 °C for 2 h and rapidly heated to 70 °C. At this temperature, the droplets grew via coalescence, such that after a 2 h incubation their average diameter was on the order of 20− 100 μm (Figure 2E). Examination of the sample through crossed Nichols polarizers failed to reveal any crystallization. Upon prolonged heating at 70 °C the droplets stopped growing. Their appearance changed, as seen in Figure 2F, which presents a micrograph of the same sample after a 30 h incubation at 70 °C. Droplets are no longer smooth. Their periphery appears rugged. Observation under polarized light revealed the blue hue of transmitted light characteristic of the presence of crystals (Figure 2G). Figure 2H, which is a composite of Figures 2G and 2F, presents evidence that crystallization occurred within the dense droplets and not in the continuous polymer-lean phase. Temperature-Induced Conformational Changes of PIPOZ Chains in Water. IR Spectra of Aqueous PIPOZ Solutions (20 °C < T < 42 °C). All measurements were performed with a 2% PIPOZ-10K solution in deuterated water (Tc = 38.3 °C, see Table 1). We confirmed independently that PIPOZ in D2O solution undergoes the liquid−liquid phase
became visible, and in a matter of seconds they grew to sizes of 10−20 μm (Figure 2B). The droplets partook in Brownian motion, and their size grew by coalescence. We were concerned by the fact that the temperature of appearance of the droplets turned out to be significantly higher than the cloud point (38.6 °C) determined by turbidimetry for a 6 wt % aqueous solution of PIPOZ-6K (see Table 1). Such a difference may be due to the rather fast heating rate (10 °C min−1) selected. We performed a control experiment, in which the stage was heated from 30 to 45 °C at a rate of 0.3 °C min−1 (Figures 2I−L). Under these conditions, droplet formation occurred between 38.5 and 40.0 °C, a temperature range that corresponds well to Tc of this solution, confirming that the heating rate affects the temperature of the liquid/liquid phase separation. However, the heating rate has no significant impact on the size of the droplets formed at a given temperature above Tc (see Figure 2L). Several experimental protocols were carried out to convince us that (i) droplets are indeed formed and (ii) this is a true reversible liquid/liquid phase separation. A sample heated at a rate of 10 °C min−1 was kept at 50 °C for 2 h. The number of smaller droplets (2−5 μm) gradually decreased. After 2 h at 50 °C, most of the droplets had a diameter of 20−50 μm, with only a few larger drops (Figure 2D). There was no further coarsening at all, although the droplet density was high enough to cause frequent collisions. To test the reproducibility of the observations and the reversibility of the liquid/liquid phase separation, we heated several samples from 20 to 50 °C and always observed droplet formation. After being kept for a few minutes at 50 °C, each sample was cooled to 20 °C. A onephase solution was recovered; no feature whatsoever could be seen by optical microscopy. The appearance of each sample was identical to the original state (Figure 2A). With several samples, we increased and decreased the sample temperature from 20 to 50 °C and back, several times, without any apparent changes in the kinetics of droplet appearance/disappearance that would indicate permanent alterations of the solution composition or microstructure. We attempted to determine the PIPOZ 3534
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spectral studies of a low molecular weight model compound, as described in the following sections. Preferred Conformations of the Model Compound EDMPAm Based on MO Calculations, Spectral Simulations, and Experimental Results. The first step in this study was to select a suitable model compound as well as an adequate MO calculation method. We considered two model compounds: TMPAm and EDMPAm (Figure 1). They correspond to the monomer and dimer units of PIPOZ, respectively. The IR spectra of the two compounds in tetrachloromethane are presented in Figure 4A, together with the spectrum of PIPOZ
separation and following crystallization in the same manner as in H2O, even though Tc of the D2O solution is slightly lower than the Tc of the H2O solution, as shown in Table 1 and discussed previously.34 Control experiments confirmed that PIPOZ crystallizes from the solution in D2O tested by IR spectroscopy heated to 70 °C, but not upon prolonged heating at 42 °C. Instrumental limitations prevented us from heating samples to 70 °C in the IR spectrometer sample compartment; hence, it was not possible to monitor PIPOZ crystallization by IR spectroscopy. The IR spectrum of aqueous PIPOZ within this temperature range presents several bands in the 1550−700 cm−1 region, arising from the vibration modes of the polymer main chain and the isopropyl side groups. It also has a strong band around 1600 cm−1, attributed to the CO stretching (νCO) mode. In the spectrum recorded at 20 °C (trace a in Figure 3), this band consists of at least two overlapping bands centered at ca. 1630 and 1605 cm−1. On the basis of previous studies,41 we assigned the former band to the vibration of CO groups with two hydrogen-bonded water molecules and the latter one to fully hydrated CO groups. Tertiary amides in water, in general, have a unimodal νCO band observed at ca. 1600 cm−1.41,42 The band at 1630 cm−1 is often present in the spectrum of hydrophilic polymers bearing tertiary amide substituents. It has been taken as an indication that the hydration of the amide CO groups is somehow inhibited, possibly due to the steric hindrance imposed by neighboring monomeric units or due to interchain association. The PIPOZ solution was heated to 40 °C, and its IR spectrum was recorded again. There were minor changes in the relative intensity of the bands at 1630 and 1605 cm−1: the intensity of the band at 1630 cm−1 increased with increasing temperature (see inset Figure 3). The IR bands in the 1500− 1400 cm−1 region also underwent slight changes. The solution temperature was raised to 42 °C, which is above Tc. There was no immediate change in the spectrum (see traces b and c in Figure 3). After a few minutes, this solution was cooled back to 20 °C. Its spectrum was identical to the initial spectrum (spectrum a in Figure 3). Subsequently, a fresh sample was heated to 42 °C and kept at this temperature for 20 h. IR spectra were recorded from time to time during incubation. As time progressed, the IR spectra underwent significant changes, as seen in Figure 3, where traces d and e are IR spectra of the PIPOZ solution incubated at 42 °C for 13 and 20 h, respectively. Qualitatively, one notices that after a 20 h incubation at 42 °C several IR bands become sharper and new bands appear at 1300, 1108, 989, 798, and 769 cm−1. The relative intensities of bands around 1430 and 1205 cm−1 also vary remarkably. At the end of the incubation, the sample was brought back to 20 °C, kept at this temperature for 12 h, and re-examined by IR spectroscopy. The spectrum recorded (trace f) was quite different from that of the original solution (spectrum a in Figure 3). It was remarkably similar to the spectrum recorded at 42 °C (spectrum e in Figure 3). Hence, the heat-induced spectral changes are irreversible, at least within the time frame probed here. The recovered solution was clear to the eye at room temperature, without visible sign of crystallization. Therefore, the spectral changes cannot be ascribed to PIPOZ crystallization. We set about to elucidate the mechanisms responsible for the remarkable changes in the IR spectrum of aqueous PIPOZ at 42 °C, with the help of MO calculations and
Figure 4. (A) IR spectra of PIPOZ, EDMPAm, and TMPAm in CCl4. (B) IR spectra of PIPOZ and EDMPAm in D2O.
in the same solvent. The IR spectrum of TMPAm is obviously different from that of the polymer. Hence, TMPAm was not considered further. The spectrum of EDMPAm reproduces reasonably well the spectrum of PIPOZ, in particular the bands near 1475, 1409, 1194, 1160, and 1088 cm−1 attributed to the s k e l e t a l v i b r a t i o n s o f t h e p o ly m e r m a i n c h a in (−NCH2CH2N−). To ascertain that EDMPAm is indeed a good model, we compared the spectra of EDMPAm and PIPOZ in aqueous solutions (Figure 4B). As in the case of the polymer, the νCO band of EDMPAm undergoes a shift to shorter wavenumbers, from 1648 (CCl4) to 1601 cm−1 (D2O). The band remains unimodal, in contrast to the case of PIPOZ in water, for which the νCO band has two contributions (see above). This was anticipated, given that the band at 1630 cm−1 in the spectrum of PIPOZ was attributed to polymer-specific steric hindrance caused by neighboring units as discussed above. The band near 1480 cm−1 in the spectrum of EMDPAm in CCl4 shifts to a higher wavenumber in the spectrum in D2O, a trend exhibited by PIPOZ as well. The B3LYP/6-31+G(d) method was selected to carry out MO calculation-based spectral simulations. A test run using TMPAm convinced us that the method is suitable (see simulated and experimental spectra shown in Figure S1 in Supporting Information). To examine the conformation effects on the stability of EDMPAm, we developed a Ramachandrantype potential energy (PE) diagram for the dihedral angles of 3535
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N−C−C−N (ϕ) and OC−N−C(−C) (ψ). The angle ϕ defines the rotational conformers of the EDMPAm backbone, while ψ reflects those of the conformers of the isobutyryl side chain. Geometry optimization in redundant internal coordinates was performed for ϕ and ψ.43 Since the effects of the relative orientation of the isopropyl group on the stability are minor, compared with those of ϕ and ψ, the rotation of isopropyl groups was not constrained in the geometrical optimization in redundant internal coordinate. The contour map of the PE diagram (Figure 5) reveals the existence of six Figure 6. Optimized geometries of the representative conformers of EDMPAm in vacuum.
Table 2 (see Table S1 also), the conformers a1, a2, and b1 are observable in a nonpolar environment, such as in the gas phase and in nonpolar solvents, while in these conditions the population of the other conformers should be low. In Figure 7, the simulated spectra of the three most stable conformers of EDMPAma1, a2, and b1are confronted to
Figure 5. Ramachandran-type potential energy diagram for ϕ and ψ of EDMPAm obtained from the MO calculations at the B3LYP/631+G(d) level. The zero point energy is not corrected. See Supporting Information for the information on scan and constraint.
minima, corresponding to the EDMPAm conformers with (ϕ, ψ) ∼ (180°, 0°) (a), (60°, 180°) (b), (−60°, 180°) (c), (180°, 180°) (d), (60°, 0°) (e), and (−60°, 0°) (f). In the vicinity of each local minimum, there are several rotational conformers depending on the relative orientation of isopropyl groups, which gives rise to a secondary effect on the stability of the conformers. Thus, we carried out the geometrical optimization without any constraint for the conformers that are assumed to be found near the six local minima of the PE diagram. The calculation results for the representative conformers are summarized in Table 2 and Figure 6.44 In the conformer designations, the letters (a, b, ...) correspond to the six minima in the PE diagram and the numbers (1, 2, ...) correspond to specific orientations of the isopropyl group. Judging from the relative energy ΔE shown in
Figure 7. Experimental IR spectra of EDMPAm (a) in CCl4 and (b) neat liquid and simulated IR spectra at the B3LYP/6-31+G(d) level for (d) a1, (e) b1, and (f) a2. Trace c is the Boltzmann-averaged spectrum of a1, b1, and a2.
the actual IR spectra of EDMPAm, neat and in CCl4. The simulated spectra of the conformers a1 and a2 correspond reasonably well to the experimental spectra.45 There are differences, however. Specifically, the bands at 1126, 837, and 538 cm−1 in the experimental spectra are conspicuously absent in the simulated spectrum. Also, the shape of the envelope near 750 cm−1 in the simulated IR spectrum of a1 or a2 is different from that in the experimental spectra. Bands at the wavenumbers mentioned above, or in close proximity to them, are present in the simulated spectrum of the conformer b1. Hence, the three conformers must coexist in EDMPAm in nonpolar solvents or neat. This conclusion is confirmed by the excellent agreement between the Boltzmann-weighted average of the simulated spectra of a1, a2, and b1 (Figure 7, trace c) and the experimental IR spectra of EDMPAm. It is generally acknowledged that Raman spectroscopy is more sensitive to skeletal conformational changes than IR spectroscopy. Therefore, as ancillary support to conclusions drawn from IR spectroscopy, we measured Raman spectra of EDMPAm neat and in CCl4 (Figure S3). We also generated simulated Raman spectra of the conformers, which are presented in Figures S3 and S4, together
Table 2. Relative Energy ΔE (kJ mol−1) from the Energy of the Most Stable Conformer (a1), ϕ, and ψ for EDMPAm Conformers Optimized in Vacuum
a
ID
ΔEa/kJ mol−1
ϕ/deg
ψ/deg
a1 a2 b1 c1 d3 e3 f4
0.00 0.446 0.0867 7.10 16.2 11.7 11.7
180.0 177.9 62.60 53.69 170.0 78.10 −78.09
11.30 14.12 182.7 158.6 175.6 16.88 −2.87
Zero point energy was corrected. 3536
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Figure 8. (right) Optimized conformations of the complex of EDMPAm and four D2O molecules. (left) Simulated IR spectra of the various conformers of the complexes calculated by the B3LYP/6-31+G(d) method.
Figure 9. (left) (a) Experimental IR spectrum of a solution of PIPOZ in D2O incubated for 20 h at 42 °C and the simulated IR spectra of (b) a-type tetramer, (c) a-type trimer, and (d) a-type dimer obtained by the B3LYP/6-31+G(d) method. (right) Optimized conformers of the multimers models.
CO group with a CO···H−O hydrogen bond for each water molecule. In the optimized structures recovered from the calculations (Figure 8), the carbonyl group is linked to only two molecules of water, but the geometry of the conformers is not modified significantly, compared to the original a1, a2, and b1 conformers. The changes in the simulated spectra, shown in Figure 8, reflect accurately the spectral differences observed experimentally (Figure 4B), a key finding which warrants the validity of the methodology. The MO calculations and spectral simulations confirm that the spectral shifts observed reflect the hydration of the amide group of EDMPAm, rather than a change in the populations of its conformers, as strongly suggested also by the Raman spectra of EDMPAm solutions in CCl4 and D2O (Figure S3). Conformation and Hydration Changes of PIPOZ in Water Concomitant with the Heat-Induced Phase Separation. As seen in Figure 3, a gradual increase in the temperature of a PIPOZ solution in water from 20 to 40 °C (below the macroscopic phase separation) is accompanied by a gradual change of the vibrational bands in the 1650−1430 cm−1 spectral window. Particularly significant is the gradual enhancement, as the temperature increases, of the intensity of the band
with the Boltzmann-weighted simulated Raman spectrum for a1, a2, and b1. There is an excellent agreement between experimental and simulated Raman spectra. Effects of Hydration on the Vibrational Spectra of EDMPAm and PIPOZ. The IR spectrum of EDMPAm in D2O differs significantly from that in CCl4 (Figure 4B). The largest changes are observed for the νCO band, which for in the aqueous solution shifts to shorter wavenumbers by ca. 40 cm−1 and for the bands at 1475 and 1409 cm−1 (CCl4), which shift by 15−18 cm−1 in the opposite direction in the spectrum of the D2O solution. The Raman spectrum of EDMPAm in water (Figure S3) hardly differs from that of EDMPAm neat or in CCl4 in the 1550−800 cm−1 spectral range, which indicates that there are no major differences in the conformer population in water compared to nonpolar solvents. The spectral region corresponding to νCO vibrations frequency, in contrast, is affected by the change in solvent. To facilitate the interpretation of the IR spectral changes observed, we carried out MO calculations for the hydrogenbonded complexes between EDMPAm and water. The input geometries were those of the conformers a1, a2, and b1 to each of which were added four water molecules placed around the 3537
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near 1630 cm−1 associated with νCO (inset, Figure 3). This indicates that the dehydration of the amide groups starts when the temperature is well below the temperature of macroscopic phase separation. As the solution temperature was brought to a temperature slightly above Tc, the overall sharpness of the spectrum increased gradually over time, and new bands appeared at 1430, 1330, 1205, 1108, 989, 798, and 769 cm−1 (see Figure 3). These bands are mainly associated with vibrations of the polymer main chain; their presence may indicate a significant change of the conformation of the polymer main chain, at least on the length scale probed by IR spectroscopy. To test this hypothesis, we carried out simulations of the IR spectra of the type-a conformers (ϕ∼180°) of the trimer and tetramer analogues of the PIPOZ monomer units (Figure 9). Their simulated IR spectra correspond much more closely to the spectrum of PIPOZ in water after prolonged incubation at 42 °C, compared to those of the dimer model. For instance, in the multimer spectra the bands at 1197 and 1163 cm−1 are prominent, compared to the band at 1281 cm−1, a key feature of the experimental PIPOZ spectrum as well. We determined that in their optimized form both multimers adopt an all-trans backbone conformation. Hence, the excellent match between the simulated trimer and tetramer IR spectra and the IR spectrum of PIPOZ solutions at 42 °C suggests that upon prolonged incubation at 42 °C the polymer main chain adopts a conformation in which the alltrans conformers (type a) become prevalent. As seen convincingly in Figure 5, for the conformational change from the b-type to the a-type to occur, it is necessary to pass though an intermediate conformer, such as the e-type or the d-type. In a nonpolar environment, these conformers seem hardly to exist under normal conditions because their relative energies are too high (Table 2). We carried out a geometrical optimization of representative conformers with the IEFPCM model using the solvent parameters for water. The results are compiled in Table 3. The most stable conformer is a1, and ΔE indicates that the
of ΔE for EDMPAm as a function of ψ and ϕ and plotted the results in Figures 10A and 10B, respectively. The energy
Figure 10. Plots of the changes of ΔE as a function of ϕ (B) and ψ(A) for EDMPAm in vacuum (circle) and in water (rectangle) calculated at the B3LYP/6-31+G(d) level.
barriers for the conformational change from b to d and from d to a are high, although the ΔE value of the d-conformer becomes very low in aqueous media. Thus, it is inferred that a thermal energy is required to induce the conformational change in the main chains of PIPOZ from b- to a-type. This may be the reason why the conformational change of PIPOZ occurs in hot water.
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Table 3. Relative Energy ΔE (kJ mol−1) from the Energy of the Most Stable Conformer (a1), ϕ, and ψ for EDMPAm Conformers Optimized in Aqueous Media
a
ID
ΔEa/kJ mol−1
ϕ/deg
ψ/deg
a1w b1w c1w d3w e3w f4w
0.000 4.67 3.90 2.90 8.23 8.44
179.94 62.02 −62.33 172.2 70.05 −70.37
4.890 172.8 −166.3 −1.38 11.65 −12.42
CONCLUSION The direct visual observation of thin layers of PIPOZ solutions confined between sealed glass slides, reported in the first part of this article, revealed the following features of the heat-driven phase separation of PIPOZ aqueous solutions and eventual crystallization of PIPOZ from water: (i) the clouding of PIPOZ solutions noted by turbidimetry corresponds to the formation of two coexisting liquid phases; (ii) the dense (polymer-rich) liquid phase is metastable with respect to the dilute solution, i.e., a barrier for its decay exists, and it is not a simple density fluctuation; and (iii) the crystallization of PIPOZ takes place by nucleation in the dense liquid phase upon input of energy in the form of heat. The second section of the article focused on the changes in PIPOZ conformation and hydration, as seen by vibrational spectroscopy and MO calculations, during the liquid/liquid phase separation and upon prolonged heating at a temperature sufficiently low to preclude irreversible polymer crystallization. This part of the work revealed that (i) the polymer chains gradually dehydrate as the solution temperature nears macroscopic phase separation and (ii) in their dehydrated form the polymer chains undergo a conformational change and adopt a mostly all-trans conformation. A pictorial representation of phenomena unveiled in the study is given in Figure 11, where we draw connections between the two aspects of the work. In cold water (T < Tc) the polymer adopts a conformation in which the trans and gauche conformers
Zero point energy was corrected.
population of a1 is prominent in aqueous media. Interestingly, ΔE between a1 and d3, which is one of the candidate intermediates, is much smaller in aqueous media than that in vacuum, presumably because the dipole interaction between the side chains is shielded by solvent molecules. It should be emphasized that ΔE of d3 is smaller than that of b1 in aqueous media, indicating that the conformational change from b- to dtype occurs spontaneously. One question still remains to be answered: why does the conformational change not occur in cold water? One reason could be that the energy barrier between the b- and d-type conformers is too high to be overcome at room temperature. To test this hypothesis, we calculated the angular dependence 3538
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Figure 11. Schematic representation of the changes in the conformation of PIPOZ in water as a function of temperature and their correlation with the macroscopic phenomena observed by optical microscopy.
coexist. The chain is decorated with a hydrogen-bonded network of water molecules forming a hydration layer. Two or more water molecules of this layer form hydrogen bonds with the amide carbonyl group of each monomer unit (gauche or trans conformers). As the solution temperature increases, the hydration layer surrounding the polymer breaks gradually via cleavage of carbonyl/water hydrogen bonds, as revealed by shifts of the carbonyl bands in the IR spectrum and detected also by microcalorimetry.21b At a specific temperature, the solution undergoes liquid/liquid phase separation, and within the polymer-rich phase, the polymer chain adopts a mostly trans conformation. This conformation is stabilized over all others and remains the predominant one upon cooling of the sample below Tc. This conformation facilitates interchain dipolar interactions between amide fragments, which may promote partial organization of the chains in the dense liquid phase and facilitate nucleation and crystallization of PIPOZ. It is worth recalling here that in the crystalline phase PIPOX has an all-trans conformation as seen in Figure 11, which was confirmed by X-ray diffraction of PIPOZ precipitated from hot water.46 In the first part of the study we observed that heating beyond 50 °C is required to induce crystallization, yet spectral data indicate that the conformational transition leading to the all-trans conformation occurs at a lower temperature. For crystallization to occur nucleation needs to take place. Our observations may indicate that nucleation requires further thermal energy and/or that it is the rate-determining step. In this context, the FT-IR measurement of the system up to 70 °C would be very interesting for understanding the nucleation process at the molecular level, if the process is indeed detectable spectroscopically. PIPOZ is often compared to its structural isomer, poly(Nisopropylacrylamide) (PNIPAM) which has a −[CH2−CH(CONH−iPr)]− repeat unit. The two polymers share many similarities from the macroscopic viewpoint. They are soluble in polar organic solvents and in cold water. Their aqueous solutions undergo a phase separation upon heating beyond a temperature in the 30−40 °C range (Tc of PNIPAM: 32 °C).47
But similarities vanish beyond such phenomenological observations. For instance, many reports on the phase separation of aqueous PNIPAM indicate that, above Tc, the polymer forms single chain globules that associate to large aggregates or mesoglobules.18 There is no evidence that a liquid/liquid phase separation occurs concomitant with the LCST-type phase separation of aqueous PNIPAM solutions. While a “coil-to-globule” collapse is often invoked in descriptions of the phase separation of aqueous PIPOZ solutions, there is no direct evidence that such a chain contraction takes place. Our results advocate that the chains adopt a more extended conformation above Tc, compared to their conformation in cold water. The dehydration mechanism also differs for the two polymers, as demonstrated for instance by analysis of vibrational spectra. As shown in this study, spectral changes in the 1650−1430 cm−1 spectral region, which is sensitive to the hydration state of the amide carbonyl, occur over a wide temperature range in PIPOZ solutions, beginning at temperatures well below Tc. In contrast, the amide I band of PNIPAM in water does not change at all upon heating until Tc is reached, at which point a band appears at a wavenumber of ∼1650 cm−1, suggesting that the molecular environment around the PNIPAM chain undergoes a discontinuous change in the vicinity of Tc. The existence of CO···H−N intrachain hydrogen bonds is believed to play an important part in the thermosensitivity of aqueous PNIPAM solutions.32 In this context, it is interesting to recall that the temperature at which the network of water of hydration surrounding peptides starts to unravel upon heating is quite constant (∼31 °C) independently of the peptide structure. A recent study suggests that this temperature is determined to a large extent by the cooperative hydration of the peptide backbone and by the Hbonds between water and the −HN−CO structural motifs.48 This unit is present in the structure of PNIPAM, which also undergoes cooperative hydration/dehydration31 around 31 °C, but in the structure of PIPOZ this unit is replaced by the >N− CO motif. Our study demonstrates that this structural difference has profound implications on the properties of the 3539
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(16) Tanaka, H. Macromolecules 1992, 25, 6377. (17) Tanaka, H. J. Phys.: Condens. Matter 2000, 12, R207. (18) (a) Wu, C.; Li, W.; Zhu, X. X. Macromolecules 2004, 37, 4989. (b) Aseyev, V.; Tenhu, H.; Winnik, F. M. Adv. Polym. Sci. 2006, 196, 1. (c) Kujawa, P.; Aseyev, V.; Tenhu, H.; Winnik, F. M. Macromolecules 2006, 39, 7686. (d) Junk, M. J. N.; Li, W.; Schlüter, A. D.; Wegner, G.; Spiess, H. W.; Zhang, A.; Hinderberger, D. J. Am. Chem. Soc. 2011, 133, 10832. (19) (a) Inaba, N.; Sato, K.; Suzuki, S.; Hashimoto, T. Macromolecules 1986, 19, 1690. (b) Tanaka, H. Phys. Rev. Lett. 1996, 76, 787. (c) Hu, H.; Shangguan, Y.; Zuo, M.; Zheng, Q. J. Polym. Sci., Part B: Polym. Phys. 2008, 46, 1923. (20) (a) Bassiri, A.; Levy, A. J. Polym. Sci., Part B 1967, 5, 871. (b) Litt, M.; Rahl, F.; Roldan, L. G. J. Polym. Sci., Part A-2: Polym. Phys. 1969, 7, 463. (c) Obeid, R.; Tanaka, F.; Winnik, F. M. Macromolecules 2009, 42, 5818. (21) (a) Uyama, H.; Kobayashi, S. Chem. Lett. 1992, 1643. (b) Diab, C.; Akiyama, Y.; Kataoka, K.; Winnik, F. M. Macromolecules 2004, 37, 2556. (c) Park, J.-S.; Akiyama, Y.; Winnik, F. M.; Kataoka, K. Macromolecules 2004, 37, 6786. (d) Park, J.-S.; Kataoka, K. Macromolecules 2007, 40, 3599. (e) Zhao, J.; Hoogenboom, R.; Assche, G. V.; Mele, B. V. Macromolecules 2010, 43, 6853. (22) Hoogenboom, R.; Thijs, H. M. L.; Jochems, M. J. H. C.; van Lankvelt, B. M.; Fijten, M. W. M.; Schubert, U. S. Chem. Commun. 2008, 44, 5758. (23) Diehl, C.; Č ernoch, P.; Zenke, I.; Runge, H.; Pitschke, R.; Hartmann, J.; Tiersch, B.; Schlaad, H. Soft Matter 2010, 6, 3784. (24) U.S. Food and Drug Administration (21 CFR 175.105) (note that the FDA approval is only valid for use as indirect food additives). (25) (a) Schlaad, H.; Diehl, C.; Gress, A.; Matthias, M.; Demirel, A. L.; Nur, Y.; Bertin, A. Macromol. Rapid Commun. 2010, 31, 511. (b) Hoogenboom, R. Angew. Chem., Int. Ed. 2009, 48, 7978. (c) Viegas, T. X.; Bentley, M. D.; Harris, J. M.; Fang, Z.; Yoon, K.; Dizman, B.; Weimer, R.; Mero, A.; Pasut, G.; Francesco, M. V. Bioconjugate Chem. 2011, 22, 976. (26) Spiess, H. W. Macromolecules 2010, 43, 5479. (27) Kujawa, P.; Aseyev, V.; Tenhu, H.; Winnik, F. M. Macromolecules 2006, 39, 7686. (28) (a) Winnik, F. M.; Ottaviani, M. F.; Bossman, S. H.; Pan, W.; Garcia-Garibay, M.; Turro, N. J. J. Phys. Chem. 1993, 97, 12998. (b) Junk, M. J. N.; Li, W.; Schluter, A. D.; Wegner, G.; Spiess, H. W.; Zhang, A.; Hinderberger, D. Angew. Chem., Int. Ed. 2010, 49, 5683. (29) Everall, N.; Griffiths, P.; Chalmers, J. M. Vibrational Spectroscopy of Polymers: Principles and Practice; Wiley: New York, 2007. (30) (a) Platé, N. A.; Levedeva, T. L.; Valuev, L. I. Polym. J. 1999, 31, 21. (b) Maeda, Y.; Higuchi, T.; Ikeda, I. Langmuir 2000, 16, 7503. (c) Maeda, Y.; Nakamura, T.; Ikeda, I. Macromolecules 2001, 34, 1391. (d) Percot, A.; Zhu, X. X.; Lafleur, M. J. Polym. Sci., Polym. Phys. Ed. 2000, 38, 907. (31) Okada, Y.; Tanaka, F. Macromolecules 2005, 38, 4465. (32) Katsumoto, Y.; Tanaka, T.; Sato, H.; Ozaki, Y. J. Phys. Chem. A 2002, 106, 3429. (33) (a) Katsumoto, Y.; Tanaka, T.; Ihara, K.; Koyama, M.; Ozaki, Y. J. Phys. Chem. B 2007, 111, 12730. (b) Katsumoto, Y.; Kubosaki, N.; Miyata, T. J. Phys. Chem. B 2010, 114, 13312. (34) Obeid, R.; Maltseva, E.; Thunemann, A. F.; Tanaka, F.; Winnik, F. M. Macromolecules 2009, 42, 2204. (35) Buswell, M.; Fleming, I.; Chosh, U.; Mack, S.; Russel, M.; Clark, B. P. Org. Biomol. Chem. 2004, 2, 3006. (36) The software, named SPINA, can be download from the following Web site: http://home.hiroshima-u.ac.jp/katsumot/spina. html. (37) (a) Becke, A. D. J. Chem. Phys. 1993, 98, 5648. (b) Lee, C.; Yang, W.; Parr, G. R. Phys. Rev. B 1988, 37, 785. (38) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, Jr., J. A.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.;
polymer in water and brings further evidence in support of the suggestion that networks of H-bonded water carry important information and may play some role in nonequilibrium biological processes.48
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ASSOCIATED CONTENT
* Supporting Information S
Calculated and observed IR spectra of TMPAm, an example for the definition of redundant internal coordinates with scan and constraint information, the simulated IR and Raman spectra for the representative nine conformers of EDMPAm, the Raman spectra of EDMPAm; sum of the electronic and zero-point energy E (hartrees) and optimized geometries of EMPAm. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Fax +81-82-424-7408, e-mail
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Grant-in-Aid for Scientific Research (No. 21750126) to Y.K. and by a grant of the Natural Science and Engineering Research Council of Canada to F.M.W. The authors thank Prof. T. Sato (University of Osaka, Japan) for stimulating discussions and useful suggestions. Dr. R. Obeid is thanked for supplying one of the PIPOZ samples.
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REFERENCES
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