Rotational Dynamics of Spin-Labeled Polyacid Chain Segments in

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Rotational Dynamics of Spin-Labeled Polyacid Chain Segments in Polyelectrolyte Complexes Studied by CW EPR Spectroscopy Uwe Lappan,* Brigitte Wiesner, and Ulrich Scheler Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Straße 6, 01069 Dresden, Germany S Supporting Information *

ABSTRACT: A nitroxide spin label has been covalently linked to the weak polyacid poly(ethylene-alt-maleic acid) (P(E-alt-MA)) to study the rotational mobility of the polyacid backbone in polyelectrolyte complexes (PEC) formed with the oppositely charged strong polycation poly(diallyldimethylammonium chloride) (PDADMAC) in dependence on the pH of the dispersion and the temperature. The rotational mobility of the polyacid chain segments has been determined by simulation of the line shape of the continuous wave (CW) electron paramagnetic resonance (EPR) spectra using the microscopic order/macroscopic disorder (MOMD) model of restricted rotational diffusion. The study has shown that the diffusion coefficient characterizing the rotational motions of the polyacid backbone is significantly smaller at low degree of dissociation at pH 4 than at high degree of dissociation at pH 7 and pH 10.

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polyelectrolyte multilayers (PEM)10 and demonstrated the application of a spin-labeled polyanion as a reporter molecule to study the complex coacervation of oppositely charged polyelectrolytes depending on the mixing ratio of the polycation and the polyanion.9 It was found that, if the spinlabeled polyanion is the excess component, the spectrum of a slow-motion component is superimposed by the spectrum of a fast-motion component. This indicates that the spin labels are located both in the core and in the shell of the PEC particles. In the opposite case, if the polycation is in excess, the spectra are dominated by a slow-motion component indicating that nearly all spin labels are located in the core. The present paper deals with the influence of pH and temperature on the rotational mobility of chain segments of a spin-labeled polyacid in complexes formed with an oppositely charged polycation.

INTRODUCTION The formation of polyelectrolyte complexes (PEC), prepared by mixing of oppositely charged polyanions (PA) and polycations (PC), is controlled by chemical structure of the polyions, concentrations, mixing ratio, mixing order, ionic strength, pH, temperature, and other factors. Stable colloidal dispersions of PEC particles are formed when the concentrations are low and one of the components is taken in excess, resulting in a net charge of the PEC particles. The state of the art of formation, structure, and properties of PEC has been the subject of some reviews.1−5 Electron paramagnetic resonance (EPR) spectroscopy is a powerful technique for the study of both structure and dynamics of paramagnetic species such as free radicals. Most polymeric materials, however, are diamagnetic and thus do not exhibit EPR signals. The absence of signals from the polymeric material provides the opportunity to apply EPR as a selective probe technique, whereby stable paramagnetic species such as nitroxide radicals are introduced artificially. These radicals are called spin labels (SL) if they are covalently linked to the macromolecules.6,7 Rotational dynamics of such spin labels on time scales between 10 ps and 1 μs can be characterized by the fast and sensitive continuous wave (CW) EPR spectroscopy, analyzing the line shape. The rotational dynamics of the spin labels is influenced by the restricted motion of the side group bearing the spin label and local polymer backbone motions at the point of the covalent linking of the spin label.8 Various papers dealing with the characterization of systems containing polyelectrolytes by EPR spin-label and spin-probe techniques have been shortly summarized in ref 9. Recently, we have shown that the EPR spin-label technique can be used to study the formation and stability of © XXXX American Chemical Society

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EXPERIMENTAL SECTION

Materials. Poly(ethylene-alt-maleic anhydride) (P(E-alt-MAn)) with Mw = 100−500 kg/mol and 4-amino-2,2,6,6-tetramethylpiperidine 1-oxide (4-amino-TEMPO) were obtained from Sigma-Aldrich Corp. (St. Louis, MO). Poly(diallyldimethylammonium chloride) (PDADMAC) with Mw = 240 kg/mol was purchased from Polysciences Inc. (Warrington, PA). A spin-labeled poly(ethylene-alt-maleic acid) (SL-P(E-alt-MA)) with 2.5 mol % of spin-labeled repeat units was synthesized by the reaction of P(E-alt-MAn) with 4-amino-TEMPO in pyridine as described previously.10 P(E-alt-MAn) (10 mmol) was introduced into a flask with 23 mL of anhydrous pyridine and stirred at 50 °C under Received: March 5, 2015 Revised: May 7, 2015

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DOI: 10.1021/acs.macromol.5b00474 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules argon for 3 h. 4-Amino-TEMPO (0.5 mmol) was dissolved in 2 mL anhydrous pyridine and added to the solution of P(E-alt-MAn). The reaction mixture was stirred at room temperature under argon for 3 h. The polymer was isolated by precipitation into n-hexane (2 L). For purification, the polymer was dissolved in anhydrous THF (60 mL) and precipitated using n-hexane (2 L). Subsequently, the polymer was dried in vacuum for 24 h at room temperature. The chemical structures of the polyanion P(E-alt-MA) (without SL) and the polycation PDADMAC are shown in Scheme 1.

microscopic order/macroscopic disorder (MOMD) model of restricted rotational diffusion13,14 was applied for calculating the slow-motion spectra of the SL-P(E-alt-MA) in the PEC dispersions. The simple models of isotropic rotational diffusion and of anisotropic rotational diffusion with an axially symmetric diffusion tensor were used here for comparison only (see Figure 2). Simulations with the MOMD model were run on a Linux cluster. The rotational diffusion of the spin labels in SL-P(E-alt-MA) was approximated by superposition of an isotropic rotational diffusion of the polymer chain segment with the rotational diffusion coefficient RS, and the internal rotation of the spin label with the rotational diffusion coefficient RI. It is assumed that RS = Rprp and RI = Rpll − Rprp, whereby Rpll and Rprp are the parallel and perpendicular rotational diffusion coefficients of the axially symmetric rotational diffusion tensor. The axis of internal rotation zR is tilted relative to the zm axis in the xmzm plane of the nitroxide axis system and specified by angle βD (Scheme 2). The restricted motion of the SL bonded to the polymer backbone is described by the orienting potential coefficients c20, c22, and c40.

Scheme 1. Chemical Structures of (a) Poly(ethylene-altmaleic acid) (P(E-alt-MA)) and (b) Poly(diallyldimethylammonium chloride) (PDADMAC)

Scheme 2. Structure of the SL-P(E-alt-MA)a

Preparation of Polyelectrolyte Complexes. In the first step, stock solutions of 7.5 mmol/L PDADMAC and 5 mmol/L SL-P(E-altMA) in Na2B4O7/NaOH (pH 10), K2PO4/Na2HPO4 (pH 7), and C6H8O7/NaOH/NaCl buffer solutions (pH 4) were prepared, with concentrations quoted with respect to the repeat units of the polymers. In the second step, stable dispersions of polyelectrolyte complexes with a mixing ratio nPC/(2nPA) = 1.5 in buffer solutions of pH 10, 7, and 4, respectively, were prepared by dropwise addition of 2.5 mL of SL-P(E-alt-MA) solution to 5 mL of PDADMAC solution under stirring. After mixing, the dispersions were further stirred for 30 min. The pH of the solutions was adjusted by buffer solutions rather than of simple acids or bases in order to keep the pH during complex formation constant. Without buffer the pH decreases by complex formation, because protons are released from the polyacid into the solution as a result of the screening of the negatively charged groups by the strong polycation. EPR Spectroscopy. CW EPR spectra were recorded on an EMXplus spectrometer (Bruker Biospin) operating at X-band, and equipped with the high-sensitivity resonator ER 4119 HS-W1, and the variable temperature unit ER4141VT. Liquid samples were loaded into glass capillaries (i.d. = 0.85 mm, Blaubrand, intraMark). Rigidlimit spectra were obtained by recording spectra of frozen solutions in quartz tubes with i.d. = 3 mm at 123 K. Acquisition parameters were microwave power of 2 mW in the case of liquid samples and 0.1 mW in the case of frozen samples, respectively, modulation frequency of 100 kHz, modulation amplitude of 1 G, sweep width of 120 G, time constant of 10.24 ms, conversion time of 40.96 ms, 64 scans, and 1024 data points. The temperature was controlled within ±1 K. All samples were allowed to equilibrate for at least 10 min after reaching the desired temperature. Simulation of CW EPR Spectra. Spectra were simulated in Matlab utilizing the functions pepper and chili of the EasySpin software package.11,12 At first, the g and 14N hyperfine tensors were determined by analyzing the rigid-limit spectra of the frozen solutions measured at 123 K. Subsequently, the slow-motion spectra of the solutions and dispersions in the temperature range from 273 to 333 K were calculated whereby the values of the g and 14N hyperfine tensor components were fixed. The simulated spectra were fitted to experimental spectra using the Levenberg−Marquart minimization algorithm, which iterates the simulations until a minimum leastsquares fit to the experimental spectra was reached. Fits were performed with different sets of initial fitting parameters, which partly result in different local minima. Average values of the parameter were calculated including all fits with a root-mean-square-deviation (rmsd) according to rmsdFit < rmsdBestfit + 0.001, where the deviation is the difference between the experimental and the simulated spectrum. The spectra are normalized to the value of the maximum height of the middle-field signal (mI = 0). For the simulation of the spectra of the free SL the simple model of isotropic Brownian rotational diffusion was used. While, the

a

The magnetic axis zm of the nitroxide axis system lies along the axis of the pz orbital of the 14N atom, the xm axis is perpendicular to zm and lies along the N−O bond direction, and the ym axis is perpendicular to these. The axis of internal rotation zR is tilted by the angle βD relative to the zm axis in the xmzm plane.

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RESULTS AND DISCUSSION Spectra of the aqueous solution of the free SL as well as the SLP(E-alt-MA) and the PEC dispersion at pH 10 and a temperature of 273 K are shown in Figure 1. On the left side, the spectra are normalized to the value of the double integral, which is proportional to the radical concentration.

Figure 1. EPR spectra of the free spin label in the presence of the polyanion (free SL) as well as the spin-labeled polyanion in absence (SL-PA) and presence of the polycation (PC/SL-PA) at pH 10 and a temperature of 273 K. The spectra are normalized to the value of the double integral (a) and the value of the maximum (b), respectively. B

DOI: 10.1021/acs.macromol.5b00474 Macromolecules XXXX, XXX, XXX−XXX

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MOMD model of restricted rotational diffusion (3). A low fraction of free SL with isotropic rotational diffusion has been included in all three cases, i.e., the complex spectrum is simulated and fitted on the basis of two components: a slowmotion main component and a fast-motion minor component, whereby the ratio of the components is also fitted. The components of the A and g tensors of the SL, which are required for the simulations in the slow-motion regime, were determined from the rigid-limit spectrum of the PEC dispersion at pH 10 measured at a temperature of 123 K: Axx = 7.7 G, Ayy = 4.8 G, Azz = 37.8 G and gxx = 2.0087, gyy = 2.0063, gzz = 2.0020. Figure 2 displays that the simulations 1 and 2 do not fit the experimental spectrum properly. The experimental spectrum of the spin-labeled polyacid is reproduced only in a simulation using the MOMD model (3). PEC dispersions were prepared at three different pH values of 10, 7, and 4. The experimental spectra of the PEC dispersions measured at seven different temperatures in the range of 273−333 K are shown in Figure 3a−c. The dependence of the line shapes on temperature is due to changes in the rotational mobility of the SL. The experimental spectra of the PEC dispersion prepared at pH 4 show a higher apparent S/N ratio than the spectra of the PEC prepared at pH 10 and 7, respectively. It indicates that the lines become broader with decreasing pH because of decreasing mobility. As mentioned, a small fraction of free SL is clearly visible in all spectra. For all simulations the same values for the A and g tensors were used because it was found that the three different pH values have no influence on the tensor components within the experimental uncertainty. The results of the simulations are also plotted in Figure 3a−c. All of the experimental spectra are well reproduced by the simulations using the MOMD model for the main component and isotropic rotational diffusion for the minor component. The simulation and fitting of the experimental spectra result in a small fraction of about 2−3 mol % free SL (see Figure 2S in the Supporting Information). For the main component the simulations result in the parallel and perpendicular rotational diffusion coefficients Rpll and Rprp, the diffusion tilt angle βD, and the orienting potential coefficients c20, c22 and c40. The diffusion coefficients RS and RI were calculated from Rpll and Rprp as described in the Experimental Section. The fitted parameters for the three PEC dispersions are shown in dependence on temperature in Figure 4 and Figure 5, respectively. Figure 4 shows that for all temperatures and pH values the coefficient RS characterizing the motion of the polymer backbone is at least 1 order of magnitude smaller than the coefficient RI describing the internal rotation of the spin label. The coefficient RI is nearly independent of temperature and pH. The coefficient RS however decreases with decreasing temperature for all three pH values. There is no difference between pH 10 and pH 7 for the coefficient RS in view of the experimental uncertainty. However, the coefficient RS is significantly lower for pH 4 than for the two other pH values. The tilt angle βD is almost constant at about 70° ± 5° as shown in Figure 4. The SL is tethered to the backbone via three bonds (C−CO, CO-NH, and NH-C). Steric constraints are assumed to be around the first bond. The second one, an amide bond, is normally fixed. Therefore, the only bond which contributes to the internal rotation of the SL is the third one, as has been discussed by Pilar et al.17 Nearly the same value of βD =64° ± 2° has been found by Pilar et al.17 for a spin-labeled

Because the three samples have the same radical concentration of 0.04 mM, Figure 1a shows the spectra with an intensity ratio as it is the result of recording the spectra using the same acquisition parameters. In order to see clearly the differences between the three spectra, on the right side the spectra are normalized to the value of the maximum. All spectra shown in the following figures are normalized in this manner. The spectrum of the free SL shows the typical three-line pattern due to coupling of the free electron to the 14N nucleus as expected for nitroxide radicals. The spectrum of the SL-P(Ealt-MA) differs significantly from the spectrum of the free SL. The three narrow lines with nearly the same amplitude of the free SL have been altered due to the covalent linkage to three broader lines with different widths. Moreover, the complex formation with the oppositely charged polycation also has an influence on the EPR line shape. The spectrum of the PEC dispersion is a typical slow-motion spectrum. The differences between the three spectra are due to changes in the rotational mobility of the SL. Figure 1b shows that there is a small fraction of free SL clearly visible in the spectrum of the PEC dispersion. It is also visible at the high-field line in the spectrum of the SL-P(E-altMA). The simulation mentioned below results in a very low concentration of about 2 mol % free SL with respect to the covalently linked SL. Furthermore, the simulation of the complex spectrum of the PEC dispersion shown in Figure 1b based on a two-component fit, indicates clearly that the line shape of the minor, fast component is nearly identical with the line shape of the free SL (see Figure 1S in the Supporting Information). Small fractions of free SL in solutions of their spin-labeld polymers have been also described by other authors.15−18 Because the spectrum of the free SL is characterized by small line widths, the free SL can be included in the simulations as a minor component without problems. Figure 2 shows once again the spectrum of the PEC dispersion at pH 10 and 273 K compared with simulated spectra which were calculated based on three different models for the motion of the main component SL-P(E-alt-MA). Models used for simulations are the simple model of isotropic rotational diffusion (1), the model of anisotropic rotational diffusion with an axially symmetric diffusion tensor (2), and the

Figure 2. Experimental and simulated EPR spectra of the PEC dispersion at pH 10 and a temperature of 273 K. The model of isotropic rotational diffusion (1), the model of anisotropic rotational diffusion with an axially symmetric diffusion tensor (2), and the MOMD model (3) are used for simulations. C

DOI: 10.1021/acs.macromol.5b00474 Macromolecules XXXX, XXX, XXX−XXX

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Figure 4. Rotational diffusion coefficients RS and RI and diffusion tilt angle βD of the main component in the PEC dispersions at given pH as a function of inverse temperature T−1.

Figure 5. Orienting potential coefficients c20, c22, and c40 of the main component in the PEC dispersions at given pH as a function of inverse temperature T−1.

⎛3 6 c 20 ⎞ cos2 Θcone = ⎜ − ⎟ 35 c40 ⎠ ⎝7

(1)

A decrease of c20 with decreasing temperature at constant c40 implies that the cone angle increases. In view of the experimental uncertainty of c20 and c40 as well as the propagation of uncertainty a dependence of the cone angle on temperature is unverifiable. The coefficient c22 characterizing rhombic distortion of the orienting potential was found to be almost constant at about 2. The effect of the orienting potential coefficients on the simulated spectrum is shown in Figure S3 in the Supporting Information for the PEC dispersion at pH 10 and a temperature of 273 K as an example. Figure S3b shows that the line shape is very sensitive to the coefficient c22, and less sensitive to the other two coefficients.

Figure 3. Experimental and simulated EPR spectra of the PEC dispersion at given temperature T and (a) pH 10, (b) pH 7, and (c) pH 4.

polystyrene (SL-PS), whereby the side group bearing the SL is identical to the side group in the SL-P(E-alt-MA) studied here. Miwa et al.19 have also studied SL-PS with the same structure of the side group and found a tilt angle βD =63° ± 3°. The values for the coefficients c20, c22, and c40 characterizing the shape of the orienting potential are shown in Figure 5. It was found that the three orienting potential coefficients are independent of the pH within the experimental uncertainty. The coefficient c20 slightly decreases with decreasing temperature. The combination of c20 and c40 coefficients with negative values for c40 indicates a conical distribution of the director axis as it is expected for spin-labeled macromolecules.14 The halfangle of the cone is determined by the ratio of c20 and c40 according to eq 1 as described by Earle and Budil:14

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CONCLUSIONS The polyacid P(E-alt-MA) has been spin-labeled and used to study the mobility of the polyacid chain segments in complexes formed with a oppositely charged polycation in dependence on pH and temperature. It was found that only the MOMD model fits the experimental spectra of the spin-labeled polyanion in the PEC dispersions well. The formation of polyelectrolyte complex coacervates is known to lead to polydisperse systems of nearly spherical particles, which consist of a charge-neutralized core, surrounded by an electrostaticcally stabilizing shell of the excess D

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Macromolecules component.2 These aggregated structures consist of many polyelctrolyte molecules, and the average particle size is in the range of 100−400 nm,5 thus the overall tumbling motion of the complex particles can be neglected. The polycation PDADMAC is in excess in the complexes prepared. For this reason, the SL-P(E-alt-MA) is only located in the core, and gives rise to a slow-motion spectrum.9 The study has shown that the diffusion coefficient RS characterizing the motion of the polymer backbone is significantly lower at pH 4 than at pH 7 and pH 10. One important property of weak polyacids such as P(E-alt-MA), which is associated with the pH, is the degree of dissociation αD. The P(E-alt-MA) is a weak diprotic polyacid and has two dissociation constants of pK01 = 4.0 and pK02 = 6.3 according to ref 20. The degree of dissociation αD is calculated based on these two constants to be 0.3 at pH 4, 0.9 at pH 7, and 1.0 at pH 10. Full dissociation at pH 10 and nearly full dissociation at pH 7 give rise to the same diffusion coefficient RS within the experimental uncertainty. At low degree of dissociation at pH 4 the coefficient RS is lower, indicating a slower rotational diffusion of the polyacid backbone. This finding may be related to the formation of inter- and intramolecular hydrogen bonds of the dicarboxylic repeat units in P(E-alt-MA). At low pH the polyacid backbone is assumed to be stabilized by hydrogen bonds, because only a part of the COOH groups is dissociated. The hydrogen bonds restrain the rotational mobility of the backbone. With increasing pH the concentration of nondissociated COOH groups decreases and hydrogen bonds are disrupted. Consequently, the polymer chains are more flexible at high pH than at low pH.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

(6) Hinderberger, D. EPR Spectroscopy in Polymer Science. In EPR Spectroscopy, Drescher, M.; Jeschke, G., Eds.; Springer: Berlin and Heidelberg, Germany, 2012; Vol. 321, pp 67−89. (7) Jeschke, G. Curr. Opin. Solid State Mater. Sci. 2003, 7, 181−188. (8) Hinderberger, D.; Jeschke, G. Site-specific Characterization of Structure and Dynamics of Complex Materials by EPR Spin Probes. In Modern Magnetic Resonance; Webb, G., Ed.; Springer: Dordrecht, The Netherlands, 2006; pp 1529−1537. (9) Lappan, U.; Wiesner, B.; Scheler, U. Macromol. Chem. Phys. 2014, 215, 1030−1035. (10) Lappan, U.; Wiesner, B.; Zschoche, S.; Scheler, U. Appl. Magn. Reson. 2013, 44, 181−188. (11) Stoll, S.; Schweiger, A. J. Magn. Reson. 2006, 178, 42−55. (12) Stoll, S.; Schweiger, A.; Hemminga, M. A.; Berliner, L. J. Easyspin: Simulating CW ESR spectra. ESR Spectrosc. Membr. Biophys. 2007, 27, 299−321. (13) Meirovitch, E.; Nayeem, A.; Freed, J. H. J. Phys. Chem. 1984, 88, 3454−3465. (14) Earle, K. A.; Budil, D. E. Calculating Slow-Motion ESR Spectra of Spin-Labeled Polymers. In Advanced ESR Methods in Polymer Research, Schlick, S., Ed. John Wiley & Sons, Inc.: Hoboken, NJ, 2006; pp 53−83. (15) Pilar, J.; Labsky, J. Macromolecules 1991, 24, 4188−4194. (16) Pilar, J.; Labsky, J. Macromolecules 1994, 27, 3977−3981. (17) Pilar, J.; Labsky, J.; Marek, A.; Budil, D. E.; Earle, K. A.; Freed, J. H. Macromolecules 2000, 33, 4438−4444. (18) Pilar, J.; Labsky, J. Macromolecules 2003, 36, 913−920. (19) Miwa, Y.; Shimada, S.; Urakawa, O.; Nobukawa, S. Macromolecules 2010, 43, 7192−7199. (20) Delben, F.; Paoletti, S.; Porasso, R. D.; Benegas, J. C. Macromol. Chem. Phys. 2006, 207, 2299−2310.

S Supporting Information *

Figure S1, two-component fit of the spectrum of PC/SL-PA at pH 10 and 273 K; Figure S2, fraction xfast of fast-motion component in the PEC dispersions at given pH as a function of inverse temperature T−1; and Figure S3, effect of parameter variation on the simulated spectrum of PC/SL-PA at pH 10 and 273 K. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.macromol.5b00474. Corresponding Author

*E-mail: [email protected]. Telephone: +49 (0)351 4658 366. Fax: +49 (0)351 4658 231 (U.L.). Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Commun. 2000, 21, 319−348. (2) Thunemann, A. F.; Müller, M.; Dautzenberg, H.; Joanny, J. F. O.; Lowen, H. Polyelectrolyte complexes. In Polyelectrolytes with Defined Molecular Architecture II; Schmidt, M., Ed.; Springer-Verlag Berlin: Berlin, 2004; Vol. 166, pp 113−171. (3) Koetz, J.; Kosmella, S. Polyelectrolytes and Nanoparticles; Springer: Berlin and Heidelberg, Germany, 2007. (4) Müller, M., Ed. Polyelectrolyte Complexes in the Dispersed and Solid State I: Principles and Theory; Springer: Berlin and Heidelberg, Germany, 2014. (5) Müller, M., Ed. Polyelectrolyte Complexes in the Dispersed and Solid State II: Application Aspects; Springer: Berlin and Heidelberg, Germany, 2014. E

DOI: 10.1021/acs.macromol.5b00474 Macromolecules XXXX, XXX, XXX−XXX