Article pubs.acs.org/Macromolecules
Hydration States of Poly(N‑isopropylacrylamide) and Poly(N,N‑diethylacrylamide) and Their Monomer Units in Aqueous Solutions with Lower Critical Solution Temperatures Studied by Infrared Spectroscopy Chihiro Hashimoto,†,* Akiyoshi Nagamoto,‡ Takashi Maruyama,‡ Naomi Kariyama,§ Yuma Irisa,§ Akifumi Ikehata,⊥ and Yukihiro Ozaki§ †
Department of Applied Chemistry and Biotechnology, Niihama National College of Technology, 7-1 Yakumo-cho, Niihama, Ehime 792-8580 Japan ‡ Kohjin Co., Ltd., 4-1-21 Nihombashi Muromachi, Chuo-ku, Tokyo, 103-0022, Japan § School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo, 669-1337, Japan ⊥ National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan S Supporting Information *
ABSTRACT: The coil-to-globule transition of poly(N-isopropylacrylamide) (PNiPA) and poly(N,N-diethylacrylamide) (PdEA) in aqueous solutions has recently received substantial interest in its drastic change in the hydration state from a hydrated random coil to a hydrophobic globule for practical applications: drug delivery and tissue engineering. In this report, the hydration states of PNiPA and PdEA in aqueous solutions were investigated by IR spectroscopy in the amide I, N−H and C−H stretching band regions as compared with those of their repeat units, N-isopropylpropionamide (NiPP) and N,N-diethylpropylacrylamide (dEP) in aqueous and cyclohexane solutions in combined with their phase diagrams. The IR spectral changes in the amide I and C−H stretching band regions of Nalkylamides and N,N-dialkylamides including NiPP and dEP in aqueous solutions with varying concentration was characteristic due to different amide−amide interaction; the amide−amide interaction for N-alkylamide (CO···H−N hydrogen bond) is stronger than that for N,N-dialkylamide (dipolar interaction). It is found that almost all amide groups of PNiPA in aqueous solution forms the intramolecular CO···H−N hydrogen bond even in the coil state and that the amide group of PNiPA is less hydrated than that of PdEA in spite of the similar degree of hydration to alkyl groups. The IR spectral changes in the amide I and C−H stretching band regions of PNiPA and PdEA in aqueous solutions with heat are ascribed to the dehydration of the amide and alkyl groups from the coil state to the globule one.
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and tissue engineering in the form of hydrogels, micelles, films and particles.2−4 Okano et al. recently showed the usefulness of PNiPA grafted layer to harvest contiguous cell sheet, in which the adhesion/detachment of cells is regulated by temperature changes.4 The coil-to-globule transition of PNiPA and PdEA is endothermic, which is attributed to the energy required to break hydrogen bonds between the hydrophilic amide group and water molecules during the dehydration of the polymer chain upon heating.5,6 Even though the rearrangement of the water molecules in the vicinity of the hydrophilic and/or hydrophobic group is considered to be essential for the LCST
INTRODUCTION Poly(N-alkylacrylamide) and poly(N,N-dialkylamides) such as poly(N-isopropylacrylamide) (PNiPA) and poly(N,N-diethylacrylamide) (PdEA) are recognized thermo-sensitive polymers with lower critical solution temperature (LCST), aqueous solutions of which exhibit phase separation upon heating at 26−39 °C.1 The phase separation behavior is also characterized by a coil-to-globule transition, where a drastic change in the hydration state from a hydrated random coil to a hydrophobic globule is observed above the transition temperature. The cross-linked thermo-sensitive polymer gel undergoes an analogous collapse transition in water, and a large volume change of gel is called volume phase transition of gel. Such thermo-sensitive polymer receives substantial interest not only from the viewpoint of theoretical understanding of LCST type phase transition but also its practical applications: drug delivery © 2013 American Chemical Society
Received: November 9, 2012 Revised: December 25, 2012 Published: January 14, 2013 1041
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Figure 1 in combined with their phase diagrams with LCSTs. The IR spectral changes in the amide I and C−H stretching
type phase transition mechanism, the role of the water molecules is still under discussion.7−9 The phase separation behavior of aqueous solution of PNiPA has been extensively studied by using various experimental techniques, including calorimetry,5,6 NMR,10 light and neutron (or X-ray) scatterings,11−13 IR spectroscopy,14 dielectric relaxation15,16 image processing17 and so on. Shikata et al. evaluated the number of hydrated water molecules per monomer unit of PNiPA in aqueous solution to be constant with a value of 11 below transition temperature by dielectric relaxation measurement.16 They also showed that each isopropylamide group of the monomer, N-isopropylacrylamide or NiPP is mainly hydrated by 5−6 water molecules so that 5−6 additional water molecules make hydrogen bond bridges between amide groups for PNiPA. Tanaka et al. insisted that the concept of “cooperative hydration” is essential to derive the flat LCST cloud-point curves on the phase diagram of aqueous solution of PNiPA.9 The cooperative hydration is caused by a positive correlation between neighboring bound water molecules due to the presence of the large hydrophobic isopropyl side groups. Wu et al. indicated that the origin of the hysterisis in the coil-to globule transition of aqueous solution of PNiPA is due to the intermolecular hydrogen bonding of amide groups in its collapsed state.11 Several research groups have synthesized stereocontrolled PNiPA homopolymers and discussed their LCST behaviors .18−21 For isotacticity-rich PNiPA homopolymer, the solubility in water decreases and the transition temperature slightly decreases with increasing isotacticity.19,20 The coil-to-globule transition of PNiPA and PdEA in water is distinctly reflected in the IR spectra especially in the CO stretching (amide I) and C−H stretching band regions.22−25 Two amide I bands are apparently observed for PNiPA and PdEA in aqueous solution and the higher wavenumber band increases with increasing temperature through the coil-toglobule transition. Maeda et al. showed that the peak-position of the higher wavenumber band of PNiPA in water is similar to that of the band of PNiPA film, and the higher and lower wavenumber bands were assigned to the hydrogen bonded C O group with N−H group of PNiPA and with O−H groups of water molecules, respectively.24 However, Katsumoto et al. showed that the formation of the CO···H−N hydrogen bonding induces a lower wavenumber shift of the amide I band by density functional theory (DFT) calculation, and the higher and lower wavenumber bands of PNiPA in water were assigned to the free CO group and the CO group which forms the CO···H−N hydrogen bonding, respectively.25 In the case of PdEA in water, Katsumoto et al. assigned the higher and lower wavenumber bands to the weakly and strongly hydrogen bonded CO groups with O−H groups of water molecules, respectively.25 Maeda et al. assigned the higher and lower wavenumber bands to mono- and dihydrogen bonding interactions of the CO group with O−H groups of water molecules from the viewpoint that the carbonyl oxygen has two lone pairs of electrons.24 It seems that the intramolecular hydrogen bonding of secondary amide groups (the CO···H− N hydrogen bonding) is considerably essential for the amide I band assignment of PNiPA in water. In the present study, the hydration states of PNiPA and PdEA in aqueous solutions were investigated by the IR spectroscopy in the amide I, N−H and C−H stretching band regions in comparison with the cases of their repeat units, Nisopropylpropionamide (NiPP) and N,N-diethylpropylacrylamide (dEP) in aqueous and cyclohexane solutions as shown in
Figure 1. Chemical structure of molecules studied in this work.
band regions of N-alkylamides and N,N-dialkylamides including NiPP and dEP (Figure 2) in aqueous solutions with concentration was characteristic due to different amide− amide interaction; the amide−amide interaction for Nalkylamide (CO···H−N hydrogen bond) is stronger than that for N,N-dialkylamide (dipolar interaction). A variety of experimental and theoretical studies has been studied extensively for N-methylacetamide (NMA) for the comprehension of the conformation and the inter- and intramolecular 1042
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d12) at several arbitrary concentrations. Deuterated N-alkylamide was obtained by evaporation of its methanol-d solution with the exchange from the N−H to N−D groups. The deuterated solvents, D2O (99.9%) and cyclohexane-d12, were purchased from Cambridge Isotope Laboratories, Inc. Measurements. For the phase diagram measurement of NiPP/ H2O, dEP/H2O/NaCl, PNiPA/H2O, and PdEA/H2O, the cloud point of the aqueous mixture in a 1 mm cell was defined as the lowest temperature at which the increase in the 90° scattered light intensity of 488 nm was detected by the fluorescence spectrometer (Hitachi F2500) with heating rate of 0.1 °C/3 min. The molar percentage (mol %) of PNiPA and PdEA in H2O was calculated using the number of monomer unit moles of PNiPA and PdEA, respectively. For the phase diagram measurement of PNiPA and PdEA gels, the weights of a gel at the given temperatures and the weight of its dry gel after drying in a vacuum were measured. The water content of a gel was calculated by Ns/Nd, where Ns and Nd are the numbers of monomer unit moles of polymers and water molecules in a gel, respectively. The molar percentage (mol %) of PNiPA or PdEA in a gel was calculated by Nd/ (Ns + Nd) × 100. The transmission and attenuated total reflection (ATR) IR spectra of N-alkylamides, N,N-dialkylamides, PNiPA, PdEA, and their solutions were measured at a 1 cm−1 resolution and accumulated 128 times using a Thermo Nicolet Magna 760 Fourier-transform IR spectrometer with a liquid-N2-cooled mercury−cadmium−telluride detector. N-alkylamides, N,N-dialkylamide and their solutions were put between two CaF2 windows (thickness of each window was 5 mm) without a spacer. The path length of the sample must be less than 6 μm, because the observed amide I band of N-alkylamides and N,Ndialkylamide was saturated by use of a spacer of 6 μm. The transmission IR spectra of N-alkylamides, N,N-dialkylamides and their H2O, D2O, and cyclohexane solutions were measured at ca. 18 °C with the exception of crystalline samples (100, 90, and 80 mol % NiPP and NMA aqueous mixtures), of which measurement temperature was increased up to ca. 70 °C to obtain their spectra in the liquid state by placing the sample cell in the dryer at 70 °C for a while just before the measurement. The aqueous solutions of PNiPA and PdEA were put on a CaF2 window, and the temporal change of the transmission IR spectra was measured on the drying process. The ATR-IR spectra of aqueous solutions of PNiPA and PdEA were measured with a horizontal ZnSe crystal with an incidence angle of 45°and 10 times reflection (Thermal A.R.K, Spectra-Tech, Inc.), of which measurement temperature was controlled by a circulating water bath (Lauda, RM6). Data processing was performed by OMNIC 6.0 (Thermo Nicolet Co.) and a program written by Y. Katsumoto.25 The wavenumbers of IR bands were determined from the zero crossing points of the first or third derivative of the IR spectra.
Figure 2. Chemical structure of molecules studied in this work.
interactions of proteins, peptides, and amide-containing polymers such as PNiPA and PdEA,26−30 whereas there are few reports about other N-alkylamides and N,N-dialkylamides. It is notable that the IR spectral change for other N-alkylamides and N,N-dialkylamides in aqueous solution with concentration is more distinctive than that of NMA in this report. Applied the band assignments of NiPP and dEP to those of PNiPA and PdEA, it is found that almost all amide groups of PNiPA forms the intramolecular CO···H−N hydrogen bond in aqueous solution and that the amide groups of PNiPA are less hydrated than those of PdEA in spite of the similar degree of hydration to alkyl groups.
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EXPERIMENTAL SECTION
Materials. Poly(N-isopropylacrylamide) (PNiPA) and poly(N,Ndiethylacrylamide) (PdEA) were prepared by free radical polymerization as reported by Idziak et al.31 N-isopropylacrylamide (NiPA; monomer) or N,N-diethylacrylamide (dEA; monomer) in methanol was stirred with azobisisobutylonitrile (initiator) at 60 °C for 30 min under N2. The solvent was evaporated in vacuo after dissolving polymer into a small amount of acetone and purified by precipitation in n-hexane. NiPA and dEA were obtained from Kohjin Chemical Co. and the other reagents were purchased from Wako Co. The weight average molecular weights of PNiPA and PdEA were 1.09 × 106 and 0.51 × 106 by the static light scattering method (ALV CGS-3, 632.8 nm), respectively. The refractive index increments, dn/dc, of PNiPA and PdEA in water at 25 °C were measured by the refractometer (Abbemat MW, 633.6 nm) as 0.1542 and 0.1627 mL/g, respectively. PNiPA and PdEA gels were synthesized as described in the previous article.17 The aqueous solution of NiPA or dEA (655 mM), N,Nmethylenebisacrylamide (cross-linker), ammonium persulfate (initiator), and N,N,N′,N′-tetramethylethylenediamine (accelerator) was undisturbed at 20 °C for 1 day and polymerized between two glass plates with a spacer; thickness was ca. 0.4 mm. The synthesized sheetlike gels were washed several times with a large amount of distilled water for about a week. The reagents except for NiPA and dEA were purchased from Wako Co. The cross-linkage density of the gel is given by 2Nc/(Nc + Nm), where Nc and Nm are molar concentrations of cross-linker and monomer, respectively, and was determined to be 8.8 × 10−3 for both PNiPA and PdEA gels. N-Isopropylpropionamide (NiPP, 99.9%) was synthesized via coupling of propyl chloride and isopropylamine in tetrahydrofuran, and it was then distilled. N-n-propylpropionamide (NnPP, 99.8%), Nisopropylacetamide (NiPA, 99.0%), and N-ethyl-N-n-propylpropionamide (EnPP, 99.4%) were synthesized in the same manner by utilizing the corresponding alkylchloride and alkylamine. All of the reagents used were purchased from Aldrich, Wako Co., or Tokyo Kasei Co. Nethylpropionamide (NEP, 99% Wako), N,N-diethylpropionamide (dEP, 99.0% Aldrich), N,N-dimethylpropionamide (dMP, 98% Wako), N,N-diethylacetamide (dEA, 99% Wako), and N,N-dimethylacetamide (dMA, 99% Wako) were dried by adding 4 Å molecular sieves without further purification. The purity of N-alkylamides and N,N-dialkylamides was analyzed by gas chromatography. PNiPA, PdEA, N-alkylamides and N,N-dialkylamides were dissolved in H2O (ultrapure water; 18.2 MΩ) or D2O at several arbitrary concentrations. NiPP and dEP were also dissolved in cyclohexane(-
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RESULTS AND DISCUSSION Phase Diagrams for Aqueous Mixtures of NiPP, PNiPA, PNiPA Gel, dEP, PdEA, and PdEA Gel. The phase diagrams for NiPP/H2O, PNiPA/H2O, dEP/H2O/NaCl, and PdEA/H2O/NaCl are shown in Figure 3. The critical point of NiPP/H2O is Tc = 34.8 °C and cc = 18 mol % (58 wt %) on account of the shape of the observed cloud point curve. PNiPA is not easy to solve into water above 3 mol % and the constant extrapolation of temperature along concentration is estimated as 30.5 °C. It seems that the critical concentration of low molecular weight PNiPA (Mw < 1 × 104) aqueous solution is obtained as cc = 40−43 wt %.32,33 Afroze et al. reported that the critical point of NiPP/H2O is Tc= 36 °C and cc= 50 wt % and that the critical temperature of PNiPA/H2O increases from 26.365 to 26.42 °C for the higher molecular weight of PNiPA from Mw = 1 × 104 to 1 × 105 at cc = 43 wt %.32 On the other hand, the critical point of PdEA/H2O is Tc= 30.0 °C and cc= 1 mol % (6.6 wt %). The mixture of dEP/H2O is transparent and homogeneous up to 90 °C, but exhibits a liquid−liquid phase 1043
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than PdEA/H2O in contrast to the case of NiPP/H2O and PNiPA/H2O. This may be linked to the LCST dependence on the molecular weight of PdEA and PNiPA as follows. The transition temperature of PdEA/H2O decreases from 37 to 28 °C for the higher molecular weight from Mw = 1 × 104 to 1.5 × 106 at cc = 1−5 wt %,6 whereas there is no significant dependence of transition temperature on the molecular weight for PNiPA/H2O except for recent researches about low molecular weight and low concentration regions in which the transition temperature of PNiPA/H2O slightly decreases for the higher molecular weight.5,35,36 This is also consistent with the case of poly(N-ethyl acrylamide) of which transition temperature decreases from 86 to 73 °C for the higher molecular weight from Mw = 5 × 103 to 34 × 103.37 The phase diagrams for NiPP/H2O, PNiPA/H2O, dEP/H2O/NaCl, and PdEA/ H2O/NaCl imply that the transition temperature of monomer unit is similar to that of its polymer, especially in the case of NiPP and PNiPA. The phase diagrams for PNiPA and PdEA gels are also shown in Figure 3. Even though the transition temperature of gel is similar to its corresponding polymer solution, the transition of PNiPA gel itself is sharper than PdEA gel. Figure 4
Figure 4. Comparison of water content as a function of temperature for PNiPA and PdEA gels.
shows that the water content change of ca. 80% occurs within 3 and 10 °C for PNiPA and PdEA gels, respectively. In addition, the polymer concentration in PdEA gel is higher than in PNiPA one above and below the transition temperature. These findings coincide with Panayiotou’s report in which PNiPA gel shows a sharper phase transition and a higher swelling ratio as compared to PdEA gel in spite of the similar cross-linker content.38 This implies that PdEA is more soluble into water than PNiPA. IR Spectra of NiPP and dEP in Water and Cyclohexane. The IR spectra of PNiPA, PdEA, NiPP, and dEP in the neat state in the 3450−2750 and 1750−1100 cm−1 regions are shown in Figure A (Supporting Information). The most prominent spectral change of NiPP and dEP in aqueous and cyclohexane solutions with varying concentration, which occurs in the amide I, N−H, and C−H stretching bands, is attributed to the solvation to the amide and alkyl groups as follows. The IR spectra of various concentrations of deuterated NiPP (DNiPP) and dEP in D2O in the amide I region and their second derivatives are shown in Figure 5. The amide I band is largely due to the CO stretching vibration of the amide group. D2O was used instead of H2O to precisely extract the characteristic
Figure 3. Phase diagrams for the aqueous mixtures of (a) NiPP, PNiPA, and PNiPA gel and (b) dEP, PdEA, and PdEA gel. The concentration of NaCl in H2O is 1 (triangle), 1.5 (rectangle), and 2 mol % (reverse triangle) for PdEA/H2O/NaCl and dEP/H2O/NaCl.
separation when NaCl is added. It has been known that the transition temperature of PNiPA and PdEA in aqueous solutions decreases by adding salt such as NaCl.34 Figure 3b shows that the transition temperature of dEP/H2O/NaCl rapidly decreases with increasing NaCl concentration as compared to the case of PdEA/H2O/NaCl, and the transition temperature of dEP/H2O is expected so high. It can be said that the transition temperature of dEP/H2O is quite higher 1044
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cm−1, and new bands emerge at 1626 and 1606−1593 cm−1 in D2O (Figure 5b). The wavenumbers of the amide I bands for D-NiPP and dEP in D2O are plotted against the concentration in Figure 6 along with the change in the peak position of the
Figure 6. Plots of the wavenumber of amide I bands of (a) D-NiPP and (b) dEP in D2O. × , ▲, and □ are the band positions determined by zero crossing points of 3rd derivative of the spectra in the amide I region. The band position at which absorbance is largest is plotted against the concentration as a solid line.
dominant band. Here, the two amide I bands of the aqueous solutions are called bands I and II in the order of descending wavenumbers, and the band of which wavenumber is higher than in neat liquid is named band I′ for D-NiPP. The peak position of the amide I band changes in the order from 1638 to 1618 (band I) to 1600 cm−1 (band II) with decreasing concentration for D-NiPP, and in the order from 1640 to 1626 (band I) to 1606 - 1593 cm−1 (band II) for dEP. The shift from the band of neat liquid to band I occurs at 50 - 60 mol % (85 91 wt %) for both D-NiPP and dEP, and the shift from band I to II occurs at 1 mol % (6 wt %) solution of D-NiPP and at 20−30 mol % (62 - 73 wt %) solution of dEP. To clarify the solute−solute interaction in solutions, in general, the IR spectral change of the solute in dilute aprotic solvents has been interpreted in terms of the isolation of solute molecules as a result of interruption of self-association through disruption of solute−solute interaction such as NMA in CCl4, C6H6, and CHCl3.26,28,39,40 Figure 7 shows the temperaturedependent IR spectra of NiPP and dEP in the neat liquid and the IR spectra of various concentrations of NiPP and dEP in cyclohexane-d12 in the amide I band region. The amide I band slightly shifts to a higher wavenumber for both NiPP and dEP with increasing temperature due to the amide−amide
Figure 5. IR spectra of various concentrations of (a) D-NiPP and (b) dEP in D2O in the amide I region and their 2nd derivatives.
amide I bands of NiPP and dEP in aqueous solutions. The amide I band of neat liquid D-NiPP is identified at 1638 cm−1, and new bands appear at 1649, 1618, and 1601 cm−1 when D2O is added (Figure 5a). Note that one of the amide I bands of D-NiPP in D2O (1649 cm−1) is observed at a higher wavenumber than in neat liquid D-NiPP (1638 cm−1). In the case of dEP, the amide I band of neat liquid is observed at 1645 1045
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Figure 8. IR spectra of various concentrations of NiPP in cyclohexane (-d12) in the N−H and C−H stretching bands region.
H stretching band at 3444 cm−1 arise from the isolated NiPP molecules that do not participate in the CO···H−N hydrogen bond formation. The CH3 asymmetric stretching band of (D-)NiPP at 2972 cm−1 changes marginally with decreasing concentration. The disruption of the CO···H−N hydrogen bonds is expected to lead to the higher wavenumber shift of the amide I and N−H stretching bands, but not the C− H stretching band. On the other hand, the amide I band of the neat liquid dEP is observed at 1645 cm−1 and gradually shifts to a higher wavenumber as the concentration of dEP decreases, then becomes constant at 1664 cm−1 below 2 mol % as shown in Figure 7b. The gradual higher wavenumber shift of the amide I band of dEP with increasing cyclohexane dilution is attributed to the isolation of dEP molecules in cyclohexane by disruption of self-association, which was also the case with NiPP. The change in peak position due to isolation occurs sharply at 0.1 mol % (0.1 wt %) dilution for NiPP, and the shift is gradual in the case of dEP and eventually becomes constant at 2 mol % (0.1 wt %) for dEP; this is because of the difference in the amide−amide intermolecular interactions of the two species. That is, the dipolar interaction of dEP is weaker than the C O···H−N hydrogen bond of NiPP. Intermolecular hydrogen bonds are expected to be formed in neat liquid NiPP, considering the following liquid structure of NMA.26,27 The hydrogen-bonded chains in neat liquid NMA are roughly linear, where a trans-NMA molecule donates and accepts one standard hydrogen bond such as CO···H−N. The intermolecular electron delocalization through the CO···H−N bond induces
Figure 7. IR spectra of (a) NiPP and (b) dEP in the neat liquid state with changing temperature and their 2nd derivatives (top panel) and their various concentrations of cyclohexane-d12 solutions (bottom panel) in the amide I region.
interaction weakened by heating. The amide I band of neat liquid NiPP is identified at 1643 cm−1, and a new band appears at 1694 cm−1 and becomes dominant with the cyclohexane-d12 dilution below 0.1 mol %. Figure 8 shows that the IR spectra of NiPP in cyclohexane (-d12) in the N−H and C−H stretching band regions by subtracting the spectrum of cyclohexane (-d12) from those of the mixture of NiPP/cyclohexane (-d12). The N− H stretching band is located at 3300 cm−1 together with a very small band at 3444 cm−1 for the neat liquid NiPP. The band at 3444 cm−1 becomes larger and dominant with the cyclohexane (-d12) dilution below 0.1 mol %. This indicates that the amide− amide interaction of NiPP in cyclohexane-d12 solution is mostly disrupted below 0.1 mol %. The transition concentration of 0.1 mol % is reasonable compared with the cases of other Nalkylamides such as NMA (
