Rb+ and Na+ Spin Relaxation in Aqueous Gellan Solutions and

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Rb+ and Na+ Spin Relaxation in Aqueous Gellan Solutions and Implication of Selective Site Binding of Alkali Metal Ions Masahiko Annaka,*,† Ryoko Takahashi,† Takayuki Nakahira,† Masayuki Tokita,‡ and Toyoaki Matsuura§ Department of Materials Technology, Chiba University, 1-33, Yayoi-cho, Inage-ku, Chiba-shi, Chiba 263-8522, Japan; Department of Chemistry for Materials, Mie University, 1515, Kamihama-cho, Tsu-shi, Mie 514-8507, Japan; Department of Ophthalmology, Nara Medical University, 840, Shijyo-cho, Kashihara-shi, Nara 634-8522, Japan Received November 7, 2000; Revised Manuscript Received April 30, 2001

NMR was applied to investigate the site binding of Rb+ ions in gellan gum gels. The temperature dependence of the transverse and longitudinal relaxation NMR relaxation rates of 87Rb+ ion and 23Na+ ion have been compared in aqueous 5% (w/v) rubidium-type and sodium-type gellan. In each sample, the relaxation rates were sensitive to the conformation (helix or random coil). In rubidium-type gellan, significant line-broadening effects (losses in intensity) were found, which is due to the presence of cation-binding sites in the ordered conformation. In sodium-type gellan, such an enhancement of the relaxation was not observed. These results indicated that gellan gum produced highly selective binding sites for alkali metal ions, in which Rb+ ion bound more strongly than Na+ ion. The 87Rb NMR line shift suggested selective site binding of ions to form the cross-linking domains in gellan gels. 87Rb

1. Introduction Gellan gum is an extracellular polysaccharide produced by Pseudomonas elodea composed of tetrasaccharide repeating units: 1,3-linked β-D-glucose, 1,4-linked β-D-glucuronic acid, 1,4-linked β-D-glucose, and 1,4-linked R-L-rhamnose (Figure 1).1,2 It is well-known that gellan gum forms a thermoreversible gel.3 Although the gelation mechanism has not been fully elucidated, the formation of ordered structure and subsequent alignment of the ordered region is considered to be responsible for gelation. Ions play an essential role in the molecular processes associated with conformational transition and gelation of gellan gum. Grasdalen and Smidsrød suggested that gel formation of polysaccharides is a twostep process, consisting first of an intramolecular transition to an ordered conformation and, second, the salt bridges between ordered segments of the chain.4 It is known that the gelation of gellan gum is markedly enhanced by the presence of group I cation in solution. The roles of group I cations in gelation and motional states in the vicinity of the sol-gel transition point are, however, not fully elucidated yet. Our previous work with sodium ion forms of gellan gum (1.5 wt %) containing NaCl, KCl, or RbCl showed that when a gellan gum solution had its temperature lowered to below the gelation temperature there was a marked apparent loss of in K+ or Rb+ NMR signal intensity whereas the NMR parameters of Na+ did not show any singular behavior.5 For K+ or Rb+ ion, this apparent loss was total and no signal * To whom correspondence should be addressed. Telephone:+81-43290-3409. Fax: +81-43-290-3401. E-mail: [email protected]. † Chiba University. ‡ Mie University. § Nara Medical University.

Figure 1. Chemical structure of the repeating unit of gellan gum.

was observed. These results were interpreted in terms of the difference in the interaction between cations and gellan gum. The line shape of K+ and Rb+ would broaden when the gelation occurs due to the restriction of the molecular motion of ions caused by a formation of junction zones. However, no conclusion was made as to the origins of these changes in motional behavior. To investigate these effects, we have studied the NMR relaxation of 87Rb (previously shown as a strongly gel-promoting ion in system that contain gellan gum5) in an aqueous sample of rubidium-type gellan together with the optical rotation, and have compared with the results with those obtained from 23Na in the in an aqueous sample of sodium-type gellan. 87Rb, despite its lower natural abundance, has more desirable NMR properties than 85Rb and, therefore, has been used almost exclusively for studies of the Rb+ ion in solutions. The quadrupole moment of this nuclei is equal to that of Na nucleus (0.12) but while the natural line width of the 23Na resonance in aqueous solution is ∼6 Hz, that of 87Rb is more than 100 Hz due to a large Steinheimer antishielding factor.6 Its relaxation in the solvated ionic state is entirely quadrupole dominated, and any interaction of the quadrupole moment with the field gradients arising from ionic charges and dipole moments in the polysaccharides will have a dominating influence on the appearance of the NMR

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spectrum.7 In addition, the large Rb+ ions are easily polarizable, giving comparatively large changes in the chemical shift when its ionic environment or its hydration shell is perturbed by binding to a polyanion. The relatively high sensitivity of 87Rb NMR, therefore, provides a convenient way of monitoring the binding of counterions to gellan gum, through observation on the counterion itself. 2. Experimental Section 2.1. Materials. Potassium-type and sodium-type gellan gum were kindly supplied by San-ei Gen FFI Co. (Osaka, Japan). NaCl and RbCl (Wako Pure Chemical Industries Ltd., Osaka, Japan) were used as supplied. 2.2. Sample Preparation. Potassium-type gellan gum was dissolved in 1.0 mol/L aqueous RbCl solutions at 70 °C, and then the solution was heated up to 90 °C to dissolve gellan completely. The gellan solution was dialyzed against water at 60 °C, and was lyophilized to give rubidium-type gellan. Rb content was estimated to be 3.43% in weight in dry matter. The powdered rubidium-type or sodium-type gellan was dissolved in salt aqueous solutions at 70 °C, and then the solution was heated up to 90 °C to dissolve gellan completely. Solutions were cooled to 25 °C and were kept at that temperature for 60 min to form gels. After gelation was completed, the gels were placed at 25 °C for 12 h. 2.3. Optical Rotation. The temperature-induced conformational transition of gellan gum investigated by NMR spectroscopy was monitored by optical rotation measurement at 589 nm using a Jasco DIP-370 polarimeter. The temperature was controlled by circulation of thermostatically regulated water through the jacketed 10 mm path length cell. 2.4. Multinuclear NMR. 87Rb and 23Na NMR spectra were obtained with a JEOL JNM-LA400 spectrometer equipped with a 9.39 T superconducting magnet. The 87Rb and 23Na resonance frequencies at this field were 131 and 106 MHz, respectively. Spectra were recorded using 8192 data points, a sweep width of 26 kHz, and a 90-pulse width of 16 µs for 87Rb, and 4096 data points, a sweep width of 3 kHz, and a 90-pulse width of 26 µs for 23Na. The probe temperature was changed from 25 to 90 °C with an interval 5.0 °C and was kept constant (0.5 °C by the passage of thermostatically regulated air during accumulation. Chemical shifts were referenced to the signal obtained from 1 mol/L aqueous RbCl (for 87Rb NMR spectra) or NaCl (for 23Na NMR spectra) solutions at 0.00 ppm as an external standard. Longitudinal relaxation rates (R1) were obtained from inversion-recovery experiments. Transverse relaxation rates (R2) were obtained from Lorentzian fits to the line-width, ∆ν1/2, using the following relation: R2 ) π∆ν1/2

(1)

The experimental errors in individual measurements are estimated to be less than 4% in both longitudinal (R1) and transverse relaxation rates (R2). 2.5. Determination of Melting Temperature. The melting temperature of gellan gum gels were determined by a falling ball method using a Teflon ball of 3.2 mm in diameter and 33 mg in weight. The melting temperature, Tm, of

Figure 2. Specific optical rotation in (a) rubidium-type gellan and (b) sodium-type gellan as a function of temperature (5% (w/v) aqueous solution, heating trace).

rubidium-type and sodium-type gellan are 75 and 35 °C, respectively. 3. Results and Discussions Figure 2 shows the temperature dependence of [R]D of 5% (w/v) aqueous rubidium-type and sodium-type gellan gums (heating process). The traces for rubidium-type (Figure 2a) and sodium-type gellan (Figure 2b) are similar: a large variation in [R]D at lower temperatures, due to the helix-tocoil transition of gellan, was followed by a plateau region where gellan molecules were completely converted into the coil conformation. However, the trace for the rubidium-type gellan displayed a shoulder in the vicinity of Tm. Therefore, the results shown in Figure 2 disclosed the following features of gellan. In the case of rubidium-type gellan, there may be the following four temperature regions (indicated in Figure 2a): region I, polymer networks are stable and insensitive to the temperature 50 °C < T), region II, the melting of double helices to form single coils, where isolated double helices converted into single coils 50 °C < T < 75 °C), region III, the dissociation of the domains composed of associated double helices 75 °C < T < 85 °C), and region IV, gellan exist as random coils (85 °C < T).3 Rubidiumtype gellan is in the gel states in regions I and II. However, the trace for sodium-type gellan did not show any shoulder, which corresponded to region II for the trace of rubidiumtype gellan. It is difficult for detail description of the conformational properties of gellan solution, which is still the subject of intense controversy.3,8-16 The small-angle X-ray scattering and the model calculation revealed the following features of gellan in aqueous KCl or CsCl solution: (i) the association of double helices of gellan was promoted at lower temperature, (ii) the high amounts of associated double helices still remained even at 60 °C, and (iii) the dissociation of these domains took place at higher temperature. The single chains, on the other hand, were the sole component at 60 °C in gellan in aqueous NaCl solution.

Rb+ and Na+ Spin Relaxation

Figure 3. Temperature dependence of the spectra of

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87Rb

and

23Na.

The step-by-step dissociation of the domains were also confirmed by the differential scanning calorimetry (DSC) as the multiple endothermic peaks above the initial melting point of double helices.17 These results indicate that the dissociation of domains composed of double helices takes place prior to the melting of the double helices into single coils. We observed the motional state of ions by means of multinuclear NMR: below the gelation temperature there was a marked apparent loss of K+ or Rb+ NMR signal intensity whereas the NMR parameters of Na+ did not show any singular behavior. The line shape of K+ and Rb+ would broaden when the gelation occurs due to the restriction of the molecular motion of ions caused by a formation of associated double helices.5 These observations are considered to explain the results shown in Figure 2, parts a and b. The temperature dependence of 87Rb and 23Na NMR spectra obtained from 5% (w/v) aqueous solutions of rubidium-type and sodium-type gellan are shown in Figure 3, parts a and b, respectively. The 23Na spectra show only slight increase in line width with decreasing temperature and no changes in intensity even at temperatures as low as 10 °C (not shown). On the other hand, 87Rb spectra show sharp changes in intensity. The behavior of the spectra is dependent on ion type. To interpret the spectra, it is necessary to consider the properties of the nuclei in some detail. 87Rb and 23Na have a spin of 3/2. The longitudinal and transverse quadrupolar relaxation processes for a spin 3/2 nucleus are biexponential.18 The decay of the transverse magnetization is given by eq 2 slow Mxy(t) ) M0[0.6 exp(-Rfast 2 t) + 0.4exp(-R2 t)]

(2)

slow are the fast and slow component rates where Rfast 2 and R2 of the transverse relaxation, respectively, which are given by eqs 3 and 4 2 Rfast 2 ) (2π /3)[J(0) + J(ω0)]

(3)

) (2π2/3)[J(ω0) + J(2ω0)] Rslow 2

(4)

where J(ω) is the spectral density function at the angular frequency ω, and ω0 is the resonance angular frequency. The

longitudinal relaxation process is often effectively exponential19 with a single relaxation rate given by eq 5: R1 ) (4π2/3)[0.2J(ω0) + 0.8J(2ω0)]

(5)

The above spectral density function is the Fourier transform of the time-correlation function that describes fluctuations in the coupling between the nuclear quadrupole moment and electric field gradients at the nucleus. In the simplest situation of an exponentially decaying correlation function, the spectral density function is a Lorentzian according to eq 6 J(ω) ) (3/10)χ2τc/[1 + (ωτc)2]

(6)

where χ is the coupling constant and τc is a correlation time that characterizes the fluctuation in the electric field gradient. In general, however, several dynamic processes on different time scales contribute to the averaging of the quadrupolar coupling. If all these processes are rapid compared to the resonance angular frequency, all spectral densities that enter the relaxation expressions become equal (the extreme narrowing case) and may be expressed in terms of an effective correlation time, τeff J(0) ) J(ω0) ) J(2ω0) ) (3/10)χ2τeff

(7)

Under extreme narrowing conditions, the transverse and longitudinal relaxation processes are both single-exponential ) Rslow ) R1). This is with the same rate constants (Rfast 2 2 generally the situation for the NMR relaxation of small ions in aqueous solutions of simple salts. For ions in macromolecular systems, however, the extreme narrowing condition does not always achieve. Large effects on the relaxation rates are observed when there is site-binding of the ion to a macromolecule, which results in long correlation time for the bound ions. For the spin 3/2 nuclei, three main spectral regions may be distinguished.20,21 These are extreme motional narrowing, where the lines are Lorentzian in shape; motional narrowing, where the spectra consist of two Lorentzian lines centered at about the same frequency;18 and a static region, where

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the spectra are very broad and generally consist of three separate lines. A further subregion may be distinguished between the extreme motional narrowing and motional narrowing regions. In this region, which is called intermediate motional narrowing, the line shape remains Lorentzian but its width becomes dependent on the Larmor frequency.22 In the motionally narrowed case, one line is generally much broader than the other and contains 60% of the total intensity.18 The remaining 40% is contained in the narrow line. The acquisition conditions used here are preferentially selected for the signal arising from the narrow component. Therefore, for rubidium-type gellan, the sharp change in intensity observed in the vicinity of melting temperature is due to a transition from the motional narrowing state to the extreme or intermediate motional narrowing situation. Lineshape changes, under the conditions of motional narrowing, occur when the spectral density at zero frequency is more intense than at the Larmor frequency. Under these conditions the line shape is a narrow Lorentzian superimposed upon a broader one, and the intensity of the latter is often lost.20 The spectra of rubidium-type gellan clearly show the presence of a broad component underlying the narrower components. This observation indicates that the condition motional narrowing holds. Belton, Morris, and Tanner indicated20,21 that line broadening can arise either from translational diffusion occurring at an appropriate rate through anisotropic regions or by interactions such as chemical bonding localizing the ion at a specific site on the polymer. The process of specific site binding implies the formation of a contact ion pair that perturbs the hydration shells. The translational diffusion process includes atmospheric condensation where a hydrated ion species is constrained to diffuse inside a certain volume surrounding the polymer.23 Chemical binding would be expected to cause large NMR chemical shifts whereas the nonspecific interaction implied by diffusion would not. Extrapolation of data to the limit of zero excess counterion gives a limiting chemical shift, relative to 87Rb ions in solution, of approximately -15 ppm. Hence it may be concluded that specific site binding occurs in rubidium-type gellan gel. The large difference in the specific binding of counterions are demonstrated by a comparison of the 87Rb spectra and 23Na spectra of gellan gum taken from all temperature regions specified above (Figure 3, parts a and b). There are large differences in both regions I (II) and III, whereas the spectra obtained in region IV are virtually identical. The spectra obtained for rubidium-type gellan gum in regions I, II, and III are composed of two components of different line widths, and consequently, there is an apparent loss of intensity. In these spectra, the width of the broad component was found to be an order of magnitude larger than that of the narrow component. In contrast, the spectra from sodium-type gellan did not display any apparent loss of intensity in any of the temperature regions. These differences in the appearance of the spectra of the two samples indicate that the significant line-broadening effects (losses in intensity) found for rubidium ions in gellan are due to the presence of cationbinding sites in the ordered conformation. The plots well

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Figure 4. Temperature dependence (on heating) of longitudinal (open circle) and transverse (solid circle) Rb+ ion and Na+ ion relaxation rates in 5 wt % samples of rubidium-type (a) and sodium-type (b) gellan. Dotted lines represent the relaxation rates in (a) 0.1 mol/L RbCl and (b) 0.1 mol/L NaCl. Vertical lines separate regions I, II, III, and IV.

correlate to the corresponding plots of [R]D (Figure 2, parts a and b) and the differences between rubidium-type and sodium-type gellan are more readily apparent in Figure 4. In the sodium-type gellan (Figure 4b), the helix-to-coil transition of gellan results in a larger decrease in both R1 and R2. And there is a larger difference between R1 and R2 as helical content increase (at lower temperatures). In contrast, above Tm, only weak temperature dependence is seen and R1 and R2 are nearly equal. Figure 4a shows R1 and R2 for rubidium-type gellan. The largest discrepancies between the relaxation rates of 87Rb and 23Na are observed for R2 within the temperature range between 50 and 75 °C, which corresponds well to the region of the transition step in [R]D observed in the region II in Figure 2. The observed transverse relaxation rates may be written as population weighted averages over the relaxation rate of bound (b) and free (f) ions according to eq 8, where pb is the fraction of bound ions. R2 ) (1 - pb)R2f + pbR2b

(8)

Equation 8 is valid in the limit of fast exchange,24 when the exchange of molecules between the bound and the free states is much faster than the difference in the respective intrinsic relaxation rates. 87Rb NMR signals from gellan gum in the gel-state indicate that the bound ions are so slow that the condition, τc . 1/2πν0 (ν0 is the resonance NMR frequency) is fulfilled, and the correlation time, τc, is greater than 10-9 s (ν0 ) 131 MHz). A motional restriction of at least 2 orders of magnitude for bound ions relative to free ions, for which

Rb+ and Na+ Spin Relaxation

Figure 5. Chemical shift of 87Rb for 5% (w/v) aqueous rubidiumtype gellan and 0.1 mol/L aqueous RuCl solution as a function of temperature. Vertical lines separate regions I, II, III, and IV, which correspond to the regions observed in Figure 2a and Figure 4a.

τc ≈ 10-11 s, is indicative of binding of the Rb+ ions at binding sites on the gellan gum, where ions reside for longer than 10-9 s. These results indicate that some of the Rb+ ions may be involved in binding sites on gellan molecules while others are relatively free.4 At this time, we cannot figure out the structure of the binding site of Rb+ ion; however, recent small-angle X-ray scattering studies and the model calculations17 suggested that these sites corresponded to cross-linking domains and structures of gellan, in which some of the functional groups arranged in such a way to fit the size of Rb+ ion. The residence time is nonetheless short enough to average out the chemical shifts for free and bound ions. The chemical shifts of 87Rb are shown in Figure 5 as a function of temperature for a sample containing 5% (w/v) aqueous rubidium-type gellan solution. Change in the 87Rb chemical shift well corresponds to the experimentally observed optical rotation and relaxation rates. Assuming the two-site model, the upfield shift relative to that at higher temperature (T < 50 °C corresponding to region I indicated in Figure 2a) may be proportional to the fraction of the bound ions, pb, which is related to the available binding site in gellan gum, i.e., the number of the cross-linking domains accompanied by site binding of Rb+ ion. Figure 6 shows the 87Rb NMR line shift as a function of the concentration of rubidium-type gellan gum at 25 °C. The upfield shift with increasing the available concentration of gellan gum is consistent with the behavior shown in Figure 5, increase in binding sites of Rb+ ion. As shown in Figure 7, the 87Rb NMR line shifts by addition of NaCl and RbCl to rubidium-type gellan gum clearly indicate the selective binding of the alkali metal. The observed downfield shift caused by the addition of NaCl suggests that the number of binding site for Rb+ ions increase. On the other hand, the upfield shift of 87Rb indicates that the addition of RbCl causes the fraction of bound Rb+ ions to decrease. The observed shift, σobs, is the weighted average of the contribution of the “bound” ions through an intrinsic shift, σ0, and of the “free” ion, whose resonance frequency is equal to that of aqueous RbCl solution and is taken as the arbitrary zero.25,26

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Figure 6. 87Rb NMR line shift as s function of the concentration of rubidium-type gellan solution at 25 °C. Equivalent Rb+ ion concentration is indicated on the top axis. The dotted line is a guide for the eyes.

Figure 7. Change in the 87Rb NMR line shifts by addition of NaCl (open circle) and RbCl (solid circle) to 5% (w/v) aqueous rubidiumtype gellan solutions at 25 °C. Dotted lines are guides for the eyes.

σobs ) )

[Rb+]bound 0 [Rb+]free σ + σRbCl [Rb+]total [Rb+]total [Rb+]bound 0 pb 0 σ ) σ RCp R

(9)

where R is the molar ratio of the Rb+ ions to charged groups on the gellan gum. To evaluate the fraction of bound Rb+ ions, pb, quantitatively one needs to know the value of σ0 for the system, which is not available on a priori grounds. The intrinsic shift for bound rubidium for complexes of Rb+ ion with various macrocyclic ligands enable us to estimate the fraction of bound ions (σ0 ≈ 50 ppm).27,28 The estimate increase in the fraction of bound ions, about 10% assuming the binding sites remain unchanged, may be explained by taking into account that Rb+ ions produce more cross-linking domain and probably more binding sites. 4. Conclusion The gelation of gellan gum produces highly selective binding sites for alkali metal ions, in which Rb+ ion binds

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more strongly than Na+ ion. The 87Rb NMR line shift suggests the selective site binding of ions to form the crosslinking domains in gellan gels. Acknowledgment. This work has been partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture. References and Notes (1) Jansson, P.; Lindberg, B.; Sandford, P. A. Carbohydr. Res. 1983, 124, 135. (2) O’Neill, M. A.; Selvenderan, R. R.; Morris, V. J. Carbohydr. Res. 1983, 124, 123. (3) Nishinari, K., Vol. Ed. Physical Chemistry and Industrial Application of Gellan Gum; Progr. Colloid Polym. Sci.; Springer: Berlin, 1999. (4) Grasdalen, H.; Smidsrød, O. Macromolecules 1981, 14, 229. (5) Annaka, M.; Honda, J.; Nakahira, T.; Seki, H.; Tokita, M. Progr. Colloid Polym. Sci. 1999, 114, 25. (6) Lindman, B.; Forsen, S. In NMR and the Periodic Table; Harris, R. K., Mann, B. E., Ed.; Academic Press: New York, 1978; Chapter 6. (7) Abragam, A. The Principles of Nuclear Magnetism; Clarendon Press: Oxford, England, 1961. (8) Ogawa, E. Macromolecules 1996, 29, 5178. (9) Ogawa, E. Progr. Colloid Polym. Sci. 1999, 114, 8. (10) Atkin, N.; Abeysekera, R. M.; Kronestedt-Robards, E. C.; Robards, A. W. Biopolymers 2000, 54, 195. (11) Bosco, M.; Miertus, S.; Dentini, M.; Segre, A. L. Biopolymers 2000, 54, 115.

Annaka et al. (12) Mazen, F.; Milas, M.; Rinaudo, M. Int. J. Biol. Macromol. 1999, 26, 109. (13) Kasapis, S.; Giannouli, P.; Hember, M. W. N.; Evageliou, V.; Poulard, C.; Tort-Bourgeois, B.; Sworn, G. Carbohydr. Polym. 1999, 38, 145. (14) Sworn, G.; Kasapis, S. Food Hydrocolloids 1998, 12, 283. (15) Milas, M.; Rinaudo, M. Carbohydr. Polym. 1996, 30, 177. (16) Lee, E. J.; Chandrasekaran, R. Carbohydr. Res. 1991, 214, 11. (17) Yuguchi, Y.; Urakawa, H.; Kitamura, S.; Wataoka, I.; Kajiwara, K. Progr. Colloid Polym. Sci. 1999, 114, 41. (18) Hubbard, P. S. J. Chem. Phys. 1970, 53, 985. (19) Halle, B.; Wennerstro¨m, H. J. Magn. Res. 1981, 44, 89. (20) Belton, P. S.; Morris, V. J.; Tannar, S. F. Int. J. Biol. Macromol. 1985, 7, 53. (21) Belton, P. S.; Morris, V. J.; Tannar, S. F. Macromolecules 1986, 19, 1618. (22) Halle, B.; Wennerstrom, H. J. Magn. Reson. 1981, 44, 89. (23) Berendsen, H. J. C.; Edzes, H. T. Ann. N.Y. Acad. Sci. 1973, 204, 459. (24) van Winkler, L.; Gutsze, A. AdV. Mol. Relax. Interact. Processes 1981, 21, 159. (25) Paoletti, S.; Delben, F.; Cesaro, A.; Grasdalen, H. Macromolecules 1985, 18, 1834. (26) Khazaeli, S.; Dye, J. L.; Popov, A. I. Spectrochim. Acta 1983, 39A, 19. (27) Shporer, M.; Luz, Z. J. Am. Chem. Soc. 1975, 97, 665. (28) Akitt, A. J. In Multinuclear NMR; Mason, J., Ed; Plenum Press: New York, 1987.

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