12964
J. Phys. Chem. B 2007, 111, 12964-12968
LCST and UCST Behavior of Poly(N-isopropylacrylamide) in DMSO/Water Mixed Solvents Studied by IR and Micro-Raman Spectroscopy Hideo Yamauchi and Yasushi Maeda* Department of Applied Chemistry and Biotechnology, UniVersity of Fukui, Fukui 910-8507, Japan ReceiVed: March 28, 2007; In Final Form: August 30, 2007
Temperature-responsive phase separations of poly(N-isopropylacrylamide) (PNiPAm)/dimethylsulfoxide (DMSO)/water mixtures have been investigated by infrared and confocal micro-Raman spectroscopy. The ternary mixtures exhibited lower critical solution temperature (LCST) and upper critical solution temperature (UCST) phenomena at low and high DMSO concentrations, respectively. The amide I band of PNiPAm consists of two components; the intensity of the 1650 cm-1 component increased, and that of the 1625 cm-1 component decreased with increasing temperature during both LCST and UCST phase transitions. Gradual red shifts of the C-H stretching and the amide II bands with increasing temperature or increasing DMSO concentration indicate a removal of water molecules from the alkyl and N-H groups. Raman microscopic measurements showed that DMSO is excluded from the polymer-rich phases upon both LCST and UCST phase separation. On the basis of the experimental results and the quantum chemical calculations, a model that explains the solvation change of the polymer during phase transitions was proposed.
Introduction An aqueous solution of poly(N-isopropylacrylamide) has a lower critical solution temperature (LCST) and exhibits a phase separation above the temperature.1 The phenomenon has attracted much attention from both fundamental and applicational aspects and has been extensively studied by means of a wide variety of techniques. These studies revealed that the macroscopic phase separation is accompanied by a large change in its conformation (coil to globule)2 and hydration state.3,4 It is generally believed that the breakage of the amide-water hydrogen bonds (H bonds) and the increase of unfavorable entropy contribution of hydrophobically hydrated isopropyl groups and backbone upon heating induce the phase separation. The effects of additives such as salts and organic solutes and solvents on the LCST behavior of PNiPAm and the swelling behavior of PNiPAm gels have also been eagerly investigated.5 The effects of salts such as metal halides on the LCST behaviors of nonionic thermosensitive polymers are rather simple. Because the ion-water interactions possess a major contribution, the phase transition temperature obeys the strengths of the interactions (the Hofmeister or lyotropic series).6-8 On the other hand, the effects of organic solvents are full of variety. The addition of small amounts of a good solvent such as methanol, ethanol, and acetone to an aqueous PNiPAm solution initially decreases the transition temperature, and a further addition increases it. Schild et al. termed the phenomenon as cononsolvency and suggested that the solvent-water interactions are preferred to the PNiPAm-water interactions at low additive concentrations.9 Recently, Costa et al. showed that PNiPAm has an upper critical solution temperature (UCST) in many mixtures of water and organic solvents including ethanol, propanol, and dimethyl sulfoxide (DMSO) at higher solvent concentrations.10 From among these organic solvents, we are interested in DMSO because of its unique properties and importance in biotechnol* Corresponding author. Fax: +81-776-27-8747. E-mail: y_maeda@ acbio2.acbio.fukui-u.ac.jp.
ogy. DMSO has a significantly high dipole moment (3.96) and boiling point (189 °C). DMSO-water mixtures exhibit a marked freezing point depression, reaching close to 60 K at a mole fraction of DMSO, nDMSO ) 0.33.11 The very low freezing point has made it a popular cryosolvent and a cryoprotective agent for a variety of cells and tissues allowing prolonged storage at subzero temperatures. It can also enhance a cell fusion and a membrane permeation12 by modulating the phase behaviors and the structural parameters of biomembranes.13 Furthermore, high concentrations of DMSO can dehydrate biological systems though the detailed molecular mechanism is not yet very clear. In short, it is of both theoretical and practical importance to understand the competitive interactions among DMSO, water, and such molecules in more detail. The phase transition of aqueous PNiPAm solutions is considered as an appropriate model for the thermal and solventinduced structural changes of proteins. It has been argued that the understanding of the molecular mechanism of both polymersolvent interactions and complexation among solvent molecules would be quite important to elucidate the observed phenomena. The combination of a vibrational spectroscopic observation and a quantum chemical simulation is a quite suitable method for this purpose. In the present study, we observe both LCST and UCST behaviors of PNiPAm in the DMSO/water mixtures by IR and confocal micro-Raman spectroscopy. To explain the experimental observation in a molecular level, we also performed a theoretical calculation based on a density functional theory (DFT). Experimental Methods Materials. PNiPAm was synthesized by radical polymerization in methanol at 70 °C and 7 h using 2,2′-azobis(isobutyronitrile) as an initiator. Polymers obtained were purified by dialysis against water and finally freeze-dried. Roughly estimated molecular weight and polydispersity of the polymer determined by gel permeation chromatography were 11 000 and
10.1021/jp072438s CCC: $37.00 © 2007 American Chemical Society Published on Web 10/20/2007
LCST and UCST Behavior of Poly(N-isopropylacrylamide) 1.9 (poly(ethyleneglycol) standard), respectively. D2O and deuterated DMSO (DMSO-d) were purchased from Aldrich. Solvent composition is shown as mole fraction of DMSO in the DMSO/water mixtures (nDMSO). Polymer concentration is shown in weight percent of the solutions. Measurements. Methods of IR and Raman spectroscopy and turbidity measurements were essentially the same as those described previously.3,4 The polymer dissolved in a solvent was placed between two CaF2 windows with a spacer (10 µm thick), and the IR spectrum was measured by using a Fourier-transform IR spectrometer (Varian, FTS-3000). The temperature of the solution was continuously raised with a circulating water bath at a rate of ∼1 °C/min, and time-resolved IR spectra were continuously collected at a resolution of 2 cm-1. DMSO-d/D2O mixtures were used as a solvent for the IR measurements with the exception of the use of DMSO-d/H2O to measure the amide II band of PNiPAm (N-H species). Raman spectra were measured by using an NRS-1000 confocal micro-Raman spectrometer (JASCO, Japan) equipped with an Ar laser operated at 514.5 nm. The laser beam was focused on the sample solution placed between two glass slips through an objective lens (×50), and backscattered light was collected by the same lens to be led to the spectrometer. The spatial resolution was approximately 2 and 4 µm to the lateral and vertical direction of the sample, respectively, and the spectral resolution was approximately 1 cm-1. DMSO-d/H2O mixtures were used as a solvent for the Raman measurements. To estimate PNiPAm concentration, we prepared calibration curves using PNiPAm/H2O mixtures of various compositions (0-100%). The curves show the ratio of the area of the ν(CH) band of PNiPAm to that of the ν(O-H) band of H2O as a function of PNiPAm concentration. To prepare the calibration curve used to determine the DMSO-d concentration, we measured the areas of the ν(C-D) band of DMSO and the ν(O-H) band of H2O using DMSO-d/H2O mixtures of nDMSO ) 0-1. Turbidity at 500 nm was measured with the solutions during continuous heating at a rate of approximately 1 °C/min. DFT Calculations. DFT calculations were performed using Gaussian 9814 at the B3LYP level with the 6-31G(d) basis set. The normal frequencies were calculated for the optimized structures and scaled down using a scale factor of 0.9613, which is accepted to be best for the level. Results Turbidimetry. First, we determined phase transition temperatures of the PNiPAm/DMSO/water ternary mixtures as a function of nDMSO by measuring the turbidity of the solutions to confirm the result reported by Costa et al.10 At nDMSO < 0.15, the mixture was clear at low temperatures and become turbid above a critical temperature (TL). TL is defined as an onset temperature of turbidity of the solution at heating. As shown in Figure 1, the value of TL gradually increases up to nDMSO < 0.025 and then decreases with increasing concentration of DMSO. At nDMSO > 0.7, the mixture exhibited a UCST-type phase transition; that is, a clear solution became turbid below a critical temperature (TU). The value of TU, which is defined as an onset temperature of turbidity at cooling, decreases steeply with increasing concentration of DMSO as shown with open circles in Figure 1. IR Spectroscopy. IR absorption spectra of PNiPAm measured in DMSO-d/D2O (nDMSO ) 0.06, 0.74) below (solid lines) and above (broken lines) the transition temperatures are shown in Figures 2a and 3a, respectively. DMSO-d and D2O were used
J. Phys. Chem. B, Vol. 111, No. 45, 2007 12965
Figure 1. Values of TL and TU of PNiPAm (0.5 wt %) measured in DMSO/H2O mixtures are plotted against nDMSO.
Figure 2. (a) IR absorption spectra at 13 (solid line) and 55 °C (broken line) and (b) IR difference spectra at 55 °C of 10% PNiPAm in DMSOd/D2O (nDMSO ) 0.06). (c) Values of ∆∆A for the amide I (b) and II (2) modes and the ν(C-H) mode (O) are plotted against temperature.
instead of DMSO and H2O to prevent their IR band from overlapping with the C-H stretching (ν(C-H)) and the amide I bands of the polymer, respectively. The polymer was deuterated at its amide group (N-D) in the solutions. It is known that PNiPAm has higher TL in D2O than in H2O by ∼1 °C because of stronger H bonding between D2O molecules,3,15 whereas it has similar TL or TU in DMSO-d/D2O as compared with those in DMSO/D2O of the same composition, meaning that the isotope effect of DMSO on the phase behaviors is small. Prominent IR bands of PNiPAm are the ν(C-H) (2900-3000 cm-1) bands, the amide I band (∼1630 cm-1, mainly due to the CdO stretching vibration), and the C-H bending (13501400 cm-1) bands. Because the amide II band is mainly due to C-N-H bending vibration, it is sensitive to the deuteration of the amide group. Hereafter, the band for the N-D species, which appears at around 1460 cm-1, is denoted as amide II′ band. In this region, several C-H bending bands also exist. Three peaks can be recognized in the ν(C-H) region, which are mainly due to the antisymmetric CH3 stretching (νas(CH3), 2980 cm-1), the CH2 stretching (2938 cm-1), and the symmetric CH3 stretching (νs(CH3), 2880 cm-1). The difference spectra for the LCST and UCST transitions are shown in Figures 2b and 3b, respectively,
12966 J. Phys. Chem. B, Vol. 111, No. 45, 2007
Figure 3. (a) IR absorption spectra at 9 (solid line) and 84 °C (broken line) and (b) IR difference spectra at 84 °C of 10% PNiPAm in DMSOd/D2O (nDMSO ) 0.74). (c) Values of ∆∆A for the amide I (b) and II (2) modes and the ν(C-H) mode (O) are plotted against temperature.
to enhance spectral changes induced by the phase transition. The differential absorbance (∆A(ν)) is obtained by subtracting the absorbance (A(ν)) at 55 (T > TL) or 84 °C (T > TU) from that at 13 (T < TL) or 9 °C (T < TU), respectively. Both a positive peak and a negative peak were observed for each vibration mode in the difference spectrum for the LCST transition, meaning that the changes in the solvation and/or the conformation of the polymer induce the shift of vibrational frequency. On the other hand, though the amide I and II bands exhibit peak shifts, the C-H stretching bands keep their positions during UCST transition. The progress of the phase transition can be followed by monitoring the intensities at the maxima and minima (∆A(ν1) or ∆A(ν2)) of the difference IR spectra. We plot the difference between them (∆∆A ) ∆A(ν1) - ∆A(ν2)) at the νas(CH3), amide I, and amide II′ modes against temperature (Figures 2c and 3c). Onset points are observed at around 30 and 60 °C for the LCST and UCST phenomena, respectively. The temperatures agree with the cloud points of these solutions determined by simultaneous measurements of the transmittance of visible light from a light emitting diode, indicating that changes in IR spectra are really related to the phase transitions. The LCST transition occurs in a narrow temperature range, but the UCST transition occurs in a broad temperature range of ∼20 °C. We can obtain information on the H bonding of the amide CdO groups from the profiles of the amide I band. As shown in our previous paper,3 the amide I band of PNiPAm in pure water consists of two components centered at 1625 cm-1 and 1650 cm-1 (the components I and II), which can be assigned to the CdO groups bound to water (CdO‚‚‚HsO) and the amide NsH group (CdO‚‚‚HsN) through H bonding, respectively. The component II appears only above TL, meaning that part of the amide groups form the amide-amide H bonds in the globule state. The amide I band observed at nDMSO ) 0.74 also contains two components at similar wavenumbers (1625 and 1652 cm-1, the components I′ and II′) though the component II′ holds a
Yamauchi and Maeda
Figure 4. (a) Baseline subtracted amide I band of 10% PNiPAm measured in (a) DMSO-d/D2O (nDMSO ) 0.06) at 20 (left) and 50 (right) °C and (b) DMSO-d/D2O (nDMSO ) 0.74) at 10 (left) and 85 (right) °C. Broken lines indicate two Gaussian components (center: 1650 and 1625 cm-1 for (a), and 1652 and 1625 cm-1 for (b)). The area fractions of (c) the component I (O) and II (b) in DMSO-d/D2O (nDMSO ) 0.06) and (d) the component I′ (O) and II′ (b) in DMSOd/D2O (nDMSO ) 0.74) are plotted against temperature.
Figure 5. Wavenumbers of (a) the νas(CH3) band of PNiPAm measured in DMSO-d/D2O mixtures and of (b) the amide II band measured in DMSO-d/H2O mixtures. Open and closed circles indicate measurements in single-phase solutions and in two-phase systems, respectively.
large part. We decompose the amide I band by using a curvefitting method and estimate the fraction of these components. As shown in Figure 4, the fraction of the components II and II′ increased with increasing temperature during both LCST (soluble-insoluble) and UCST (insoluble-soluble) transitions in the mixed solvents of nDMSO ) 0.06 and 0.74, respectively. We can know the solvation of the alkyl groups of PNiPAm through the positions of the C-H stretching band. The wavenumber of the νas(CH3) band is plotted against nDMSO in Figure 5. Open and closed circles in the figure show the wavenumbers for 1-phase and 2-phase systems, respectively. The νas(CH3) band shifts downward upon the LCST phase transition. In general, a C-H stretching frequency shift upward as the corresponding C-H group interacts with water.3 The red shift of the νas(CH3) band during LCST transition, therefore, indicates a dehydration of the polymer chain. The band also shifts toward lower wavenumbers with an increase in DMSO concentration, indicating a gradual replacement of water molecules surrounding the alkyl groups by DMSO. On the other hand, the νas(CH3) frequency does not change during UCST phase transition (nDMSO
LCST and UCST Behavior of Poly(N-isopropylacrylamide)
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Figure 6. Raman spectra of 20 wt % PNiPAm in a DMSO-d/H2O (nDMSO ) 0.06) mixed solvent at 45 °C. The upper and lower spectra were measured in the polymer-rich phase and the solvent-rich phase, respectively.
Figure 8. Optimized structures of 1:1 complexes.
TABLE 1: Interaction Energies for the 1:1 Complexes Obtained by DFT Calculation (in kcal/mol) donor
Figure 7. Temperature dependences of the concentrations of PNiPAm (O) and DMSO-d (b) in the domain phases (T > TL or T < TU) or homogeneous solutions (T < TL or T > TU) of (a) 20 wt % PNiPAm in DMSO-d/H2O (nDMSO ) 0.06) and (b) 10 wt % PNiPAm in DMSOd/H2O (nDMSO ) 0.74).
) 0.74). This suggests that an environment around the methyl group is identical, although the solvation of the amide group change during phase transition. Figure 5b shows the wavenumber of the amide II band measured in DMSO-d/H2O mixtures. In H2O, the amide II band of PNiPAm appears at 1540-1565 cm-1, and no other band overlaps with it. The amide II band also gradually shifts downward with increasing concentration of DMSO. In addition, the band shifts downward with increasing temperature on both LCST and UCST phase transitions. Micro-Raman spectroscopy. We measured Raman spectra at both domain and matrix phases separately at T > TL and T < TU at low and high DMSO concentrations, respectively. Figure 6 shows Raman spectra of 20% PNiPAm in the DMSOd/H2O mixture (nDMSO ) 0.74) measured in the domain and matrix phases at above TL. The C-H stretching bands of PNiPAm (3050-2850 cm-1), the C-D stretching bands of DMSO-d (2265 and 2140 cm-1), and the O-H stretching bands of H2O (3700-3120 cm-1) are observed. Figure 7 shows the changes in the polymer and DMSO concentrations in the domain phases at cooling. While PNiPAm concentration increased with increasing temperature, DMSO concentration decreased. On the other hand, PNiPAm concentration increased and DMSO concentration decreased with decreasing temperature during
acceptor
H2O
DMSO
(H-N)PNiPAm
H2O DMSO PNiPAm(CdO)
-12.2 -20.4 -16.5
-10.2 -13.5
-11.4 -15.1 -12.0
UCST transition. In both of the cases, DMSO is excluded from the polymer-rich phases. DFT Calculation. H bond is important to discuss the molecular interactions among PNiPAm, water, and DMSO. These molecules accept H bond at the CdO, H-O, and SdO groups, respectively. In addition, PNiPAm and water have the N-H and O-H groups as a donor, respectively. The C-H group of DMSO also acts as a donor, which is weaker than ordinal donors such as O-H and N-H groups. Hereafter, we indicate a H bond between an acceptor and a donor as acceptor‚ ‚‚donor, for example, PNiPAm(CdO)‚‚‚H2O and H2O‚‚‚(HN)PNiPAm. We estimate the interaction energies of 1:1 complexes of these molecules by using a DFT calculation. The optimized structures of the complexes are shown in Figure 8, and the interaction energies are compiled in Table 1. Among these sorts of the 1:1 complexes, the DMSO‚‚‚H2O complex is the strongest. It is known that the DMSO and water form very strong complexes,17 which could be responsible for a very low freezing point at nDMSO ) 0.25-0.33. This is consistent with the fact that the lifetime of water-DMSO H bonds is longer than that for water-water H bonds.16 Discussion Taking the present experimental and theoretical results into consideration, we discuss the solvation changes of PNiPAm during LCST and UCST phase transitions. First, we consider the LCST phenomenon of the PNiPAm/H2O binary mixture. In the mixture, both CdO and N-H groups of PNiPAm are considered to form a H bond with water. A typical structure is shown in the model a in Figure 9. The interaction concerning the amide group of PNiPAm is solely considered in these models, and the calculated amide I frequencies are compared with observed ones. In the model a, the CdO group accepts two H bonds from two water molecules, and the N-H group forms a H bond with an oxygen atom of water. The amide I frequency for the model a is 1624 cm-1 and is comparable to
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Yamauchi and Maeda contribute to the component II′ to some degree. The component I′ may result from the doubly H bonded CdO groups (model a). Therefore, larger amount of water is retained in the polymerrich phase than that expected from the solvent composition at T < TU (Figure 7b). On heating, PNiPAm(CdO)‚‚‚H2O is partially broken as shown in Figure 5d, and the mixture becomes homogeneous. Conclusions
Figure 9. Models of solvated PNiPAm in (a) PNiPAm/water and PNiPAm/water/DMSO (low DMSO content) and (b) PNiPAm/water/ DMSO (high DMSO content). The numbers indicated beside the Cd O groups are the frequencies (cm-1) of the amide I mode calculated based on DFT.
the observed frequency for the component I of the amide I band (1625 cm-1, Figure 4). During phase separation, part of the polymer-water H bonds are broken, and the polymer-polymer H bonds (PNiPAm(CdO)‚‚‚(H-N)PNiPAm) are formed. This situation is represented by the model b, which gives the amide I band at 1652 cm-1 and is responsible for the component II. However, the result shown in Figure 4c indicates that a large part of the CdO groups retain water even at T > TL. Whereas, judging from the ν(C-H) wavenumber of the alkyl groups of PNiPAm, the water molecules surrounding them are mostly removed in the phase-separated states at T > TL. When a small amount of DMSO is added to the PNiPAm/ H2O mixture, the system becomes a ternary mixture. Because the DMSO‚‚‚H2O interaction is stronger than the PNiPAm(Cd O)‚‚‚H2O and the H2O‚‚‚(H-N)PNiPAm interactions, DMSO removes water molecules from PNiPAm, induces dehydration of the polymer, and finally reduces TL of the mixture. Because DMSO is a weaker H bond donor than water, a large part of the CdO groups still form H bonds with water, whereas the water molecules bound to the C-H and N-H groups are removed or replaced by DMSO as shown in Figure 5. While water molecules are separated from the polymer chain and form a water-rich phase during phase separation, DMSO tends to exist in the phase because of a strong DMSO-water interaction (Figure 7a). The solvation of PNiPAm in the ternary mixtures at low DMSO concentration is essentially the same as that in the PNiPAm/water binary system with the exception that some N-H groups form H bond with DMSO and the number of water molecules surrounding the C-H groups is reduced. This discussion is also applicable to explain the dehydration effect of DMSO on biological systems such as proteins and biomembranes. The stronger interaction between water and DMSO makes DMSO better than amide and amine groups in forming H bonds with water. Next, we consider the PNiPAm/DMSO binary mixture. In the mixture DMSO‚‚‚(H-N)PNiPAm, a weak H bond between the CdO group of PNiPAm and the methyl group of DMSO may be formed (model d). Because PNiPAm(CdO)‚‚‚H2O is stronger than the latter interaction, water replaces DMSO to form a H bond with the CdO group (the model c). The model c has the amide band at 1654 cm-1, which may be responsible for the component II′. The polymer-polymer H bonds may also
The phase transition of PNiPAm in the DMSO/water mixed solvents has been investigated by IR and confocal micro-Raman spectroscopy. The ternary mixtures exhibit the LCST and UCST phenomena at low and high DMSO concentrations, respectively. The amide I band of PNiPAm contains two components in the ternary mixtures, and the ratio of the component II (1650 cm-1) against the component I (1625 cm-1) increased with increasing temperature at both low and high DMSO concentrations. Though the solvation of the CdO group in the ternary mixtures is similar to that in PNiPAm/water binary mixtures, water molecules bound to the C-H and N-H groups are gradually removed or replaced by DMSO molecules with increasing DMSO concentration. Confocal micro-Raman spectroscopic measurements showed that DMSO is excluded from the polymer-rich phases both above the LCST and below the UCST. The result indicates that the interaction between PNiPAm and DMSO is not strong. The density functional theory was used to estimate the interaction energies and the amide I frequencies for the 1:1 complexes. By comparing the experimental result with the theoretical one, a model that explains the solvation change of the polymer during LCST and UCST phase transition was proposed. Acknowledgment. This work was supported by a Grantin-Aid for Scientific Research (17550115) from Japan Society for the Promotion of Science. References and Notes (1) Fujishige, S.; Kubota, K.; Ando, I. J. Phys. Chem. 1989, 93, 3311. (2) Wang, X.; Qiu, X.; Wu, C. Macromolecules 1998, 31, 2972. (3) Maeda, Y.; Higuchi, T.; Ikeda, I. Langmuir 2000, 16, 7503. (4) Maeda, Y.; Yamamoto, H.; Ikeda I. Macromolecules 2003, 35, 5055. (5) Van Durme, K.; Rahier, H.; Van Mele, B. Macromolecules 2005, 38, 10155. (6) Hofmeister, F. Arch. Exp. Pathol. Pharmakol. 1888, 24, 246. (7) McBain, J. W. In Colloid Science, Heath: Boston, MA, 1950. (8) Robinson, D. R.; Jencks, W. P. J. Am. Chem. Soc. 1965, 87, 2470. (9) Schild, H. G.; Muthkumar, M.; Tirrell, D. A. Macromolecules 1991, 24, 948. (10) Costa, R. O. R.; Freitas, R. F. S. Polymer 2002, 43, 5879. (11) Rasmussen, D. H.; MacKenzie, A. P. Nature 1968, 220, 1315. (12) Yu, Z. W.; Quinn, P. J. Biosci. Rep. 1994, 14, 259. (13) Tristram-Nagle, S.; Moore, T.; Petrache, H.; Nagle, J. F. Biochim. Biophys. Acta 1998, 1369, 19. (14) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B. G.; Chen, W.; Wong, M. W.; Andres, J. L.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98; Gaussian, Inc.: Pittsburgh, PA, 1998. (15) Shirota, H.; Kuwabara, N.; Ohkawa, K.; Horie, K. J. Phys. Chem. B 1999, 103, 10400. (16) Rablen, P. R; Lockman, J. W.; Jorgensen W. L. J. Phys. Chem. A 1998, 102, 3782. (17) Vishnyakov, A.; Lyubartsev, A. P.; Laaksonen, A. J. Phys. Chem. A 2001, 105, 1702.
