Attenuated Total Reflectance/Fourier Transform Infrared Studies on the Phase-Separation Process of Aqueous Solutions of Poly(N-isopropylacrylamide)

Transcription

1 Attenuated Total Reflectance/Fourier Transform Infrared Studies on the Phase-Separation Process of Aqueous Solutions of Poly(N-isopropylacrylamide) ORY RAMON, 1 ELLINA KESSELMAN, 1 RONEN BERKOVICI, 2 YACHIN COHEN, 2 YARON PAZ 2 1 Department of Food Engineering and Biotechnology, Technion, Haifa Israel 2 Department of Chemical Engineering, Technion, Haifa Israel Received 6February 2001; revised 9April 2001; accepted 27 April 2001 Published online 00 Month 2001 ABSTRACT: Temperature-induced phase separation of poly(n-isopropylacrylamide) in aqueous solutions was studied by attenuated total reflectance (ATR)/Fourier transform infrared spectroscopy. The main objectives of the study were to understand, on a molecular level, the role of hydrogen bonding and hydrophobic effects below and above the phase-separation temperature and to derive the scenario leading to this process. Understanding the behavior of this particular system could be quite relevant to many biological phenomena, such as protein denaturation. The temperature-induced phase transitionwaseasilydetectedbytheatrmethod.asharpincreaseinthepeaksofboth hydrophobic and hydrophilic groups of the polymer and adecrease in the water-related signalscouldbeexplainedintermsoftheformationofapolymer-enrichedfilmnearthe ATR crystal. Deconvolution of the amide Iand amide II peaks and the OOH stretch envelope of water revealed that the phase-separation scenario could be divided, below the phase-separation temperature, into two steps. The first step consisted of the breaking of intermolecular hydrogen bonds between the amide groups of the polymer and the solvent and the formation of free amide groups, and the second step consisted of an increase in intramolecular hydrogen bonding, which induced a coil globule transition. No changes in the hydrophobic signals below the separation temperature could be observed, suggesting that hydrophobic interactions played adominant role during the aggregation of the collapsed chains but not before John Wiley &Sons, Inc. JPolym Sci Part B: Polym Phys 39: , 2001 Keywords: Fourier transform infrared (FTIR); hydrogen bonding; poly(n-isopropylacrylamide); phase separation INTRODUCTION Correspondence to: Y. Paz ( paz@tx.technion.ac.il) Journal of Polymer Science: Part B: Polymer Physics, Vol. 39, (2001) 2001 John Wiley &Sons, Inc. The solution properties of many nonionic watersoluble polymers are temperature-dependent and exhibit phase separation of the lower critical solution temperature (LCST) type. During the process,themacromoleculesexhibitdramaticconfor- mationalchangessuchascoil globule,coil helix, and association to supramolecular assembly. 1 In acoil globuletransition(cgt),anexpandedflexible polymer chain turns into acollapsed globule 2 that precipitates on aggregation. The CGT phenomenon has been studied extensively both theoretically and experimentally. 3 Several studies concentrated on polystyrene in various organic solvents, 4 determining the molecular parameters related to the initial and final states, without clarifying in detail the crucial role of intermolec- 1665

2 1666 RAMON ET AL. ular and intramolecular interactions in polymer solvent systems. An investigation of CGT in water-soluble synthetic polymers can be very useful in elucidating the mechanisms governing denaturation (folding) of large proteins. 5 A common model polymer for such studies is poly(n-isopropylacrylamide) (PNIPA), a polyvinyl polymer, containing hydrophilic amide groups and hydrophobic isopropyl groups. 6 Because of its side chains, the polymer is soluble in polar solvents (e.g., water) and lowpolarity solvents such as tetrahydrofuran. 5 The crosslinked PNIPA gel in water undergoes a reversible volume-phase transition due to changes in temperature and solvent composition. 7 These thermoreversible phase-separated systems were studied for their potential use as intelligent materials in controlled drug delivery, enzyme immobilization, and separation processes. 8 It has been proposed 9 that phase separation in PNIPA solutions is a two-step mechanism, initiated by the collapse of single chains and advanced by aggregation. According to this explanation, PNIPA chains in aqueous solutions form expanded coils at room temperature that undergo a CGT when the temperature is increased to C (ca. the temperature). The relative roles of hydrogen bonding 10 and hydrophobic interactions 11 in promoting the phase transition are a matter of some debate. Although the gross scenario of PNIPA solution phase separation is well understood, the detailed scenario of the process is still under debate. More specifically, the role of water PNIPA hydrogen bonding versus the role of intramolecular hydrogen bonding is not yet fully understood. Many methods, including cloud-point determination, 12 light scattering, 13 thermal analysis, differential scanning calorimetry (DSC) and microcalorimetry, 14 rheology, 15 fluorescence, 16 and NMR, 17 have been used to study the phase-separation phenomena. However, these methods lack the ability to provide detailed information on a molecular level. Pioneering studies of aqueous polymer solutions by direct absorption IR spectroscopy 18,19 suggest that the water structure around a solute is different than that in bulk water. However, such measurements are hampered at the fundamental transitions by the exceptionally high absorbency of water. Implementation of the attenuated total reflectance (ATR) setup has given a way to overcome this obstacle. Quantitative analyses of changes in these spectra with the temperature or Hydrogen/Deuterium (H/D) isotopic ratio have led to a novel description of the structure and dynamic of water at a molecular level. 20 The same technique was found to be beneficial for extracting information on polymers, proteins, and their interactions with water. 21 In two pioneering works, ATR/Fourier transform infrared (FTIR) was used in studies related to the temperature-dependent phase separation of PNIPA gels 22 or PNIPA water solutions. 23 In the latter study, relevant information at a molecular level was derived on the role of intermolecular and intramolecular hydrogen bonding and the enhancement of the hydrophobic interactions in this system. In this study, the ATR technique was used to investigate the phase separation of well-defined aqueous PNIPA solutions. The main objective was to clarify the mechanisms leading to the phase-separation phenomenon by interpretation, on a molecular level, of the role of hydrogen bonding and hydrophobic effects below and above the phase-separation point and by elucidation of the role of water in the phase-transition process. EXPERIMENTAL Preparation of PNIPA PNIPA samples were prepared with a redox polymerization in an aqueous solution at 4 C with a protocol similar to that used by Otake et al. 24 The reagents were N-isopropylacrylamide (NIPA) monomer (Aldrich Chemical Co., United States), ammonium persulfate (APS; Merck, Darmstadt, Germany), and N,N,N,N -tetramethylethylenediamine (TEMED; Aldrich Chemical). The NIPA monomer was recrystallized from a 1:1 toluene/petrol-ether mixture as described elsewhere. 25 The polymerization was conducted according to the following procedure: ml of NIPA monomer was dissolved in doubly distilled water, and 2 ml of 4% APS was dissolved separately. Then, the two solutions were mixed and cooled to 4 C, and nitrogen was bubbled over the solution to remove oxygen. At that stage, 0.48 ml of TEMED was added, and polymerization continued for 3 h under a nitrogen atmosphere. The polymer solution was then dialyzed for 6 days at 4 C [the molecular cutoff was a weight-average molecular weight (M w ) of 14,000] to remove nonreacted monomers, initiator, and catalyst, and then the unfractionated polymer was freeze-dried (Martin Christ, Alpha 1, Germany). The molecu-

3 ATR/FTIR STUDIES OF PNIPA 1667 Figure 1. DSC peak of an aqueous solution of PNIPA (2 wt %). lar weight (7 1 MDa) was evaluated from the determination of the intrinsic viscosity of the PNIPA aqueous solutions with an Ubbelohde capillary viscometer (at 20 C) and the expression [ ] M w 0.51 (cm 3 /g), developed by Kubota et al. 5,7 Determination of the Phase-Transition Temperature The phase-transition temperature was determined by two thermal analysis techniques. The first, DSC, was done with a PerkinElmer DSC-7 apparatus at a scanning rate of 5 C/min. Figure 1 presents the thermogram of a 2% (w/w) aqueous solution of PNIPA. The figure shows an endothermic peak (0.81 J/g) at 36.2 C, with an onset at 35.1 C. The second technique, microcalorimetry, was performed with a VP-DSC microcalorimeter (Micro-Cal Inc., Northampton, MS). Solutions of 0.5 wt % PNIPA in water were degassed and transferred to a sample cell (0.5 ml) with a calibrated syringe. The thermograms were obtained at heating rates of 1.5 C/min. Polymer-free solutions (water) were used in the reference cell. ATR Spectroscopy Studies ATR/FTIR measurements of PNIPA aqueous solutions (1 3.5 wt %) were performed with a Bruker IFS55 machine equipped with a temperature-controlled circle cell accessory [circle cell attenuated total reflection (CCATR)], as shown in Figure 2. The accessory was made of a ZnSe cylindrical crystal (75 mm, 6-mm diameter, 45 edges), fixed by a leak-proof O-ring at the center of a temperature-controlled stainless steel vessel (60 mm long, 15-mm inner diameter) containing the PNIPA solution. To increase the signal/noise ratio, the IR beam was first decollimated and then redirected onto the crystal edges in a circular manner that enabled the probing of the entire circumference of the embedded crystal, with a reflecting angle of 45. The IR beam that impinged on the edges of the crystal, outside of the vessel, propagated through the crystal and exited through the other end, where it was redirected by another set of mirrors toward the IR detector. The short distance from which the signal was collected by this method (a few micrometers) minimized problems related to the high absorption coefficient of water. The temperature of the sample solution was monitored by a thermocouple inserted into the vessel. The ATR/FTIR spectra were taken in the range of C with a resolution of 1 C. The circle cell was equipped with a transparent poly(methyl methacrylate) cover, allowing visual observation of the temperatureinduced transition from a clear solution to a turbid solution.

4 1668 RAMON ET AL. Figure 2. CCATR/FTIR setup. All measurements were taken with the empty circle cell as a background. Because the IR spectrum of water is known to change as a function of temperature, mainly because of the breaking of hydrogen bonds, 26 care was taken to record a set of temperature-dependent spectra of water (20 48 C), later to be subtracted from the temperature-dependent spectra of the aqueous polymer solutions. band peaking at 2125 cm 1, a peak that is known to be a good indication for water quantity. 27 Another reason for using this peak as a subtraction RESULTS Phase-Separation Effect as Probed by the PNIPA IR Peaks Figure 3 presents the ATR raw spectra of a 2.5% aqueous solution of PNIPA at 22, 34, 35, 36, 37, and 45 C. As mentioned in the Experimental section, the background here was an empty cell, so the spectra are dominated by the water signals and, more specifically, the OOH stretch envelope of the solvent at cm 1, the HOH bending mode at 1635 cm 1, and the wide combination band peaking at 2125 cm 1. At temperatures greater than 35 C (the phase-separation temperature as measured by DSC; Fig. 1), a series of peaks seem to emerge in the spectra. These peaks could easily be identified as those of the polymer. To obtain better insight into the temperatureinduced changes in the spectra, we subtracted the spectra of water at the corresponding temperatures from that of the raw data. To account for the effects of dilution, changes in penetration depth, and interaction between the solvent and the polymer, we fitted a multiplication constant to the water spectra. Fitting was based on obtaining a perfect elimination of the water wide combination Figure 3. Spectra of a 2.5% PNIPA solution at six different temperatures. The polymer signals can hardly be resolved because of the dominance of the waterrelated peaks. Note the difference between the 35 C spectrum and the 36 C spectrum.

5 ATR/FTIR STUDIES OF PNIPA to 2878 cm 1, the CH 2 (a) peak shifted from 2939 cm 1 to 2936, and the CH 3 (a) peak shifted from 2982 to 2975 cm 1. Line-shape fitting of these peaks revealed a gradual decrease in their width below the phase-transition point, followed by an increase in their width at the phase-separation temperature. Above the phase-separation point and after oversubtraction of water, another three weak peaks could be resolved. These were the amide II overtone at 3070 cm 1, the hydrogen-bonded NOH stretch at 3310 cm 1 and another peak at 3550 cm 1 (free NOH stretch?) with intensities of 5, 10, and 7% of that of the amide II, respectively. The qualitative description of Figure 4 is given in a more quantitative manner in Figure 5. Here, the absorbance, integrated over the envelope of each peak, is presented as a function of temperature. According to the figure, the temperature dependency of the integrated absorbance of all peaks could be divided into four regions: 1. Below 31 C, a region of constant intensity. Figure 4. Subtraction spectra of a 2.5% PNIPA solution at six different temperatures obtained by the subtraction of the water contribution from the crude spectra presented in Figure 3. guide was the fact that this spectral range is clear of polymer-related peaks, unlike, for example, the HOH bending peak that partially overlaps the amide I peak of PNIPA. For a 2.5% solution, the multiplication factor below the phase-transition temperature ranged between 0.98 and 0.99, whereas above the phase-separation point, the value of this factor was no more than The subtraction spectra are presented in Figure 4. The phase transition is clearly indicated by the intensity enhancement in the amide I (mostly CAO stretch) band and amide II (mostly NOH in-plane deformation) band at 1628 and 1556 cm 1, respectively, 28 and in the coupled band of COH and CH 3 at 1460 cm 1. In parallel, an increase in the signal of the isopropyl group 22 at cm 1 was observed, together with that of the CH 2 antisymmetric mode and the CH 3 symmetric and antisymmetric stretching modes. Here, a sharp change in the location of the peaks toward lower frequencies was observed with the phase transition: the CH 3 (s) peak shifted from Figure 5. Changes in the integrated intensity of the PNIPA peaks as a function of temperature: ( ) amide I (1628 cm 1 ), (Œ) amide II (1556 cm 1 ), (F) CH 3 and CH 2 stretch envelope ( cm 1 ), ( ) isopropyl I ( cm 1 ), and (E) isopropyl II ( cm 1 ).

6 1670 RAMON ET AL. Figure 6. Deconvoluted spectra of the amide I and amide II peaks at (A) 25 and (B) 40 C, showing the intermolecular, intramolecular, and free components of each band. 2. At C, a pretransition region in which the intensity of some of the peaks is reduced slightly, whereas the intensity of others (such as the amide I and II peaks) is enhanced slightly. 3. At C, a phase-transition region, indicated by a sharp increase in the absorbance of all the PNIPA-related peaks. 4. Above 37 C, a posttransition region, manifested by a very gradual increase of all peaks. It is well known that the solubility of PNIPA in water is due to the hydrophilic amide groups that possess the capability of hydrogen bonding either with the surrounding water molecules or with other amide groups. It is further known that, for polyamides, the band contour of the amide I and amide II peaks are conformationally sensitive. 21(a) In agreement with the treatment of Lin et al., 23 the amide I peak (mostly CAO stretch) in the subtraction spectra was deconvoluted into three subbands: an intramolecular hydrogen-bonded subband at 1630 cm 1, an intermolecular hydrogen-bonded subband at 1620 cm 1, and a free form of non-hydrogen-bonded subband at 1643 cm 1. Likewise, the amide II band (mostly NOH deformation) was deconvoluted into an intramolecular hydrogen-bonded subband at 1551 cm 1, an intermolecular hydrogen-bonded subband at 1565 cm 1, and a free form of non-hydrogenbonded subband at 1535 cm 1. These wave numbers are in line with the notion that hydrogen bonding tends to make steeper the potential surface of deformational modes while flattening the potential surface of stretching modes. 29 In the deconvolution, peak positions and peak shapes (20% Lorentzian and 80% Gaussian) were taken as constant parameters, whereas the intensity and width of the amide I and II subbands were regarded as free variables. Figure 6 presents the deconvoluted IR spectra of amide I and amide II peaks of a 2.5% PNIPA solution at 25 and 40 C. A difference in the peak shapes is clearly observed. A detailed, quantitative examination of the temperature-dependent relative contribution of each of the amide II components is presented in Figure 7. The figure reveals that changes in the relative intensity of the subbands exist at temperatures lower than the phase-transition temperature and may provide a hint about the collapse scenario. Four different regions can be observed: 1. Below 31 C, increasing temperature causes a moderate decrease in the intermolecular hydrogen bonds and a moderate increase in the free amide population. The intramolecular hydrogen-bond population hardly changes. 2. At C, there is an abrupt decrease in the intermolecular hydrogen-bond popula-

7 ATR/FTIR STUDIES OF PNIPA 1671 Figure 7. Changes in the three subbands of the amide II peak as a function of temperature for a 2.5% PNIPA solution: (Œ) intermolecular hydrogen-bonding subband, (F) free amide groups, and ( ) intramolecular hydrogen-bonding subband). tion and an abrupt increase in the intramolecular hydrogen bond and the free amide populations. This region is characterized also by an abrupt increase in the width of the free amide subband. 3. At C, there is an increase in the relative amount of the intermolecular hydrogen-bond population and a decrease in the integrated absorbance and width of the free amide subband. 4. Above 40 C, there is a moderate increase in the relative population of the free amide, coupled with a moderate decrease in the intramolecular hydrogen-bond and intermolecular hydrogen-bond populations. The same trends were observed in an analysis of the temperature-dependence graphs of the integrated intensity of the amide I subbands, although amide I graphs were found to be less smooth than the amide II graphs, probably the result of some remaining overlap with the HOH bending. The same four regions were also found in aqueous solutions containing PNIPA at other concentrations. Although the relative integrated absorbance should not be taken as representing the relative quantities of the different populations (because of a possible difference in the transition moment of the three populations), it is still possible to obtain some quantitative information. For example, the fact that the relative integral of the intramolecular hydrogen-bonding subband increased four times from about 7% to about 28% implies that at low temperatures the percentage of amide groups engaged in intramolecular hydrogen bonding does not exceed 25% of the total number of amide groups. Phase-Separation Effect as Manifested by the Water-Related IR Peaks In the mid-ir range, water has three bands: an HOH bending mode at 1640 cm 1, a wide combination band ( cm 1 ) peaking at 2125 cm 1, and a strong OOH stretch band at cm 1. The IR spectrum of water is known to change with increasing temperature because of the reduced extent of hydrogen bonding. The main changes are the shifting of the OOH envelope toward higher frequencies and the narrowing of the HOH bending peak, accompanied by a small change in this peak toward lower frequencies. It was already mentioned that the cm 1 peak was used for internal calibration to get an accurate subtraction of the water spectra from that of the PNIPA solution. On the basis of this analysis, an apparent sharp decrease in the water signal was observed with the phase transition. This complementary section discusses changes in the temperature-induced behavior of water due to interaction with solvated PNIPA.

8 1672 RAMON ET AL. Figure 8. Integrated intensity of water-related peaks in (E, ) pure water and (F, Œ) a 2.5% PNIPA solution: (A) HOH bending mode (1635 cm 1 ) and (B) OOH stretch envelope ( cm 1 ). The integrated absorbance of the HOH bending mode (1640 cm 1 ) of water and water containing dissolved PNIPA is presented in Figure 8(A) as a function of temperature. We calculated the area of the HOH peak of the PNIPA solution by subtracting the area of the amide signals from the overall area of the peak. As shown in the figure, the integrated intensity of pure water increased slightly with temperature. In contrast, the integrated intensity of this peak in the PNIPA solution exhibited a maximum at the phase-transition temperature and decreased at temperatures higher than the LCST. As shown in Figure 8(B), the area of the coupled intramolecular and intermolecular OOH stretch band ( cm 1 ) in pure water decreased as a function of temperature because of the breaking of hydrogen bonds. Such monotonic behavior was observed also in the PNIPA solution, although in the latter case, a sharp decrease at the phase-transition temperature was observed. Furthermore, a change in the slope of the graph, to a more pronounced decrease, was observed at temperatures above the transition point. In accordance with the procedure of Sammon et al., 30 the OOH stretch band of pure water and water containing PNIPA was deconvoluted into four subbands, reflecting four distinct types of water environments, that differed by the degree of hydrogen bonding (Fig. 9). Although this classification is somehow artificial, it does follow experimental evidence showing that strong hydrogen bonding skews the cm 1 envelope toward lower wavenumbers, whereas a lesser extent of hydrogen bonding skews that envelope toward higher wavenumbers. In the deconvolution, the subbands were (a) 3223 cm 1, strong hydrogen bonding; (b) 3395 cm 1, mediumstrength hydrogen bond; (c) 3525 cm 1, weak hydrogen bond; and (d) 3612 cm 1, free water. In a fashion similar to the procedure taken during the deconvolution of the amide I and amide II peaks, the peak positions and peak shapes (20% Lorentzian and 80% Gaussian) were taken as constant parameters, whereas the intensity and width of the subbands were regarded as free variables. To verify that the NH stretch bands of the PNIPA and its amide II overtone did not alter the analysis, we took care to include these peaks as well, but their contribution was found to be negligible, compared with the water signal. The relative areas of the OH stretch subbands of water and the relative areas of the OH stretch subbands of water containing 2.5% PNIPA are presented, as a function of temperature, in Figure 10. As shown in the figure, at a temperature of 25 C, the area of subband a was close to 55% of the total area of the peak, whereas the areas of subbands b d were 33, 10, and 2%, respectively. Water in the PNIPA solution was found to differ from pure water by a lower extent of strong hydrogen bonds and a higher extent of medium-strength hydrogen bonds. In contrast, hardly any difference could be observed in the extent of weak hydrogen bonds or free water. The lower amount of strong hydrogen-bonding subbands found for the PNIPA solution correlates well with the claim that PNIPA behaves like a structure breaker, because the water must reorient around the nonpo-

9 ATR/FTIR STUDIES OF PNIPA 1673 Figure 9. Deconvoluted spectrum of the OH stretch envelope of water, showing the four types of populations. lar regions of the solute. 31 At low temperatures, the differences between pure water and PNIPA solutions can hardly be observed by an inspection of the crude data (Fig. 3) because of the small concentration of solute. However, these differences are easy to observe if one inspects the subtraction spectra (Fig. 4), where a negative peak is observed at the locus of the strong hydrogenbonding subband. As the temperature of the liquid is increased, a decrease in the 3223-cm 1 subband of pure water could be observed, together with an increase in the 3395-cm 1 subband. Slight changes were found also in the other two subbands (the cm 1 subband decreased, and the 3612-cm 1 subband increased); however, these changes were smaller than the deconvolution error. A similar behavior was found also for water containing PNIPA. At the phase-transition point, however, an abrupt decrease in the relative amounts of the strong hydrogen-bonding subband and weak hydrogen-bonding subband coupled with an abrupt increase in the relative amount of mediumstrength hydrogen-bonding subband was observed. At higher temperatures, a subtle increase in the slope of the temperature-dependent relative areas could be observed. Effect of PNIPA Concentration on Phase-Transition Phenomena as Measured by ATR As already mentioned, the measurements were performed at various concentrations of PNIPA (1 3.5%). The phase-transition temperature was found to be the same, within an experimental error of 1 C, for all solutions. Qualitatively, the spectral changes were similar, regardless of polymer concentration. These changes included an increase in the PNIPA IR signals, a decrease in water-related signals, and a similar behavior for deconvoluted amide peaks. An interesting point was the increase of the PNIPA peaks with the phase transition. Below the transition temperature, the integrated absorbance of the PNIPA peaks increased, more or less, linearly with the concentration, as could be expected. In contrast, the integrated absorbance at higher temperatures was almost constant, regardless of the PNIPA concentration. This constant absorbance as a function of concentration occurred for all the PNIPA-related peaks. DISCUSSION Reduction in Water Concentration near the ATR Crystal As described in the previous sections, the phase transition was characterized by a sharp increase in the ATR signal of all the PNIPA-related peaks (Fig. 4). This increase at the phase-transition temperature was coupled with a decrease in all the water-related peaks: the HOH bending at 1640 cm 1 [Fig. 8(A)], the OH stretch envelope at cm 1 [Fig. 8(B)], and the wide combi-

10 1674 RAMON ET AL. nation band at 2125 cm 1 (see the description on the subtraction procedure). The aggregation of molecules is known to have strong effect on band intensity. However, in this case it is expected that each band will respond to aggregation in a different manner, according to the change in its induced dipole moment. In particular, it could be expected that the extent of the increase would differ significantly between NH-related peaks and COH-related peaks, or between stretch modes and deformation modes. The fact that the area of all PNIPA peaks, including those related to the hydrophobic groups, were found to increase at the transition approximately by the same factor (for a given concentration) suggests that the observed increase in the signal was mostly due to an increase in the average concentration of the polymer close to the crystal surface. This suggestion is supported by the decrease in all the waterrelated signals at the phase-transition temperature. Furthermore, the fact that the area of the PNIPA peaks was, above the transition temperature, independent of polymer concentration in the solution may suggest that a film with a constant PNIPA concentration is formed with the phase transition. If indeed this is the case, the average PNIPA concentration near the crystal (at a distance of a few micrometers) should be 7 10%, based on calibration according to the IR signals of the PNIPA bands as measured below LCST. This value is further supported by the water peak data. Figure 10. Relative area of the four subbands in the OH stretch envelope for pure water (empty symbols) and a 2.5% PNIPA solution (filled symbols): (, ) strong hydrogen-bonding subband (3223 cm 1 ), (,Œ) medium-strength hydrogen-bonding subband (3395 cm 1 ), (, ) weak hydrogen-bonding subband (3525 cm 1 ), and (E,F) free-water subband (3612 cm 1 ). Phase-Transition Scenario The temperature-induced changes in the relative areas of the three subbands in the amide II peak (Fig. 7) imply a four-stage process of phase separation in the PNIPA solution, which will be termed the early (A), intermediate (B), aggregation (transition temperature), relaxation (C), and postrelaxation (D) stages. The early stage, below 31 C, is characterized by the domination of the water PNIPA intermolecular hydrogen bonding, which contributes to approximately 60% of the overall amide II signal measured at this stage. As the temperature is raised, the relative contribution of this subband is gradually reduced, whereas that of the subband corresponding to non-hydrogen-bonded amides (free amides) is increased. Because of the increased number of free amides, the movement of the side chains becomes less restricted, a factor that may have an influence on the flexibility of the backbone of the polymer. The required increase in entropy during this stage could result from a decrease in the number of ordered water molecules due to the breaking of the thin shell of ordered molecules around the polymer. However, it is possible that during this pretransition stage, the required entropy comes from structural changes in the backbone of this relatively flexible 32 polymer, as water, considered to be a good solvent at low temperatures, becomes a solvent at 32 C. Indeed, both the radius of gyration and the hydrodynamic radius are known to decrease moderately at this stage, 33 approaching maximal entropy at the temperature.

11 ATR/FTIR STUDIES OF PNIPA 1675 The intermediate stage (31 35 C) is characterized by a sharp decrease in the relative intensity of the intermolecular hydrogen-bonding subband. This decrease is coupled with a sharp increase in the amide amide hydrogen-bond population in the lower temperatures of this stage and an increase in the population of the free amide groups at the higher temperatures of this stage. The phase separation, as indicated by DSC measurements and visual inspection, occurred quite abruptly at the end of this stage, that is, at 35 C. The behavior below the phase-separation temperature suggests that the phase-separation process is triggered by the formation of free amide groups. Once a critical number of side chains are free to change their conformation, they begin to form amide amide hydrogen bonds. It is likely that at this stage the amide amide bonds are intramolecular because, otherwise, one could have expected this scenario to be concentrationdependent. The structural changes involved in the formation of the intramolecular hydrogen bonds trigger a very fast decrease in the intermolecular water amide bonds, thus further increasing the amount of free amides. At this point, the polymer side chains are expected to be very flexible, thereby able to form hydrophobic interactions between their isopropyl groups and/or their backbone. Because in PNIPA the hydrophobic isopropyl groups are located adjacent to the amide groups, one could assume that the formation of the intramolecular hydrogen bonding at 33 C should be coupled with the formation of hydrophobic interactions, however, no change in the intensity or vibration frequency of methyl or methylene groups could be observed at temperatures below the transition temperature. Phase separation (i.e., aggregation of the collapsed chains) occurred at a point where the amount of free amide groups was at its maximum. This point was characterized also by a shift in the vibrational band of the hydrophobic groups, probably reflecting the formation of intermolecular hydrophobic interactions. As can be deduced from analyzing the water-related peaks, this aggregation stage is characterized also by the repulsion of water from the vicinity of the chains and the formation of polymer-rich regions on the surface of the ZnSe crystal. This scenario is in line with claims made by other groups, according to which the PNIPA water system is thermodynamically stable at low temperatures, and it becomes unstable at the onset of the phase-separation stage. 34 It has been suggested that at this stage the collapsed globules contain less water 22,34 and expose their isopropyl groups to the solvent, which becomes less polar in the vicinity of the polymer, as demonstrated by Winnik. 35 This hydrophobically driven aggregation mechanism does not contradict the mechanism suggested by us but rather is complementary to the one presented here. Above the transition temperature and up to 40 C, a decrease in the amount of free amide groups, coupled with an increase in the intermolecular component (i.e., water amide hydrogen bonding), could be observed. It is believed that at this relaxation stage hydrogen bonds are formed between water molecules that were trapped inside the polymer globules and amide groups that were free. Above the transition temperature, the relative amount of water molecules belonging to the weak hydrogen-bonding population is decreased, whereas that of the medium-strength hydrogenbonding population is increased (Fig. 10). It is not clear whether this difference is due to water trapped within the globules or water molecules at the interface between bulk water and polymer aggregates. A true equilibrium is achieved only above 40 C, as demonstrated in Figure 7. Hence, it can be said that although phase separation in PNIPA is well defined and abrupt, the scenario of this process can be quite complex and occurs over a wide range of temperatures. It is interesting to compare these findings with those of other groups who used different techniques. On the basis of an analysis of the temperature dependence of the ratio between the radius of gyration and the hydrodynamic radius, as obtained from light scattering experiments, it has been claimed that, before aggregation, the chains go through a four-stage scenario where the chains contract, crumple, rearrange, and collapse. 33,34 CONCLUSIONS This article has demonstrated that the ATR/FTIR technique can provide detailed information on the process of temperature-induced phase separation in aqueous solutions of hydrophobic polymers, such as PNIPA. Based on the ATR/FTIR results, the following scenario for the temperatureinduced collapse of PNIPA is proposed. At low temperatures, the polymer is solvated in such a manner that the hydrophilic groups form relatively strong hydrogen bonds with water. As the temperature is raised, more and more hydrogen bonds between amide groups and water are

12 1676 RAMON ET AL. broken. On average, the free amides do not form new intramolecular hydrogen bonds as long as their number is low. Once a critical number of bonds are broken, intramolecular hydrogen bonds are formed, at the expense of the free amide group population and the intermolecular hydrogen-bond population. As a result, the polymer begins to transform itself to a more compact conformation. This promotes the breaking of more intermolecular hydrogen bonds, which eventually causes the CGT of the chain. The exposed hydrophobic moieties induce aggregation due to hydrophobic interactions. The collapsed, aggregated polymer tends to adhere to the ATR crystal, so an increase in the intensity of all polymer-related peaks is observed, together with a decrease in the intensity of all water-related peaks. On the basis of the decrease in the free amide signal and the increase in the signal of the intermolecular hydrogen bonds above the phase-separation temperature, it is believed that, with phase separation, water molecules that were entrapped within the collapsed areas form hydrogen bonds with free amide moieties. The detailed role of water in the phase-transition process of PNIPA solutions has been revealed (to our knowledge) for the first time on a molecular level by the CCATR/FTIR technique. At present, this technique is used to study the effect of low molecular weight solutes (salts) on that process. A forthcoming article on that subject is now under preparation. This work was performed under contract number of the Israel Science Foundation. The authors thank Professor Rolfe Herber (Hebrew University) for lending the circle cell and for a critical reading of the manuscript. REFERENCES AND NOTES 1. Piculell, L.; Nilsson, S. Prog Colloid Polym Sci 1990, 82, Stockmayer, W. H. Makromol Chem 1960, 35, (a) Grosberg, A. Y.; Kuznetsov, D. V. Macromolecules 1993, 26, ; (b) Yamakawa, H. Macromolecules 1993, 26, ; (c) Park, I. H.; Wang, Q.; Chu, B. Macromolecules 1987, 20, ; (d) Chu, B.; Park, I. H.; Wang, Q. W.; Wu, C. Macromolecules 1987, 20, Tanaka, T. Polymer 1979, 20, Kubota, Ki.; Fujishige, S.; Ando, I. Polym J 1990, 22, For a comprehensive review on PNIPA, see Schild, H. G. Prog Polym Sci 1992, 17, Shibayama, M.; Tanaka, T. J Adv Polym Sci 1993, 109, (a) Cole, C.-A; Schreiner, S. M.; Priests, J. H.; Munji, N.; Hoffman, A. S. Am Chem Soc Symp Ser 1987, 350, ; (b) Priest, J. H.; Murrey, S. L.; Nelson, R. J.; Hoffman, A. S. Am Chem Soc Symp Ser 1987, 350, ; (c) Matsukata, M.; Aoki, T.; Sanui, K.; Ogata, N.; Kikuchi, A.; Sakurai, Y.; Okano, T. Bioconjugate Chemistry 1996, 7, Yamamoto, I.; Iwasaki, K.; Hirotsu, S. J Phys Soc Jpn 1989, 58, (a) Walker, J. A.; Vause, C. A. Sci Am 1987, 253, 98; (b) Hirotsu, S. J Phys Soc Jpn 1987, 56, (a) Tanford, C. The Hydrophobic Effect, 2nd ed.; Wiley: New York, 1973; Chapters 1 3; (b) Ulbrich, K.; Kopecek, J. J Polym Sci Polym Symp 1979, 66, 209; (c) Ben-Naim, A. Hydrophobic Interactions; Plenum: New York, (a) Heskins, M.; Guillet, J. E. J Macromol Sci Chem 1968, 2, ; (b) Schild, H. G.; Tirell, D. A. J Phys Chem 1990, 94, (a) Ricka, J.; Meenes, M.; Nyffenegger, R.; Binkert, T. Phys Rev Lett 1990, 65, ; (b) Meewes, M.; Ricka, J.; Le Silva, M.; Nyffeneger, R.; Binkert, T. Macromolecules 1991, 24, (a) Inomata, H.; Goto, G.; Saito, S. Macromolecules 1990, 23, ; (b) Tiktopulo, E. I.; Bychkova, V. E.; Ricka, J.; Ptitsyn, O. B. Macromolecules 1994, 27, ; (c) Boutris, C.; Chatzi, E. G.; Kiparissides, C. Polymer 1997, 38, Fujishige, S.; Kubota, K.; Ando, I. J Phys Chem 1989, 93, (a) Winnik, F. M. Macromolecules 1989, 22, ; (b) Schild, H. G.; Tirrell, D. A. Langmuir 1991, 7, ; (c) Schild, H. G.; Tirrell, D. A. Macromolecules 1992, 25, ; (d) Armentrout, R. S.; Hu, Y.; McCormick, C. L. Polymer Preprints 1996, 37, (a) Tokuhiro, T.; Amiya, T.; Mamada, A.; Tamaka, T. Macromolecules 1991, 24, ; (b) Zeng, F. Z.; Feng, H. Polymer 1997, 38, Scarpa, J. J.; Mueller, D. D.; Klotz, I. M. J Am Chem Soc 1967, 89, Snyder, W. D.; Klotz, I. M. J Am Chem Soc 1975, 89, (a) Marechal, Y. J Mol Struct 1994, 322, ; (b) Rossi, A. V.; Dawanzo, C. V.; Tubino, M. J Braz Chem Soc 1996, 7, (a) Skrovanek, D. J.; Howe, S. E.; Painter, P. C.; Coleman, M. M. Macromolecules 1986, 19, ; (b) Skrovanek, D. J.; Painter, P. C.; Coleman, M. M. Macromolecules 1986, 19, ; (c) Durrani, C. M.; Prystupa, D. A.; Donald, A. M. Macromolecules 1993, 26, ; (d) Harris, P. I.; Chapman, D. Trends Int Biol Sci 1992, 17,

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