Micellization and Micellar Structure of a Poly ... - ACS Publications

Nov 15, 1997 - Romanian Academy, Institute of Physical Chemistry “I. G. ... 202, 77208 Bucharest, Romania, Institute of Organic Chemistry “C. D. N...
0 downloads 0 Views 282KB Size
Download PDF
6912

Langmuir 1997, 13, 6912-6921

Micellization and Micellar Structure of a Poly(ethylene oxide)/Poly(propylene oxide)/Poly(ethylene oxide) Triblock Copolymer in Water Solution, As Studied by the Spin Probe Technique Agneta Caragheorgheopol,*,† Horia Caldararu,† Ileana Dragutan,‡ Heikki Joela,§ and Wyn Brown| Romanian Academy, Institute of Physical Chemistry “I. G. Murgulescu”, Splaiul Independentei 202, 77208 Bucharest, Romania, Institute of Organic Chemistry “C. D. Nenitzescu”, 77208 Bucharest, Romania, University of Jyva¨ skyla¨ , Department of Chemistry, FIN-40351 Jyva¨ skyla¨ , Finland, and University of Uppsala, Department of Physical Chemistry, S-75121 Uppsala, Sweden Received May 2, 1997. In Final Form: September 17, 1997X The micellization of the triblock copolymer Pluronic P85 (poly(oxyethylene)27/poly(oxypropylene)39/poly(oxyethylene)27) in aqueous solution was followed vs temperature and addition of aliphatic alcohols, using the spin probe technique. Different types of probes properly chosen (spin-labeled (SL) poly(oxyethylene(4))nonylphenol, SL-Pluronic L62, TEMPO-laurate, TEMPO-hexanoate, CAT 4, CAT 8, CAT 11, CAT 16, and 5-, 7-, 10-, and 12-doxylstearic acids) provided information about the micellar structure (polarity, viscosity, and order degree) at different radial locations. Micellization was found to be low at room temperature, even for 10% aqueous solutions, but strongly increasing with temperature increase to about 323 K. Hydration of the poly(oxyethylene) (PEO) chains in the shell was found to diminish toward the interior and with increasing temperature. At an intermediate region of the shell, a hydration number ([H2O]/EO unit) close to 3 was found at 293 K, which decreased to ∼1 at 323 K. No hydration was found in a region corresponding to 4-6 EO units from the core. At the PPO/PEO interface, a further decrease in polarity occurred in the 323-373 K temperature range, probably due to an extension of the hydrophobic core with increasing temperature, with prevalence of the nonpolar conformations of the PPO and PEO chains. At 323 K, the order degree of the PEO chains decreased rapidly toward the interior of the shell. Addition of medium chain aliphatic alcohols to the aqueous solution of the polymer substantially enhanced micellization at room temperature. The effect increased with the alcohol chain lenght in the C4-C6 series. The addition of alcohols (C5, C6) was found to have similar effects with temperature increase, i.e., to promote micellization and to reduce the hydration and the order degree in the shell. In both cases the effects are due to an increased hydrophobe character of the core.

Introduction Recently, much attention has been devoted to the study of nonionic triblock copolymers of the poly(oxyethylene)x/ poly(oxypropylene)y/poly(oxyethylene)x (ExPyEx) type, especially regarding the influence of molecular structure, concentration and temperature on their aggregation behavior.1-7 Two recent reviews give thorough and penetrating coverages of the topics.2,8 It is now widely accepted that both the critical micellization concentration (cmc) and critical micellar temperature (cmt) decrease with an increase in PPO content or molecular weight. Complete phase diagrams have been established for typical members of this class, such as L649,10 and 25R411 in ternary †Romanian Academy, Institute of Physical Chemistry “I. G. Murgulescu”. ‡ Romanian Academy, Institute of Organic Chemistry “C. D. Nenitzescu”. § University of Jyva ¨ skyla¨, Department of Chemistry. | University of Uppsala, Department of Physical Chemistry. X Abstract published in Advance ACS Abstracts, November 15, 1997.

(1) Brown, W.; Schillen, K.; Almgren, M.; Hvidt, S.; Bahadur, P. J. Phys. Chem., 1991, 95, 1851. (2) Alexandridis, P.; Hatton, T. A. Colloids Surf. A 1995, 96, 1. (3) Brown, W.; Schillen, K.; Hvidt, S. J. Phys. Chem. 1992, 96, 6038. (4) Mortensen, K.; Brown, W. Macromolecules 1993, 26, 4128. (5) Mortensen, K. Europhys. Lett. 1994, 19, 599. (6) Gilatter, O.; Scherf, G.; Schillen, K.; Brown, W. Macromolecules 1994, 27, 6046. (7) Caragheorgheopol, A.; Pilar, J.; Schlick, S. Macromolecules 1997, 30, 2923. (8) Almgren, M.; Brown,W.; Hvidt, S. Colloid Polym. Sci. 1995, 273, 2. (9) Wu, G.; Zhou, Z.; Chu, B. Macromolecules 1993, 26, 2117.

S0743-7463(97)00450-2 CCC: $14.00

copolymer/water/xylene systems, disclosing a rich phase behavior with normal micellar, hexagonal, lamellar, bicontinous cubic, reverse hexagonal, and reverse micellar phasessresembling those of low molecular weight poly(oxyethylene) alkyl ethers (CiEj). However, at variance with the CiEj surfactants, the difference in hydrophile/ hydrophobe character of the two blocks is much lower and the resulting interface is broad and less well defined. The amphiphilic character of these copolymers changes substantially with temperaturesat 298 K the poly(oxypropylene) (PPO) part is no longer soluble in watersa fact that leads to interesting phase behavior in aqueous solution. One of the best investigated symmetrical triblock copolymers of this series is E27P39E27, commercially designed as Pluronic P85. There is a fairly complete c-T phase description of this polymer in water,8 resulting from small angle neutron scattering (SANS) and small angle X-ray scattering (SAXS) studies, ultrasound, and low sheer viscosity measurements.4,5 Extensive SANS and dynamic light scattering (DLS) studies have shown the coexistence of unimers, micelles, and clusters in proportions that depend critically on temperature and concentration.4 Micelles are formed at about c ) 5% over a broad temperature range above 298 K.1 At 313 K and above this temperature, micelles are present at all concentrations above 0.3%.1 The cmc and cmt values determined by (10) Alexandridis, P.; Olsson, U.; Lindman, B. Macromolecules 1995, 28, 7700. (11) Alexandridis, P.; Olsson, U.; Lindman, B. J. Phys. Chem. 1996, 100, 280.

© 1997 American Chemical Society

Micellar Structure of a PEO/PPO/PEO Triblock Copolymer

Alexandridis et al.12 using a dye solubilization technique are in accord with these data; the cmc at 298 K is 8.695 mM, which corresponds to 4% (w/w) P85. Analysis of the scattering function of the aggregates in terms of a hard-sphere interacting micelle4 has led to the determination of a core radius, Rc, which increases with temperature from 35 Å at 293 K to 50 Å at 323 K and a hard sphere interaction radius, Rhs, which is longer by 18 Å, the difference representing the thickness of the PEO shell. The hard sphere volume fraction vs T dependence for different concentrations has shown that, at low temperature the polymer is dissolved as a unimer, while above a critical micellization temperature, Tcm1, the volume fraction increases linearly with temperature until a saturation value is reached at Tcm2. Within the temperature range Tcm1-Tcm2, micelles are in thermodynamic equilibrium with unimers, while above Tcm2 unimers are probably present only in a very small number density. It was found also that close to Tcm1 the aggregation number is very small, close to unity, and increases following a N ∼ (T - Tcm1)0.6 relationship4 to N ∼ 200. It is the aim of this paper to explore the possibilities of the spin probe technique in the study of micellization and micellar structure of triblock copolymers, using as an example the rather well described Pluronic P85. The problem is more complicated as compared to other nonionic surfactants, such as CiEj, due to the small difference in hydrophilic/hydrophobic properties of the chemically different parts of the block copolymer and to the strong temperature dependence of these characteristics. Therefore, our approach was to first do a wide scale search for the best suited nitroxides to serve as spin probes in the P85 micelles; we have also used these data for a qualitative description of the temperature dependence of micellization. An important goal was to get structural information (polarity, viscosity, and order degree) on the micelles, using the selected spin probes. While there is some information on the polarity of the hydrophobic core,2 data on the local polarity (hydration) in the PEO chains and on their order in micelles of PEO type surfactants are not available. We have also followed the effects of medium chain alcohols on the micellization of P85 and measured the structural changes induced by the solutes. Medium chain alcohols have been found to preferentially dissolve in C12E5 bilayers, rendering them more hydrophobic.13 On the other hand, solubilization data of aromatic solutes in aqueous solutions of Pluronic block-copolymers suggested an aggregation promotion effect.14 High sensitivity differential scanning calorimetry measurements have evidenced the promoting effect of 1-butanol and 1-propanol on the micellization of Pluronic block copolymers.15,16 Experimental Part Materials. The block copolymer Pluronic P85 was obtained from Serva AG, Heidelberg, Germany, and used without further purification. It has a declared molecular weight of 4500 and 2200 for the PEO component and 2300 for PPO. Thus, the resulting formula is E27P39E27. The PEO used as reference samples were Carbowax 200 (Aldrich) and Carbowax 300 (Aldrich) (average mol wt ) 200 (12) Alexandridis, P.; Holtzwarth, J. F.; Hatton,T. A. Macromolecules 1994, 27, 2414. (13) Jonstromer, M.; Strey, R. J. Phys. Chem. 1992, 96, 5993. (14) Gadelle, F.; Koros, W. J.; Schechter, R. S. Macromolecules 1995, 28, 4883. (15) Cheng, Y.; Jolicoeur, C. Macromolecules 1995, 28, 2665. (16) Armstrong, J.; Chowdhry, B.; Mitchell, J.; Beezer, A.; Leharne, S. J. Phys. Chem. 1996, 100, 1738.

Langmuir, Vol. 13, No. 26, 1997 6913 and 300, respectively), and the PPO was from Aldrich (average mol wt ) 2000). The following alcohols have been used as additives: n-butanol (C4OH), n-pentanol (C5OH), and n-hexanol (C6OH). They were reagents of laboratory grade purity. Freshly prepared deionized water has been used. The following nitroxides have been used as spin probes. SpinLabeled L62, L62-NO. L62 is a triblock copolymer of the same family as P85, having the formula E6P30E6. The spin probe was prepared by labeling both terminal OH groups of L62 as follows.

L62–NO

Prior to spin labeling the commercial surfactant Pluronic L62 was purified by azeotropic distillation with benzene, followed by chromatography on neutral alumina (eluent: hexane/benzene, 1:1 (v/v), then chloroform) to remove water and a carbonyl impurity, respectively. Labeled surfactant L62-NO was obtained by reacting 3-(chlorocarbonyl)-2,2,5,5-tetramethylpyrroline-1oxyl17 and purified L62 (molar ratio 3:1) in benzene/chloroform (4:1 (v/v); 15 mL/g of L62) containing pyridine as the hydrogen chloride acceptor. After stirring for 3 h at room temperature and standing overnight the mixture was filtered with suction, the filtrate was chromatographed on basic alumina to remove the unreacted paramagnetic acid chloride (eluent: chloroform/ benzene, 20:80 (v/v)), and the eluate was concentrated in vacuo to give a yellow, viscous oil (yield 81%). IR spectrum (CCl4): υCsO 1105 (vs) and υCdO 1730 cm-1 (s); the absorption at υCdO (1775 cm-1) of the acid chloride was not present. NMR spectrum (δ (ppm), CCl4): 1.10 (d, 90 H, CH(CH3), 1.28 (s) (12 H) and 1.40 (s) (12 H) (pyrrolinic methyls), 3.42 (m) and 3.57 (m) (130 H, CH2O and (CH3)CHO). Spin-labeled poly(oxyethylene(4))nonylphenol, NPE4NO, was synthesized as previously described.18

NPE4 –NO

TEMPO-hexanoate, C6-NO, and TEMPO-laurate, C12-NO, were prepared according to the general procedure described by Waggoner et al.19

C6 –NO

C12 –NO

The series of CAT n (4-[N,N-dimethyl-N-(methylene)nammonio]-2,2,6,6-tetramethylpiperidine-1-oxyl iodide, where n ) 4, 8, 11, or 16)) spin probes, were purchased from Molecular Probes, Inc.

CAT n

x-Doxylstearic acids (where x ) 5, 7, 10, or 12) were purchased from Aldrich. (17) Rozantsev, E. G. Free Nitroxyl Radicals; Plenum Press: New York, London, 1970. (18) Caldararu, H.; Caragheorgheopol, A.; Dimonie, M.; Donescu, D.; Dragutan, I.; Marinescu, N. J. Phys. Chem. 1992, 96, 7109. (19) Wagonner, A. S.; Keith, A. D.; Griffith, O. H. J. Phys. Chem. 1968, 72, 412.

6914 Langmuir, Vol. 13, No. 26, 1997

Caragheorgheopol et al.

x–doxyl

Sample Preparation. Samples of P85 in water with concentrations between 5 and 20% (w/w)sthe concentration region characterized in the phase diagram to yield micelles (Figure 18)shave been prepared. Measured volumes of spin probe stock solutions in alcohol were carefully evaporated in glass vials; then adequate amounts of the aqueous solutions of P85, with the concentrations indicated in the text, were added so as to yield spin probe concentrations of ∼5 × 10-4 M. Solubilization of the probes was achieved by mechanical stirring and sometimes by gentle heating. With x-doxyl probes the surfactant solution was prepared in 0.1 M NaOH instead of water (see discussion in the text). Alcohols were added to the surfactant solution in the indicated proportion, on a volumetric basis. For calibration purposes a series of PEO/water mixtures have been also prepared, with w (w ) [H2O]/EO unit molar ratio) varying from 0.2 to 5. The introduction of the spin probes in these mixtures has followed the same procedure as above. ESR Measurements. The ESR spectra were recorded on an ESP 300 (Bruker) and on a JES-3B (JEOL) spectrometer, with 100 kHz field modulation in the X-band frequency. Variable temperature measurements were carried out with the instrument attachments. Precautions regarding the microwave power, as well as field modulation amplitude, have been taken to avoid line-broadening artifacts. The 14N isotropic hyperfine (hf) splittingsthe polarity sensitive parametershas been measured in comparison with that of Fremy’s salt (aN ) 13.0 G). Spectra at 150 K have been measured after quenching the samples in liquid nitrogen. The ESR measurements of the micellization effect of C4-C6 alcohols in 5% (w/w) aqueous solution of P85 at 293 K (Table 6), have been carried out in quantitative conditions, i.e., using equal amounts of spin probe in all samples, calibrated capillary sample tubes, and adequate instrumental precautions. The integrated intensity of the M ) -1 nitrogen hf line of the C12-NO spectrum in different samples (calculated as I∆H2 , where I is the peak height in the first derivative spectrum and ∆H is the peak-topeak width of the same line) was used to compare the amounts of solubilized C12-NO. The concentrations of the micelles were considered to be proportional to the concentration of solubilized C12-NO (Nm in Table 6) and were compared with a standard solution of the same radical in ethanol (Net in Table 6). Care was taken to avoid line-broadening by radical-radical interaction. The isotropic rotational correlation time τc has been calculated according to the formula20

τc ) (6.51 × 10-10)∆H(0){[h(0)/h(-1)]1/2 + [h(0)/h(1)]1/2 - 2}s where ∆H(0) is the line width in Gauss and h(-1), h(0), and h(1) are the peak heights of the M ) -1, 0, and +1 lines, respectively. This formula has been used even in those cases when the peak height ratio indicated departure from the isotropic tumbling, considering that for the purpose of qualitative description it is a fair approximation of the average τc ) (τ|τ⊥)1/2. The DebyeStokes-Einstein equation: τc ) 4πηr3/3kT (where r is the hydrodynamic radius of the tumbling entity) is considered to relate τc with the local viscosity, η. The order parameter S, for doxyl spin probes, is defined in terms of observed spectral parameters as

S)

A| - A⊥ Azz - (Axx + Ayy)/2

where Azz, Axx, and Ayy are the principal elements of the A tensor in the absence of molecular motion and A| and A⊥ are derived from experimental spectra. Order parameters corrected for (20) Stone, T. J.; Buckman, T.; Nordio, P. L.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1965, 54, 1010.

Figure 1. Schematic phase diagram for the P85/water system.8 differences in polarity21 were calculated using the following values for 5-doxylstearic acid:22 Azz ) 33.5 G, Axx ) 6.3 G, and Ayy ) 5.8 G.

Results and Discussion Micellization vs Increasing Temperature. Labeled Surfactants. Spin Probes L62-NO and NPE4 -NO. When L62-NO (5 × 10-4 M) is dissolved in a 10% aqueous solution of P85, an ESR spectrum at 283 K is observed (Figure 2 and Table 1) whose assignment to L62-NO in water is straightforward. At 303 K a composite spectrum appears, indicating that the probe is distributed between two locations, the exchange rate being slow on the ESR time scale (υex < 106 s-1). CuCl2 was added to the samples in order to “quench” the spectrum in water18 (Figure 2). The ESR parameters of the remaining spectrum are reported in Table 1. Comparison with the spectrum at 323 K of the same spin probe in poly(ethylene oxide) (PEO) (Carbowax 200) shows identical spectral parameters (Table 1). Thus, the second spectrum corresponds to the probe in the micelle, with its nitroxide group in the PEO part. In the PPO part a lower value is expected, since in pure PPO 2000 at 323 K the aN value is 0.2 G lower than that found in PEO. At 303 K micelles are present in a significant proportion. With increasing temperature, there is an obvious increase of the micelle spectrum/water spectrum ratio (Figure 2). Since the spin probe concentration is fixed, the described evolution of the spectra indicates a strong increase of the micelle concentration with increasing temperature. Considering the chemical structure of L62, with the PPO block of almost the same length as in P85, but with a much shorter PEO chain, we expect the PPO block of L62-NO to be intercalated in the PPO core with the label localized in the PEO shell, close to the PPOPEO interface. With NPE4-NO similar results were observed regarding both the temperature effect and the aN value (Figure 3, Table 1). This is not surprising if one considers the hydrophobic part of the spin probe to be associated with the PPO core. Then the label is in the same region as the nitroxide group of the L62-NO probe. The aN values of both spin probes, very sensitive to polarity changes, show no signs of hydration in this region and are constant over the 323-373 K temperature range. Spin Probes C6-NO and C12-NO. The data for C6-NO in Table 1 and Figure 4 show an evolution in 10% aqueous (21) Seelig, J. J. Am. Chem. Soc. 1970, 92, 3881. See also: Seelig, J. In Spin Lebaling I; Berliner, L. J., Ed.; Academic Press: New York, San Francisco, London, 1976; p 373. (22) Gaffney, B. J. In Spin Labeling I; Berliner, L. J., Ed.; Academic Press: New York, San Francisco, London, 1976, p 567.

Micellar Structure of a PEO/PPO/PEO Triblock Copolymer

Langmuir, Vol. 13, No. 26, 1997 6915 Table 1. ESR Spectral Parameters of the L62-NO, NPE4-NO, C6-NO, and C12-NO Spin Probes in Aqueous Solution of P85, as a Function of Temperature sample

spin probe

T (K)

aN (G)

P85 10%

L62-NO

Carbowax 300 P85 10%

NPE4-NO

P85 10%

C6-NO

Carbowax 200 Carbowax 300 P85 10%

C12-NO

283 323 348 373 323 283 323 348 373 283 293 298 303 323 363 373 303 303 283 293 303 323 348 373 323

15.9 14.7a 14.7a 14.7a 14.7 16.3 14.7a 14.7a 14.7a 16.9 16.9 a 15.8a 15.6 15.5 15.5 15.8 15.8 17.1b 16.0 15.9 15.7 15.5 15.5 15.7

Carbowax 300

τc (10-10 s) 2.3 2.9a 1.4a 0.6a 3.0 1.3 3.5a 1.4a 1.3a 1.4 1.2 a 4.6a 2.5 0.9 0.8 5.2 5.0 7.5 5.7 2.9 1.0 1.0 2.3

aTwo overlapped spectra; parameters measured after “quenching” the water spectrum with CuCl2. bWeak spectrum of three lines superposed on a broad line.

Figure 2. ESR spectra of the L62-NO spin probe in a 10% (w/w) aqueous solution of P85: (a-c) measured at the indicated temperatures; (d) the same as (b) after “quenching” with CuCl2 the spectrum of the probe in water.

P85 similar to that obtained by using L62-NO, with the difference that this spin probe has a higher tendency to associate with micelles than the labeled surfactants. Thus, starting from 298 K, the spin probe is distributed between water and micelles, the two spectra appearing in approximately a 1:1 ratio, while above 303 K, a single spectrumsin the micellesis observed, “quenching” with CuCl2 being unnecessary in the latter case. The aN value measured at 303 K indicates the same polarity as in Carbowax 200, but with increasing temperature, a slight decrease is noted. The observed spectral shape, with the height ratios h(+1) > h(0) of the nitrogen lines, characteristic for preferential rotation around the N-O axis,23 was observed for this nitroxide in high-viscosity media. A similar spectrum has been observed in Carbowax 200 at 303 K (Figure 4). Spin probe C12-NO is scarcely soluble in water, or in 10% aqueous P85. At 283 K, with very few micelles present, C12-NO forms its own micelles,24 yielding a characteristic broad line in the middle of the three-line spectrum (Figure 5), due to spin-spin interaction between closely packed radicals. A very weak three-line spectrum (23) Nordio, P. L. Chem. Phys. Lett. 1970, 6, 250. (24) Baglioni, P.; Ferroni, E.; Martini, G.; Ottaviani, F. J. Phys. Chem. 1984, 88, 5107.

(aN ) 17.1 G) is superposed, originating from the radicals in solution, the concentration corresponding to the low water solubility of the probe. With increasing temperature, the spectrum of the probe in P85 micelles appears (Figure 5). The ESR parameters are given in Table 1. At 323 K the spectral parameters are identical with those in a Carbowax 300 sample at the same temperature (Figure 4). At 303 K the line shape is practically identical with that of C6-NO, but at 348 K C6-NO has a somewhat more isotropic motion than C12-NO. The ESR parameters for the two probes are very close. The same decrease of aN value with increasing temperature is observed. At variance with L62-NO and NPE4-NO, the C6-NO and C12-NO probes show a gradual polarity decrease with increasing temperature, starting from the value corresponding to Carbowax at 303 K, to a significantly lower value at 373 K (aN ) 15.5 G), reaching a value close to the one found in PPO (aN ) 15.5 G at 323 K and aN ) 15.4 G at 373 K). The effect surpasses the polarity decrease of neat PEO with temperature (aN ) 15.8 G at 303 K, aN ) 15.7 G at 373 K), which was assigned to the prevalence of nonpolar conformations at higher temperatures. These results justify the assumption that both C6-NO and C12NO probes have their alkyl chains associated with the PPO core, resulting in the label location in the PPO/PEO interface region. A smooth polarity decrease in the core region with increasing temperature was also reported by Alexandridis et al.2 This observation might be related to the core radius increase, deduced from SANS and DLS data.4 A theoretical approach25 also predicts for Pluronic micelles an extension of the hydrophobic core with increasing temperature, due to advanced segregation of EO/PO segments, a mechanisms that should be very active in the PPO/PEO “border” region, where the labels are supposed to be located. On the other hand, this effect is not sensed at some radial distance from the interface, corresponding to the effective length of 4-6 EO units, where the polarity was found to be constant with tem(25) Linse, P. Macromolecules 1993, 26, 4437.

6916 Langmuir, Vol. 13, No. 26, 1997

Figure 3. ESR spectra of the NPE4-NO spin probe in a 10% (w/w) aqueous solution of P85: (a-e) measured at the indicated temperatures; (f) the same as (d) after “quenching” with CuCl2 the spectrum of the probe in water.

perature, as measured by both L62-NO and NPE4-NO probes. The consistency of these results confirms the assumptions regarding the locations of the spin probes. Polarity and Order Profiles in the Micellar Shell. x-Doxylstearic Acid Probes. The x-doxylstearic acid spin probes were used to monitor and measure the order degreesif presentsof the surfactant chains in the micellar shell. With the 5-, 7-, 10-, and 12-doxylstearic acids used, this information refers to the local order at a number of points at increasing depth along the micellar radius. For small micelles, rotation of the micelle as a whole,26-28 as well as lateral diffusion of the probe on the curved surface of the shell26-28 may interfere and alter order degree measurements. With P85, these effects can be discarded and the measured value corresponds to the “real” order degree. When using stearic acid probes one has to consider the pH-dependent dissociation equilibrium of the carboxyl group and the fact that the dissociated and the undisso(26) Wikander, G. G.; Johansson, L. B.-A. Langmuir 1989, 5, 728. (27) Lasic, D. D.; Hauser, H. J. Phys. Chem. 1985, 89, 2648. (28) Haering, G.; Luisi, P. L.; Hauser, H. J. Phys. Chem. 1988, 92, 3574.

Caragheorgheopol et al.

Figure 4. ESR spectra of the C6-NO spin probe: (a-d) in a 10% (w/w) aqueous solution of P85, measured at the indicated temperatures; (e) in Carbowax 300 at 303 K.

ciated forms prefer different locations.28,29 Thus, in 10% aqueous P85 5-doxylstearic acid yields composite spectra, consisting of a slower and a faster component. The “slow” spectrum is the only one that appears in alkaline solutions and is consequently ascribed to the anionic form; the “fast” spectrum seems to have the label in the core region (aN ) 14.3 G, as in PPO) and is correspondingly attributed to the less polar, undissociated form-an assignment supported also by the spectrum of 5-doxyldecane in the same system, which was practically identical to the fast component. In order to get reproducible results and to avoid composite spectra, we chose to work with alkaline solutions (0.1 N NaOH aqueous solutions). When comparison between neutral and alkaline solutions was possible, the differences found in the spectral parameters were not important. Phase boundaries may, however, be affected. In Table 2 the parameters of the spectra of 5-doxylstearic acid in 10% alkaline aqueous P85 solution, as well as the calculated order parameter,21 S, are reported, as a function of temperature. The spectra are unique, the spin probe detects well-ordered surfactant chains in the aggregates formed in a 10% surfactant solution, at room temperature (29) Caldararu, H.; Caragheorgheopol, A.; Vasilescu, M.; Dragutan, I.; Lemmetyinen, H. J. Phys. Chem. 1994, 98, 5320.

Micellar Structure of a PEO/PPO/PEO Triblock Copolymer

Langmuir, Vol. 13, No. 26, 1997 6917

Figure 6. ESR spectra of the x-doxyl probes in a 20% (w/w) aqueous solution of P85, measured at 323 K. Figure 5. ESR spectra of the C12-NO spin probe: (a-c) in a 10% (w/w) aqueous solution of P85, measured at the indicated temperatures; (d) in Carbowax 300 at 323 K.

Table 3. ESR Parameters of the Anisotropic Spectra of x-Doxyl Probes in a 20% (w/w) Aqueous Alkaline Solution of P85

Table 2. ESR Parameters of the Anisotropic Spectra of 5-Doxylstearic Acid in a 10% (w/w) Aqueous Alkaline Solution of P85 and S, the Order Parameter, as a Function of Temperature T (K)

A| (G)

A⊥ (G)

S

298 303 313 323 333 343 353 373

23.0 23.0 22.0 21.3 20.6 20.4 19.8 19.3

10.5 10.9 11.5 11.7 12.2 12.4 12.6 12.8

0.49 0.45 0.39 0.36 0.31 0.29 0.27 0.24

(298 K). As expected, S decreases with increasing temperature. The fact that slow motional spectra are observed is an indication that at all measured temperatures, the micellar rotation and/or lateral diffusion of the probe is an order of magnitude slower than 10-8 s. This is expected for micelles whose radii exceed 20-25 Å.26-28 The high-temperature (323 K) spectra of x-doxyl probes in 20% aqueous solution of P85 are typical of anisotropic rotation of the probe, with decreasing order in the 5-, 7-, 10-, 12-doxyl series (Figure 6, Table 3). At 293 K and below, however, the 2A| value of the x-doxyl probes in the same sample is almost constant, even

spin probe

A| (G)

A⊥ (G)

aN (G)

S

293 K 5-doxyl 7-doxyl 10-doxyl 12-doxyl

23.5 23.5 23.9 23.3

5-doxyl 7-doxyl 10-doxyl 12-doxyl 5-doxyl decane

21.3 20.3 17.9

10.1 10.1 9.7 9.7

a a a a

a a a a

323 K

c

11.7 12.0 12.5

14.9b 14.7b 14.3b 14.6c 14.5c

0.36 0.31 0.21

aNot calculated (see text). b Calculated as a ) 1/3(A + 2A ). N | ⊥ Measured directly.

increasing from the 5- to 7- and 10-doxyl probe (Figure 7, Table 3). Regardless of the position of the probe along the micellar radius, the appearance of the spectra corresponds to a slowing down of the motion about the long molecular axis of the probe.30,31 It has been shown30 that when the rate of rotation of the spin probe decreases under a certain limit, the order parameter formalism can no longer be (30) Mason, R. P.; Polnaszek, C. F.; Freed, J. H. J. Phys. Chem. 1974, 78, 1324. (31) Cannon, B.; Polnaszek, C. F.; Butler, K. W.; Eriksson L. E. G.; Smith, I. C. P. Arch. Biochem. Biophys. 1975, 167, 505.

6918 Langmuir, Vol. 13, No. 26, 1997

Caragheorgheopol et al.

Figure 8. Isotropic hf splitting, aN (at 370 K) vs Azz (at 150 K), for the 5-doxyl spin probe in PPO and various POE/water mixtures: (A) PPO; (B) PEO; (C) PEO/H2O, w ) 0.5; (D) PEO/ H2O, w ) 1. The 5d, 7d, 10d, and 12d points result from the Azz values of the corresponding x-doxyl spin probes, measured at 150 K, in a 20% (w/w) aqueous solution of P85, after quenching from 323 K.

Figure 7. ESR spectra of 5-, 7-, 10-, and 12-doxylstearic acids in a 20% (w/w) aqueous solution of P85, measured at 293 K.

used. Therefore, in this case, values for the order parameter, S, have not been calculated (Table 3). The rather high temperature at which this “freezing” of the surfactant chain mobility occurs (as compared to C12E5, for instance32 ), might be the result of the longer PEO chains with stronger hydration at room temperature and below, the H2O molecules acting as binders between EO chains; at higher temperatures, partial dehydration occurs33-36 resulting in a hydration profile (see below), and a decreasing order profile is observed (Table 3). Investigations in progress on other PEO type surfactants, with EO chains of different lengths, have shown this effect to be specific for surfactants with longer chains (Pluronic L64 (E13P30E13),37 Triton X-10032). With regard to the considerations above, the importance of determining local hydration values becomes obvious. An attempt was made to use for this purpose the aN (aN ) 1/3(A| + 2A⊥)) values extracted from the anisotropic doxyl spectra. However, there is a certain error connected with measuring the A⊥ from experimental spectra,21,22 which increases as the resolution of the spectra decreases, as happens in the 5-, 7-, 10-, and 12-doxyl series. Thus, the aN values in Table 3 are affected by an increasing error in the series 5-, 7-, and 10-doxyl. An alternative approach for the determination of aN values from anisotropic spectra relies on the presumed (32) Caldararu, H.; Caragheorgheopol, A.; Schlick, S. To be published. (33) Nilsson, P-G.; Wennerstro¨m, H.; Lindman, B. J. Phys. Chem. 1983, 87, 1377. (34) Kalstro¨m, G. J. Phys. Chem. 1985, 89, 4962. (35) Kalstro¨m, G.; Carlsson, A.; Lindman, B. J. Phys.Chem. 1990, 94, 5005. (36) Lindman, B.; Carlsson, A.; Kalstro¨m, G.; Malmsten, M. Adv. Colloid Interface Sci. 1990, 32, 183. (37) Caragheorgheopol, A.; Schlick, S. Submitted for publication.

Table 4. ESR Parameters of the 5-Doxyl Probe, Measured at 150 and 370 K, in the Calibration Samples and of x-Doxyl Probes, Measured at 150 K, in a 20% (w/w) Aqueous Solution of P85, after Quenching from 323 K sample

spin probe

Azz (G)a

aN (G)

weffd

PPO PEO PEO/H2O, w ) 0.5 PEO/H2O, w ) 1.0 P85 20%

5-doxyl 5-doxyl 5-doxyl 5-doxyl 5-doxyl 7-doxyl 10-doxyl 12-doxyl 5-doxyldecane

33.0 33.4 34.0 34.4 34.0 33.8 33.3 33.0 33.0

14.5b 14.7b 14.9b 15.0b 14.9c 14.8c 14.6c 14.5c 14.5c

0.5 0.2 0.0 e e

a Measured at 150 K. b Measured at 370 K. c Values from the calibration curve (Figure 8). d Values resulting from calibration curve (Figure 9). e aN value as in PPO.

proportionality between aN and Azz, the low-temperature limit of the A| value in the actual sample.38 For this purpose, the ESR spectra of the 5-doxyl probe in a series of PEO/water mixtures were measured at 150 K after rapid freezing in liquid nitrogen. A calibration curve (straight line) was obtained, relating high-temperature (370 K) aN values of 5-doxylstearic acid in PEO/water mixtures with different water contents with the Azz values of the same samples at 150 K (Figure 8 and Table 4). The Azz values of x-doxyl probes in 20% aqueous P85 were measured at 150 K too, after quenching from 323 K, and the corresponding aN values inferred from the calibration plot (Table 4). The delicate point of this procedure is related to the possible modification of the system at freezing. However, the fact that different aN values have been obtained for 5-, 7-, and 10-doxyls in the same micellar sample seems to be a strong indication that the nonuniform (38) Griffith, O. H.; Jost, P. C. In Spin Labeling I; Berliner, L. J., Ed.; Academic Press: New York, San Francisco, London, 1976; p 453.

Micellar Structure of a PEO/PPO/PEO Triblock Copolymer

Figure 9. Calibration curves of the isotropic hf splitting, aN, of 5-doxyl (O) and of CAT 4 (0) spin probes vs w for various POE/water mixtures.

water distribution was preserved. The freezing procedure also finds support in similar results on the water distribution obtained by Baglioni and Kevan with ESEM spectroscopy in frozen micellar systems.39 Compared to aN values obtained from the anisotropic spectra (Table 3), the values deduced from the aN vs Azz plot (Table 4) are rather close; the agreement is best for 5-doxyl, where the anisotropic spectrum is well resolved. Good agreement is also obtained for 12-doxyl and 5-doxyl decane, probes that have isotropic spectra at 323 K. These facts increase the degree of confidence in the measurement procedure. The aN values thus obtained can be used further to evaluate local hydration degrees (weff ) [H2O]/ EO unit), by using the calibration curve of aN vs. w for 5-doxyl in various PEO/water mixtures (Figure 9). The inferred weff values are listed in Table 4. The hydration reported by the 5-doxyl probe represents only 0.5 H2O/EO unit and decreases rapidly along the stearic acid chain (weff ∼ 0.0 at 10-doxyl location). These hydration values are surprisingly low, if one considers the COO- group anchored at the exterior limit of the shell, as in aggregates of ionic surfactants.21 At this point it should be mentioned that very similar results are obtained in the aggregates of other PEO type surfactants with long EO chains (Pluronics L64, Triton X-100), regardless of the chain length.32,37 In the case of C12E6 micelles with much shorter PEO chains, the 5-doxyl and 7-doxyl groups were found to be located at 4-5 Å and, respectively, at 1-2 Å from the core, while the 10-doxyl group was found to be 1-2 Å inside the core,39 all the probes sensing an almost apolar environment. All these observations led us to question the positioning of the doxyl probes in the aggregate of PEO type surfactants. PEO type surfactants have extended polar heads and, unlike the ionic surfactants with a narrow water/shell interface, they expose in their aggregates a broader region of hydrated PEO chains. Thus, the possibility appears for the doxylstearic acid probes, with their 18 carbon hydrocarbon chains, to have the COO- group closer to the (39) Baglioni, P.; Kevan, L. Heterog. Chem. Rev. 1995, 2, 1.

Langmuir, Vol. 13, No. 26, 1997 6919

limit of the hydrated region toward the nonpolar part, in order to avoid the nonfavorable contact of the hydrocarbon chain with the hydrated PEO chains. This positioning would explain the similarity of the order and hydration profiles, regardless of the PEO chain length for surfactants with longer PEO chains. The question then arises about the possible existence of more hydrated exterior regions of the micellar shell, missed by the doxyl probes, and about the way to measure the polarity in those regions. Cationic Spin Probes. A possible alternative solution might be to use as probes the nitroxides of the homologous CAT n series. The positioning of these amphiphilic probes is a result of the fine balance between the interactions of the cationic head group and those of the hydrocarbon “tail”. Since the head group is always the same and the “tail” increases in a regular way with n, their relative positioning is obvious. As compared to the 5-doxylstearic acid probe, even CAT 16 is expected to place the nitroxide moiety at a more exterior radial position; the shorter-chain CAT n probes even more so. Other advantages of using these probes: (i) there is no need for using alkaline solutions (see above); (ii) the isotropic hf splitting, aN, is directly measured from the spectra. Since the CAT n probes are water soluble, the problem of their distribution between water and micelles arises. There are no obvious line splittings on the spectra, but the appearance might be misleading for overlap of spectra whose parameters are close, as would be the case here. Therefore, the spectra were measured at three different concentrations (5, 10, and 20% (w/w)) of surfactant, i.e., at different concentrations of micelles, at 323 K, when most of the surfactant is micellized (Figure 10), and at room temperature (293 K). The corresponding aN values are presented in Table 5. At 323 K, in the CAT 16 spectrum, there is no variation of aN value with P85 concentration. For CAT 11, at 10% and 5% there seems to be a slight asymmetry in the M ) -1 nitrogen line. CuCl2 was added to quench the water component. After quenching, the aN value in the 5% solution became equal to the value in the 20% solution; this value will be considered the micellar aN value. In the case of CAT 8, the aspect of the resolved spectrum in 20% solution is characteristic for overlap of water and micellar spectra: the hf resolution details on the M ) 0 line appear different from that of the M ) +1 line, a fact which is not observed in water. The exchange between the two species is in the slow limit, since the hf lines are not broadened. The spectrum of CAT 4 strongly resembles the water spectrum, which is probably prevailing. For the hydration profile data only the aN values for CAT 16 and CAT 11, have been considered. The corresponding weff values (Table 5) obtained from the calibration curve (Figure 9) are 0.7 and 1.1, respectively. Thus, indeed, the doxyl nitroxides do not probe the most hydrated external micellar regions (compare weff values in Tables 4 and 5). At 293 K, in 20% (w/w) solutions, micelles are present in measurable proportions, as has been shown above, with doxyl probes. The spectrum of CAT 16 shows a slight increase of aN to 16.1 G as compared to that at 323 K (Table 5). More spectacular changes are shown by CAT 11: an apparent aN ) 16.65 G value for this probe (as compared to a value of 16.2 G at 323 K), a value close to that measured in solution, while the τc ) 5.8 × 10-10 s clearly indicates association with micelles. A slight asymmetry on the M ) -1 line also indicates overlap of the micellar and water spectrum. After subtraction of the latter, the measured micelle parameters are aN ) 16.45 G and τc ) 8.4 × 10-10 s. This result represents an important experimental evidence for a much stronger

6920 Langmuir, Vol. 13, No. 26, 1997

Figure 10. ESR spectra of the CAT n spin probes in a 20% (w/w) aqueous solution of P85, measured at 323 K.

hydration of the outer regions of the micellar shell at room temperature (weff ) 3.3) as compared to that at higher temperatures (weff ) 1.1). Dehydration with increasing temperature is a fact generally accepted for PEO type surfactants and recently measured by SAXS for a series of Pluronic surfactants.40 The data conveyed by even more hydrophilic probes (CAT 8 and CAT 4) at 293 K, similar to those found at 323 K, are affected by the contribution of a predominant water component. The fact, however, that the spectra have the appearance of unique lines is an indication that their parameters in the two media are very close. Thus, the locations of the monitor group in CAT 8 and CAT 4, respectively, in the micelles are even more hydrated. Effects of Alcohol Addition. Micellization Effect. Aliphatic alcohols with medium chain length and in different quantities enhance micellization. This effect is best revealed with the C12-NO spin probe in 5% P85 aqueous solution at 293 K. Since this probe is dissolved only in the micelles, its relative concentration (Nm/Net), as calculated from the micellar spectra, was used to measure the progress of the micellization process (Table 6). In the absence of the alcohols, a very low micellization level was observed. In the presence of alcohols, the spectrum of the spin probe in micellar aggregates appears with various intensities, depending on the alcohol used and its concentration. (40) Alexandridis, P.; Zhou, D.; Kahn, A. Langmuir 1996, 12, 2690.

Caragheorgheopol et al.

With gradual addition of n-hexanol, the micellar spectrum of C12-NO increases in intensity up to 2% (v/v) alcohol, when a saturation value is obtained. Samples with 3% of normal medium chain alcohols from C4-C6 were compared too. The data in Table 6 show a significant increase of the micellization effect in the C4, C5, C6 series. Structural Effects. The effects of alcohol solubilization on the micellar parameters sensed by the C12-NO probe consisted of a small gradual decrease of the polarity and a more pronounced decrease of viscosity (Table 6). The effects become more pronounced as the alcohol alkyl chain increases. This is probably related to the positioning of the alcohol molecules with the alkyl chain in the PPO core (C6-OH for instance, being similarly located as the C6-NO spin probe; see above) having a loosening effect on the packing of the chains in the PPO/PEO border region. Structural effects of the solubilized alcohols have been followed also with the CAT 16 and CAT 11 probes. In the micelles of 5% (w/w) P85 with 2% (v/v) hexanol, CAT 16 yields only a micellar spectrum, slightly different from that in the absence of alcohol, while CAT 11 presents two overlapping spectra; i.e., it is distributed between water and micelles, the exchange rate being slow. The micellar aN valuesmeasured after subtraction of the water spectrumsappears to be substantially lowered (from 16.45 to 16.10 G) (Table 5) in the presence of hexanol. This is an indication that in the presence of alcohol a very important dehydration effect occurs at the intermediate levels of the PEO shell. Once again, the addition of alcohol has effects similar to those of temperature increase. Effect of Alcohol Addition on the Order Degree. The data in Tables 7 and 8 show the influence of various amounts of linear alcohols added on the order degree, S, in the micelles formed. A very significant decrease of S with the amount of added alcohol is observed, denoting a loosening effect of the dissolved alcohol molecules on the ordering of the PEO chains. The presence of alcohols in the micelles also determines at room temperature (298 K) a decreasing order profile (Table 8), the “freezing” of the surfactant chains (see above) being relieved. The values of the order parameter S measured at 298 K with the highest content of n-hexanol are very close (Table 7) to those measured at a higher temperature (323 K) in the micelles without alcohols (Table 3). Thus, the loosening effect sensed by the C12-NO probe at the PPO/PEO border is transmitted farther into the shell. The polarity decrease reported by CAT 11 supports the hypothesis that these effects are also due to the dehydration of the PEO chains in the shell. Considering the hydrophile/hydrophobe character of the components and the information provided by the various spin probes, a model with the alkanols dissolved in the PPO core emerges; they produce an important loosening effect on the packing of the polymer chains in the core and at some distance from the PPO/PEO interface. There is also an important reduction of hydration in the intermediate region of the shell, as a result of alcohol solubilization. The addition of medium chain alcohols (C5, C6) seems to play a role similar to that of temperature increase, i.e., promotes micellization and reduces hydration of the shell. These results are in line with previous findings on the promotion effect of 1-propanol and 1-butanol on the micellization of Pluronic block copolymers.15,16 Results on reverse micelles of Pluronic L64 in binary aqueous and ternary L64/p-xylene/water systems7 have evidenced the important role of water in promoting micellization and the influence of the organic solvent on increasing water segregation in the PEO core. In an analogous way, hexanol promotes micellization in water, by increasing the hydrophobe character of the nonpolar

Micellar Structure of a PEO/PPO/PEO Triblock Copolymer

Langmuir, Vol. 13, No. 26, 1997 6921

Table 5. Isotropic hf Splitting, aN, and Rotational Correlation Time, τc, of CAT n Spin Probes in Aqueous Solutions of P85 of Different Concentrations and with Addition of 2% n-Hexanol and Effective Hydration Numbers, weff, Measured from the Calibration Curve (Figure 9) CAT 4 sample

aN (G)

τc

CAT 8

(10-10

s)

aN (G)

τc

CAT 11

(10-10

s)

aN (G)

τc

(10-10

CAT 16 s)

weff

aN (G)

τc (10-10 s)

weff

323 K P85 20% P85 10% P85 5%

16.65 16.70

c c

16.50 16.65 16.65

c c c

P85 20% P85 5% +C6OH

16.70

c

16.60

2.0

16.20 16.20a 16.20a

6.0 5.3a 5.3a

1.1 1.1 1.1

16.05 16.05 16.05

6.6 7.1 7.4

0.7 0.7 0.7

16.45b 16.10b

8.4b 10.1b

3.3 0.8

16.10 16.10

13.8 11.7

0.8 0.8

293 K

a

After quenching with CuCl2. b After subtraction of the “water” spectrum. c Resolved proton hfs; in this case τc was not determined.

Table 6. Isotropic hf Splitting, aN, and Rotational Correlation Time, τc, of the C12-NO Spin Probe (Measured at 293 K) in a 5% (w/w) Aqueous Solution of P85 with Addition of Medium Chain n-Alkanols alcohol added

% (v/v)

[alcohol]/[P85]a

aN (G)

τc (10-10 s)

Nm/Netb

C6OH

0.0 0.5 1.0 2.0 3.0 3.0 3.0

3.6 7.1 14.2 21.3 24.8 29.5

16.0 15.9 15.8 15.8 15.8 15.9 16.0

7.5 7.0 5.6 4.1 3.9 4.9 5.5

0.05 0.23 0.55 1.00 0.95 0.70 0.40

C5OH C4OH

a Molar ratio. b N /N represents the ratio of the intensities of m et C12NO spectrum in the micelle and in a 3 × 10-4 M ethanol solution.

Table 7. ESR Parameters of the Anisotropic Spectra of 5-Doxylstearic Acid in a 5% Aqueous Alkaline Solution of P85, Measured at 298 K, as a Function of Added Alcohol Concentration alcohol added C6OH

C5OH C4OH a

(%v/v) 0.0a 0.5 1.0 2.0 3.0 3.0 3.0

A| (G)

A⊥ (G)

S

23.0 22.7 21.9 21.7 21.5 21.3 22.8

10.5 10.5 11.3 11.5 11.8 11.8 11.3

0.47 0.46 0.42 0.38 0.36 0.35 0.42

Measured in 10% P85.

Table 8. ESR Parameters of the Anisotropic Spectra of x-Doxyl Probes in a 20% (w/w) Aqueous Alkaline Solution of P85 with Addition of 4% (v/v) of C6OH, at 298 K spin probe

A| (G)

A⊥ (G)

S

5-doxyl 7-doxyl 10-doxyl

21.7 20.9 a

11.3 11.4 a

0.39 0.36 a

a Spectra of three asymmetrical lines; parameters not measurable from this spectrum.

PPO part, and “repels” hydration water away from the PPO/PEO border. Conclusions All spin probes used confirm the c-T phase description of earlier studies. Thus, at room temperature with the most hydrophobic probes, micelles are first observed in a 10% solution. Their concentration strongly increases with

temperature up to about 323 K, when micelles are predominant. Addition of aliphatic alcohols C4-C6 promotes micellization, so that micelles are prevailing already at room temperature. Longer chain alcohols have a stronger effect. The different spin probes used report about the polarity and order degree at different radial positions in the micelles, yielding a consistent picture. Thus, 5-doxyldecane and 12-doxylstearic acid have the label located in the PPO core; the C6-NO and C12-NO spin probes are close to the PPO/PEO “border” and sense the polarity of unhydrated PEO, which decreases to the value found in PPO, when temperature increases. The L62-NO and NPE4-NO spin probes, with the label localized further from the PPO/PEO interphase, also sense the polarity of neat PEO, which remains constant in this region up to 373 K. With x-doxylstearic acid probes, the polarity profile at 323 K indicates local hydration corresponding to weff ) 0.5 H2O molecules/EO unit for the 5-doxyl probe, decreasing rapidly to 0.2 and 0.0 H2O/EO for the 7- and 10-doxyl probe, respectively. A decreasing order degree along the PEO chain is also noticed for these spin probes at the same temperature. However, with the more hydrophilic CAT 11 and CAT 16 spin probes, regions with higher degrees of hydrationswith values of 0.70 and 1.1 H2O/EO unit at 323 K, respectivelyshave been monitored. At 293 K the hydration of the shell is considerably larger: CAT 11 measures a local hydration value of 3.3 H2O/EO. Our data seem to indicate that the alcohols added are solubilized in the PPO core, since they modify the polarity and viscosity, as sensed by the C12-NO probe. Their presence is, however, also felt in the shell, reducing the order degree of the surfactant chains. A significant dehydration effect is also observed: at 293 K the weff measured by CAT 11 decreases from 3.3 to 0.8 H2O/EO. The addition of alcohols (C5, C6) plays a role similar to that of temperature increase, i.e., promotes micellization and reduces hydration of the shell. In both cases the effects are due to an increased hydrophobe character of the core. Acknowledgment. This contribution is a result of cooperation programs of the Romanian Academy with the Academy of Finland and with the Royal Swedish Academy of Sciences. Financial support from the Romanian Academy (Grant 127/1995) is gratefully acknowledged. LA970450D