Pressure-Dependence of Poly(N ... - ACS Publications

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Pressure-Dependence of Poly(N‑isopropylacrylamide) Mesoglobule Formation in Aqueous Solution Bart-Jan Niebuur,† Kora-Lee Claude,† Simon Pinzek,† Coleman Cariker,‡ Konstantinos N. Raftopoulos,†,§ Vitaliy Pipich,∥ Marie-Sousai Appavou,∥ Alfons Schulte,*,‡ and Christine M. Papadakis*,† †

Physik-Department, Fachgebiet Physik weicher Materie, Technische Universität München, James-Franck-Str. 1, 85748 Garching, Germany ‡ Department of Physics and College of Optics and Photonics, University of Central Florida, 2385 Central Florida Boulevard, Orlando, Florida 32816, United States ∥ Jülich Centre for Neutron Science (JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ), Forschungszentrum Jülich GmbH, Lichtenbergstr. 1, 85748 Garching, Germany S Supporting Information *

ABSTRACT: Above their cloud point, aqueous solutions of the thermoresponsive polymer poly(N-isopropylacrylamide) (PNIPAM) form large mesoglobules. We have carried out very small-angle neutron scattering (VSANS with q = 0.21−2.3 × 10−3 Å−1) and Raman spectroscopy experiments on a 3 wt % PNIPAM solution in D2O at atmospheric and elevated pressures (up to 113 MPa). Raman spectroscopy reveals that, at high pressure, the polymer is less dehydrated upon crossing the cloud point. VSANS shows that the mesoglobules are significantly larger and contain more D2O than at atmospheric pressure. We conclude that the size of the mesoglobules and thus their growth process are closely related to the hydration state of PNIPAM.

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may serve as a simple model system to investigate the effect of pressure on the hydration behavior and its implications on the mesoscopic behavior at the phase boundary since they neither contain charged groups nor form secondary structures, but are restricted to the coil-to-globule transition with subsequent aggregation.6 Fourier-transform infrared spectroscopic experiments showed that the thermoresponsive polymer poly(N-isopropylacrylamide) (PNIPAM) dehydrates when heated through the cloud point (CP) at atmospheric pressure.7−9 However, it does not phase-separate macroscopically, but forms mesoglobules, typically in the size range of 50 nm to 1 μm with the size depending on the conditions.10−18 In contrast, PNIPAM does not dehydrate at the CP when the phase separation is induced by increasing pressure at constant temperature.7 The present study describes the previously unexplored structure of phase-separated aqueous PNIPAM solutions at high pressure. Using pressure as a tool to tune the degree of hydration, we aim to elucidate the relation between the hydration state and the mesoglobule size. This may further improve the understanding of the growth process of the mesoglobules and their composition, which is related to their

ater-soluble polymers, such as polyelectrolytes, biopolymers, and thermoresponsive polymers, may feature charged groups, hydrophilic groups and hydrophobic groups. The interplay between ionic interactions, H-bonds, and hydrophobic interactions governs their solubility, secondary structure, responsivity to changes of pH, ionic strength, or temperature and their aggregation behavior. At this, the hydrophobic interaction is of special importance as it displays interesting behavior when high pressure is applied. In proteins, for instance, it is the main driving force for stabilizing the native state at atmospheric pressure.1 The clathrate-like structure of water formed around hydrophobic groups enhances the entropic contribution to the free energy, hindering the hydration of hydrophobic groups. In contrast, at high pressure, bulk water changes its state from an open tetrahedral structure to a more ordered hexagonal one, that is, ordered water around hydrophobic groups becomes more similar to bulk water.2 A second effect of pressure concerns the compressibility of the hydration shell around a sequence of hydrophobic groups, which is significantly larger than the compressibility of bulk water and of hydration water around hydrophilic groups. With increasing pressure, exposure of the hydrophobic groups to an aqueous environment leads to the possibility to further compress water in the newly formed hydration layers, which, for instance, favors the denatured, unfolded state of proteins.3−5 Nonionic thermoresponsive polymers featuring lower critical solution temperature (LCST) behavior in aqueous solution © XXXX American Chemical Society

Received: July 29, 2017 Accepted: October 6, 2017

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ACS Macro Letters interaction potential. To this end, we study a 3 wt % solution of PNIPAM (36.0 kg mol−1) in D2O using Raman spectroscopy to investigate the hydration state and very small-angle neutron scattering (VSANS) to characterize the size of the mesoglobules as well as their composition. D2O was chosen as a solvent to maximize the scattering contrast in VSANS and to avoid band overlap in Raman spectroscopy. Employing these techniques, temperature scans were carried out around the respective CPs at atmospheric pressure, ∼80 and ∼113 MPa. These were determined as described in the Supporting Information (Table S1). A close relation between the hydrophobic hydration, the water fraction of the aggregates, and their size is found. Temperature-dependent Raman spectra at these pressures around the respective CPs give insight into the hydrophobic hydration (Figure 1). Four bonds are present in the CH stretching region of PNIPAM, namely, at 2884, 2923, 2947, and 2990 cm−1, which are assigned to symmetric stretching of the CH3 groups in the side group (νs(CH3)), stretching of the CH groups (ν(CH)), antisymmetric stretching of the CH2 groups in the backbone (νas(CH2)), and antisymmetric stretching of the CH3 side groups in the side group (νas(CH3)).8,9,19 The differences at Tcp are most pronounced at atmospheric pressure, mainly due to the decrease in intensity of the νas(CH2) peak and the frequency shifts of the νs(CH3) and νas(CH3) bands. In contrast, hardly any changes are discernible at 79 and 114 MPa. The peak frequencies of the two CH3bands are given in Figure 2. The peak frequencies of both the symmetric and the antisymmetric CH3 stretching bands are constant below Tcp(p) and decrease with increasing temperature above. This points to a change of hydrogen bonding between the CH3 groups in the isopropyl substituent of PNIPAM and D2O as the two-phase state is entered. For systems containing methyl groups, it was observed that the C−H bond length increases when they are hydrated, i.e. the hydrogen bonds between CH3 groups and D2O are improper.20−23 The decrease of the peak frequency with pressure can thus be attributed to a dehydration of the CH3 groups at Tcp(p). However, whereas the decrease in frequency at Tcp(p) is abrupt at atmospheric pressure, it is smooth at 79 and 114 MPa. We conclude that the change of the hydration state at Tcp(p) is much less pronounced at high pressure than at atmospheric pressure, that is, the CH3 groups stay hydrated to a certain degree even in the two-phase state. The reduced dehydration of the CH3 groups at elevated pressure reflects the increased compressibility of water around these groups. Because the CH3 groups dehydrate to a certain degree, the hydrophobic interaction still plays a role at temperature-induced phase separation at the investigated pressures. Also, the stretching frequency in the one-phase region depends on pressure. The larger vibrational frequency reflects the increased hydration of the chains at elevated pressures. The peak frequencies of the antisymmetric CH2 stretching band also decrease with temperature above Tcp(p), but there are no pronounced frequency shifts between the three pressures. We conclude that the changes of the hydration state of the backbone of PNIPAM are not as significant as the ones of the CH3 groups in the side groups with pressure. Optical microscopy provides first insights into the mesoglobular structure formed by PNIPAM in the segregated state at high pressure. Examples are shown in Figure 3. Whereas the solution is transparent at atmospheric pressure below Tcp

Figure 1. Raman spectra of the 3 wt % PNIPAM solution in D2O in dependence on temperature at (a) atmospheric pressure, (b) 79 MPa, and (c) 114 MPa. Temperatures are given in the graphs. Tcp(p) is the CP measured using in situ optical microscopy at the respective pressure (Table S1 in the SI). Red lines: below Tcp(p), green lines: above Tcp(p). For clarity, the curves were smoothed using the Savitzky-Golay algorithm and shifted vertically.

(Figure 3a), a phase-separated structure can be identified above (Figure 3b). However, the optical resolution is insufficient for detailed characterization of the mesoglobules. The measurement at 79 MPa (Figure 3c) shows pronounced mesoglobules with diameters of ∼4 μm that are weakly correlated. VSANS is a more suitable method to characterize the size of the mesoglobules, because of its ability to probe smaller length scales than optical microscopy. Furthermore, the dependence of the water fraction of the mesoglobules on pressure can be resolved. Representative data from temperature scans at 0.1 MPa, 80 MPa and 112.5 MPa are displayed in Figure 4. In the q range investigated, the curves below Tcp(p) show only weak scattering, as expected for a homogeneous solution (chain scattering is expected at higher q values).24,25 Above Tcp(p), the 1181

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Figure 2. Peak frequencies of (a) the symmetric and (b) the antisymmetric CH3 stretching bands, νs(CH3) and νas(CH3), in dependence on temperature. Black squares: atmospheric pressure, red circles: 79 MPa, blue triangles: 114 MPa.

Figure 4. Representative VSANS data of the 3 wt % PNIPAM solution in D2O from temperature scans at (a) atmospheric pressure, (b) 80 MPa, and (c) 112.5 MPa. Red curves: below Tcp(p), green curves: above Tcp(p). Tcp(p) is taken from the sudden decrease of the neutron transmission (Table S1 in the SI). Black lines: fits, see text. Figure 3. Optical microscopy images at atmospheric pressure just below CP (a) and just above CP (b) and at 79 MPa just above CP (c).

CP: The initial large PNIPAM-rich domains rearrange and thereby shrink. However, far away from the CP, the radii of gyration are still significantly larger at 80 MPa and 112.5 MPa than at atmospheric pressure. The Porod exponent, m, indicative of the surface structure28 of the PNIPAM-rich domains, depends only weakly on pressure (Figure 5b). At atmospheric pressure, m increases from ∼3.7 to ∼4.2, meaning that water molecules in the outer layer of the initially rough aggregates diffuse out, resulting in a concentration gradient at the surface. We attribute this to the formation of a dense PNIPAM shell, which confines water molecules within the mesoglobules. At high pressure, m increases from ∼3.5 to ∼4, meaning that the initially rough aggregates become smooth with increasing temperature. Since PNIPAM stays hydrated above the CP (as follows from Raman spectroscopy), no dense shell is formed, enabling the composition of the mesoglobules to be homogeneous. To characterize the fraction of D2O in the PNIPAM domains, the invariant Q* (eq S4 in the SI) was calculated from the fitted Beaucage functions (Figure 5c). It increases with temperature and becomes constant at ∼1 K above Tcp(p). The values at high pressure are a factor of ∼100 lower than at atmospheric pressure. Following eq S5, we assign this difference to the compositions of the PNIPAM domains: While they are rather pure at atmospheric pressure, they contain a significant amount of D2O at high pressure. This result is in consistency with the dehydration at atmospheric pressure observed by Raman spectroscopy, which is much stronger than the one at higher pressures.

scattering curves feature a clear shoulder which strongly shifts to lower q values with increasing pressure, in accordance with the increased sizes of the mesoglobules at high pressure found with optical microscopy: It reaches out to ∼2 × 10−3 Å−1 at atmospheric pressure, to ∼1 × 10−3 Å−1 at 80 MPa, and to ∼8 × 10−4 Å−1 at 112.5 MPa. Although correlations between mesoglobules are probably present (as suggested by optical microscopy), the correlation length cannot be extracted since the expected peak of the structure factor lies below the lower limit of the accessible q range. Therefore, the empirical Beaucage model (eq 1 in the Experimental Section) is employed to model the scattering curves.26,27 It yields information on the size and surface structure of the mesoglobules; correlation between mesoglobules is not accounted for. Excellent fits to the I(q) curves above Tcp(p) are obtained (Figure 4; Fits of models for disperse spheres did not give satisfactory results). The scattering curves reveal large differences in morphology between atmospheric and high pressure, shown in Figure 5. At atmospheric pressure, the radius of gyration of the mesoglobules, Rg, increases slightly from ∼0.4 μm right above Tcp(p) to ∼0.55 μm at 0.3 K above Tcp(p) (Figure 5a). At 80 MPa, it increases to 1.4 μm, and at 112.5 MPa, it is constant at ∼1.5 μm. Thus, the domains are much larger at high pressures than at atmospheric pressure. At all pressures, the value of Rg decreases as temperature is increased further above Tcp(p), which may be due to a nonequilibrium state after crossing the 1182

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EXPERIMENTAL SECTION

Materials. Poly(N-isopropylacrylamide) with Mn = 36.0 kg mol−1 and a dispersity of 1.26 (end groups carboxylic acid and a hydrogen atom, respectively) was purchased from Sigma-Aldrich. It was dissolved in D2O at a concentration of 3 wt %. The solutions were stirred for at least 24 h before the measurements. Raman spectroscopy was performed with a LabRam HR 800 system (JY Horiba) at a spectral resolution of 2 cm−1. The excitation source was a frequencydoubled Nd:YAG laser with a wavelength of 532 nm. The laser power at the sample position was less than 3 mW at a spot size of 1.5 μm. The sample solutions were contained in a fused silica micro capillary high pressure cell, withstanding pressures up to 300 MPa. It was connected to a pressure generator (High Pressure Equipment Company), using ethanol as the pressure transmitting medium. The sample solution was probed near the end of the capillary (∼30 cm away from the sample-ethanol interface) so that no contamination could occur.30 The capillary was anchored in a Cu block that was attached to a circulating bath thermostat. The sample temperature was measured as described in the SI. Following each temperature change the sample was equilibrated for 10 min, and spectra were acquired with an integration time of 300 s. After dark count subtraction the Raman spectra in the C−H stretch region (2850 cm−1 to 3050 cm−1) were deconvoluted using a superposition of four Lorentzian lines attributed to the CH−, CH2−, and CH3− groups in PNIPAM (Figure S1 in the SI). Optical Microscopy. Optical microscopy was conducted using the same microcapillary cell as for Raman spectroscopy. Images were acquired with a CCD camera on a port of an Olympus X41 microscope. For details, see the SI. Very Small-Angle Neutron Scattering. Very small-angle neutron scattering (VSANS) experiments were carried out at KWS-3 at the Heinz Maier-Leibnitz Zentrum, Garching, Germany.31 The neutron wavelength was λ = 12.8 Å with a spread Δλ/λ = 0.18. The sample−detector distance was 9.4 m, resulting in a q range of 2.1 × 10−4 − 2.3 × 10−3 Å−1. q = 4π × sin(θ/2)/λ is the momentum transfer. A temperature-controlled pressure cell withstanding pressures up to 500 MPa based on the one presented in ref 32 was used. A Viton ring separated the sample from the pressure-transmitting medium. The sample thickness was 2 mm, independent of pressure. Temperature scans were carried out in steps of 0.1 K. At each temperature, four measurements of 5 min were carried out. The background from the empty cell as well as the dark current measured using boron carbide were subtracted from the data. A Plexiglas measurement was used to determine the detector sensitivity and the direct beam was used to bring the data to absolute scale. These operations as well as azimuthal averaging were carried out using the software QtiKWS. Analysis of the VSANS Curves. The VSANS curves were fitted by the Beaucage model:26,27

Figure 5. (a) Radius of gyration of the mesoglobules, Rg, (b) Porod exponent, m, and (c) invariant scattering, Q*. Black squares: atmospheric pressure, red circles: 80 MPa, blue triangles: 112.5 MPa.

We conclude that high pressure provides a mean to alter the hydration state of PNIPAM above the CP, which is reflected in the overall water fraction in the formed mesoglobules, in their size and in their surface structure. At atmospheric pressure, significant dehydration of the CH3-groups in the isopropyl side group of PNIPAM occurs, the water fraction in the mesoglobules is low, their size is rather small, and their surface layer is rich in PNIPAM, in accordance with previous results.29 In contrast, the dehydration at high pressure is less pronounced, the mesoglobules have higher water fraction, their size is larger, and their composition is homogeneous. These observations point to a difference in growth process of the mesoglobules. The dehydrated, collapsed PNIPAM chains at atmospheric pressure form aggregates that cannot grow easily, possibly due to low chain mobility. In contrast, the less dehydrated PNIPAM chains at high pressures form larger aggregates that can grow more readily, possibly because rearrangements during merging are not hindered. These nonionic thermoresponsive polymers may constitute a simple model system to investigate the relation between the hydrophobic interaction and the aggregation behavior without complications due to other interactions and the formation of secondary structures that may obscure the effect. To the best of our knowledge, this is the first comprehensive structural study of mesoglobules at high pressure, which becomes possible by extending SANS to very low momentum transfers.

⎫m ⎧ ⎛ qR ⎞⎤3 ⎪ ⎛ q 2R 2 ⎞ ⎪⎡ g g ⎟ + B⎨⎢erf⎜ ⎬ + Ibkg I(q) = G exp⎜⎜− ⎟⎥ /q⎪ ⎪ 3 ⎟⎠ ⎝ ⎩⎣ ⎝ 6 ⎠⎦ ⎭

(1)

where G and B are scaling factors, Rg is the radius of gyration, m is the Porod exponent, and Ibkg is the incoherent background that was kept fixed during fitting at 300 cm−1. erf(x) denotes the error function. Smearing effects due to the divergence of the neutron beam and the wavelength distribution were taken into account following standard procedures.33

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00563. Determination of cloud points; Deconvolution of Raman spectra; Modeling of VSANS curves; and Calculation of the invariant (PDF). 1183

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(11) Chan, K.; Pelton, R.; Zhang, J. On the Formation of Colloidally Dispersed Phase-Separated Poly(N-isopropylacrylamide). Langmuir 1999, 15, 4018−4020. (12) Chuang, J.; Grosberg, A. Y.; Tanaka, T. Topological Repulsion Between Polymer Globules. J. Chem. Phys. 2000, 112, 6434−6442. (13) Wu, C.; Li, W.; Zhu, X. X. Viscoelastic Effect on the Formation of Mesoglobular Phase in Dilute Solutions. Macromolecules 2004, 37, 4989−4992. (14) Aseyev, V.; Hietala, S.; Laukkanen, A.; Nuopponen, M.; Confortini, O.; Du Prez, F. E.; Tenhu, H. Mesoglobules of Thermoresponsive Polymers in Dilute Aqueous Solutions Above the LCST. Polymer 2005, 46, 7118−7131. (15) Kujawa, P.; Aseyev, V.; Tenhu, H.; Winnik, F. M. TemperatureSensitive Properties of Poly(N-isopropylacrylamide) Mesoglobules Formed in Dilute Aqueous Solutions Heated above Their Demixing Point. Macromolecules 2006, 39, 7686−7693. (16) Balu, C.; Delsanti, M.; Guenoun, P.; Monti, F.; Cloitre, M. Colloidal Phase Separation of Concentrated PNIPAm Solutions. Langmuir 2007, 23, 2404−2407. (17) Maresov, E. A.; Semenov, A. N. Mesoglobule Morphologies of Amphiphilic Polymers. Macromolecules 2008, 41, 9439−9457. (18) Halperin, A.; Kröger, M.; Winnik, F. M. Poly(N-isopropylacrylamide) Phase Diagrams: Fifty Years of Research. Angew. Chem., Int. Ed. 2015, 54, 15342−15367. (19) Sun, B.; Lin, Y.; Wu, P. Structure Analysis of Poly(Nisopropylacrylamide) Using Near-Infrared Spectroscopy and Generalized Two-Dimensional Correlation Infrared Spectroscopy. Appl. Spectrosc. 2007, 61, 765−771. (20) Hobza, P.; Havlas, Z. Blue-Shifting Hydrogen Bonds. Chem. Rev. 2000, 100, 4253−4264. (21) Scheiner, S.; Kar, T. Red- Versus Blue-Shifting Hydrogen Bonds: Are There Fundamental Distinctions? J. Phys. Chem. A 2002, 106, 1784−1789. (22) Kar, T.; Scheiner, S. Comparison of Cooperativity in CH···O and OH···O Hydrogen Bonds. J. Phys. Chem. A 2004, 108, 9161− 9168. (23) Joseph, J.; Jemmis, E. D. Red-, Blue-, or No-Shift in Hydrogen Bonds: A Unified Explanation. J. Am. Chem. Soc. 2007, 129, 4620− 4632. (24) Shibayama, M.; Isono, K.; Okabe, S.; Karino, T.; Nagao, M. SANS Study on Pressure-Induced Phase Separation of Poly(Nisopropylacrylamide) Aqueous Solutions and Gels. Macromolecules 2004, 37, 2909−2918. (25) Meier-Koll, A.; Pipich, V.; Busch, P.; Papadakis, C. M.; MüllerBuschbaum, P. Phase Separation in Semidilute Aqueous Poly(Nisopropylacrylamide) Solutions. Langmuir 2012, 28, 8791−8798. (26) Beaucage, G.; Schaefer, D. W. Structural studies of complex systems using small-angle scattering: a unified Guinier/power-law approach. J. Non-Cryst. Solids 1994, 172−174, 797−805. (27) Beaucage, G. Approximations Leading to a Unified Exponential/ Power-Law Approach to Small-Angle Scattering. J. Appl. Crystallogr. 1995, 28, 717−728. (28) Koberstein, J. T.; Morra, B.; Stein, R. S. The Determination of Diffuse-Boundary Thicknesses of Polymers by Small-Angle X-ray Scattering. J. Appl. Crystallogr. 1980, 13, 34−45. (29) Kyriakos, K.; Philipp, M.; Adelsberger, J.; Jaksch, S.; Berezkin, A. V.; Lugo, D. M.; Richtering, W.; Grillo, I.; Miasnikova, A.; Laschewsky, A.; Müller-Buschbaum, P.; Papadakis, C. M. Cononsolvency of Water/ Methanol Mixtures for PNIPAM and PS-b-PNIPAM: Pathway of Aggregate Formation Investigated Using Time-Resolved SANS. Macromolecules 2014, 47, 6867−6879. (30) Park, S. H.; Arora, S.; Schulte, A. Micro-Spectroscopy of Biomolecules and Cells at Variable Pressure in a Micro-Capillary. Biophys. J. 2010, 98 (3), 742a. (31) Heinz Maier-Leibnitz Zentrum et al. KWS-3: Very Small Angle Scattering Diffractometer with Focusing Mirror. Journal of Large-Scale Research Facilities 2015, 1, A31. (32) Kohlbrecher, J.; Bollhalder, A.; Vavrin, R.; Meier, G. A High Pressure Cell for Small Angle Neutron Scattering up to 500 MPa in

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Vitaliy Pipich: 0000-0002-3930-3602 Alfons Schulte: 0000-0003-0824-8572 Christine M. Papadakis: 0000-0002-7098-3458 Present Address §

Konstantinos N. Raftopoulos, C-4 Department of Chemistry and Technology of Polymers, Cracow University of Technology, Warszawska 24, 31−155 Kraków, Poland. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

K.N.R. acknowledges a generous fellowship (TUFF) by the TUM University Foundation. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is based on experiments performed at the KWS-3 instrument and SANS high pressure cell operated by JCNS at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany.

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REFERENCES

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