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Detection of Intramolecular Associations in Hydrophobically Modified Pectin Derivatives Using Fluorescent Probes A. Fischer,*,† M. C. Houzelle,† P. Hubert,† M. A. V. Axelos,‡ C. Geoffroy-Chapotot,§ M. C. Carre´,§ M. L. Viriot,§ and E. Dellacherie† Laboratoire de Chimie-Physique Macromole´ culaire, UMR CNRS 7568, ENSIC-INPL, 1, rue Grandville, BP 451, 54001 Nancy Cedex, France, Laboratoire de Physico-Chimie des Macromole´ cules, INRA, BP 71627, 44316 Nantes Cedex 3, France, and De´ partement de Chimie-Physique des Re´ actions, GRAPP, UMR CNRS 7630, ENSIC-INPL, 1, rue Grandville, BP 451, 54001 Nancy Cedex, France Received December 11, 1997. In Final Form: May 18, 1998 Hydrophobically modified pectin derivatives were prepared by immobilization of long alkyl chains (C12C18) at various substitution ratios, using two different synthetic pathways, one affording covalent fixation and the other one leading to a mere ionic association. These derivatives display an associative tendency in semidilute as well as in dilute aqueous solutions. This phenomenon, which stems from both intra- and intermolecular interactions between hydrophobic groups, results in the formation of hydrophobic microdomains. The latter can be characterized, especially in the dilute regime, thanks to fluorescence spectroscopy. Fluorescent molecular rotors, as well as pyrene, a classical fluorescence probe of widespread use, witness the variations of the medium polarity. In addition, they can also provide further information, particularly about the local cohesion of the microenvironment of the probe, without performing any complementary experiment, for example, the addition of quenchers together with the fluorescent probe in the polymer solutions. CAC values derived from polarity changes (CACpolarity), using the molecular rotor as well as pyrene as the fluorescence probes, are significantly different from those determined from the cohesion of the microenvironment (CACcohesion), accessible only with the molecular rotor. This latter type of fluorophore may therefore enable us to determine more accurately the actual critical aggregation concentration.
Introduction Among water-soluble polymers, hydrophobically modified derivatives of polyelectrolytes have triggered growing concern for the last 20 years. As a matter of fact, owing to their tendency to self-associate in aqueous medium due to both intra- and intermolecular interactions of the hydrophobic groups, these polymers offer outstanding properties as rheology modifiers. Among other applications, the use of amphiphilic associating polymers as thickening agents directly stems from these physicochemical properties.1,2 In our group, different studies dealing with solutions of hydrophobically modified propylene glycol alginate derivatives have already been reported.3-6 We focus here on pectin, another polysaccharide (Figure 1). As far as its chemical structure is concerned, pectin is predominantly a succession of 1 f 4 linked R-D-galacturonic acid monomer units, some of which are esterified by methyl * To whom correspondence should be addressed. † UMR CNRS 7568. ‡ INRA. § UMR CNRS 7630. (1) Schulz, D. N., Glass, J. E., Eds. Polymers as Rheology modifiers; ACS Symposium Series 462; American Chemical Society: Washington, DC, 1991. (2) Shalaby, S. W., McCormick, C. L., Buttler, G. B., Eds. WaterSoluble Polymers. Synthesis, Solution Properties and Applications; ACS Symposium Series 467; American Chemical Society: Washington, DC, 1991. (3) Sinquin, A.; Hubert, P.; Dellacherie, E. Langmuir 1993, 9, 3334. (4) Sinquin, A.; Hubert, P.; Dellacherie, E. Polymer 1994, 35, 3557. (5) Sinquin, A.; Hubert, P.; Marchal, P.; Choplin, L.; Dellacherie, E. Colloids Surf., A 1996, 112, 193. (6) Sinquin, A.; Houzelle M. C.; Hubert, P.; Choplin, L.; Viriot, M. L.; Dellacherie, E. Langmuir 1996, 12, 193.
Figure 1. Schematic structure of high-methoxyl citrus pectin (DM ) 78).
groups.7 This linear backbone is interrupted by some rhamnogalacturonan segments carrying neutral sugars as side chains.8 Originally a commercially available hydrophilic substance extracted from apple and citrus pomace, pectin can be transformed into an amphiphilic polymer by covalent fixation of long alkyl chains on the polysaccharide backbone. In dilute aqueous solutions, the modified (7) Cros, S.; Garnier, C.; Axelos, M. A. V.; Imberty, A.; Perez, S. Biopolymers 1996, 39, 339. (8) Renard, C. M. G. C.; Cre´peau, M. J.; Thibault, J. F. Carbohydr. Res. 1995, 275, 155.
S0743-7463(97)01362-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/15/1998
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Figure 2. Chemical formula of the molecular rotor 1,1-dicyano(4′-N,N-dimethylaminophenyl)-1,3-butadiene (DMAC).
polymer gives rise to intramolecular hydrophobic interactions resulting in the formation of microdomains. The aim of our study was to highlight this associative tendency thanks to fluorescence spectroscopy, a technique which has long proved efficient for the characterization of hydrophobic organized structures (micelles, polymer microdomains, ...) (for a review see for instance refs 9 and 10). Several chromophores have been used so far as fluorescent probes, being either merely introduced in the solutions, independently of the polymer, or directly attached to the latter. Among them, no doubt, pyrene has been the most investigated compound. Yet, for a few years, fluorescent probes of a new type have emerged, which also display an excellent ability to detect hydrophobic microdomains, that is molecular rotors.11,12 Fluorescent molecular rotors give indications about the polarity but also about the local cohesion of the microenvironment.13,14 The present article investigates the formation of hydrophobic microdomains in pectin and some of its hydrophobically modified derivatives prepared by two different synthetic pathways and compares their detections using two different fluorescence probes. Experimental Section Materials. High-methoxyl citrus pectin (degree of methylation (DM) ) 78; accordingly, the remaining carboxyl groups, approximately 22%, are a mixture of COOH and COO-Na+, the proportion of which has not been determined) ([η]0.025 M NaCl ) 577 mL/g) was provided by SBI (Baupte, France). Pyrene was purchased from the Community Bureau of Reference (No. 177). The molecular rotor 1,1-dicyano-(4′-N,N-dimethylaminophenyl)1,3-butadiene (DMAC, abreviation for the trivial name dimethylamino cinnamylidene) (Figure 2) was synthesized by a Knoevenagel’s reaction between 4-(N,N-dimethylamino)cinnamaldehyde and malononitrile with a 84% yield after recrystallization from ethyl acetate (mp ) 147 °C; lit.15 146-148 °C). Hydrophobically modified pectin derivatives were obtained by two different synthetic pathways: (1) For the pathway adapted from that of Della Valle,16,17 an aqueous solution (2% w/v) of pectin (pH 3.2) was neutralized by tetrabutylammonium hydroxide (TBA+OH-) up to pH 7. After freeze-drying, the resulting pectin salt was dissolved in dimethyl sulfoxide (2% w/v) and a long-chain (C12, C16, or C18) alkylbromide was introduced and left to react for 24 h. The reactivity yield was 45% for C12 chains and approximately 30% for C16 and C18 chains. The reaction mixture was allowed to dialyze for 7 days against water containing sodium azide as a bactericide. Freezedrying was carried out before the final product was left to react (9) Winnik, F. M. Chem. Rev. 1993, 93, 567. (10) Winnik, F. M.; Regismond, S. T. A. Colloids Surf, A 1996, 118, 1. (11) Benjelloun, A.; Brembilla, A.; Lochon, P.; Adibnejad, M.; Viriot, M. L.; Carre´, M. C. Polymer 1996, 37, 879. (12) Damas, C.; Adibnejad, M.; Benjelloun, A.; Brembilla, A.; Carre´, M. C.; Viriot, M. L.; Lochon, P. Colloid Polym. Sci. 1997, 275, 364. (13) Loutfy, R. O. In Photophysical and Photochemical Tools in Polymer Science; Winnik, M. A., Ed.; D. Reidel Publishing Company: Dordrecht, The Netherlands, 1986; p 429. (14) Rettig, W. Topics in Current Chemistry Springer-Verlag: Berlin, Heidelberg, 1994; Vol. 169, 253. (15) Matsuoka, M.; Takao, M.; Kitao, T.; Fujiwara, T.; Nakatsu, K. Mol. Cryst. Liq. Cryst. 1990, 182A, 71. (16) Della Valle, F. U.S. Patent 4,965,353, 1990. (17) Della Valle, F. European Patent, application no. 92400352-8, 1992.
Langmuir, Vol. 14, No. 16, 1998 4483 with sodium chloride (1 M in 70% ethanol, 4 °C, 24 h) in order to ensure a complete exchange of the residual TBA+ by Na+ ions. After filtration, the resulting derivative was washed with ethanol until any remnant of chloride ion was eliminated and finally washed with acetone. The resulting polymer was left to dry out under ambient temperature and pressure. The substitution ratio was determined after alkaline hydrolysis of an aliquot, followed by gas chromatography measurements. The nomenclature we use for the polymers prepared according to this pathway is PCnSx. P stands for pectin, Cn stands for the given immobilized alkyl chain (C12, C16, or C18), S stands for dimethyl sulfoxide, and x is the substitution ratio (in moles per 100 galacturonic units). (2) The other synthetic pathway, in dimethylformamide, was as follows, after Yalpani and Hall’s procedure.18 It consisted of the nucleophilic displacement of some of the pectin’s methyl esters by a given long chain alkylamine. The reaction was carried out in a heterogeneous medium, for 90 min at room temperature, in freshly distilled anhydrous dimethylformamide, with a 1/1 amine/ sugar unit molar ratio. The resulting polymers were purified by dispersion of the reaction mixture in absolute ethanol, followed by extensive washings with, successively, absolute ethanol, dioxane, and acetone, until a reliable constant substitution ratio, as determined by gas chromatography analysis of an aliquot after acidic hydrolysis, was obtained. In the resulting product, about 11% of the total galacturonic units were substituted. As evidenced by various controls, the description of which is out of the scope of the present article, it turns out, unexpectedly, that the immobilization of the considered alkylamine is mostly ionic (10% ionic association, 1% amide bond formation). The nomenclature we use for the polymers prepared according to this pathway is PCnFx with P, Cn, and x as above. F stands for dimethylformamide. Polymers named PS and PF are controls. They have experienced the entire reaction and purification procedures, except for the addition of the long chain alkylbromide or alkylamine, respectively, in the reaction mixture. They are aimed at investigating the intrinsic effect of experimental conditions (solvents, dialysis, freeze-drying, ...) on the physicochemical properties of the final products, irrespective of the hydrophobic substitution itself. A schematic representation of polymers prepared by both routes is given in Figure 3. Polymer solutions were prepared by dissolution of the polymer in ultrapure water (Milli-Q water purification system, Millipore). They were left under vigorous stirring for 18 h before the fluorescent probe was added and for 18 h afterward as well, to ensure the completion of the incorporation of the molecular rotor in microdomains. Methods. Absorption spectra were recorded on a PerkinElmer (lambda 5) UV/vis spectrophotometer. Fluorescence emission spectra were recorded on a Spex-fluorolog-2 spectrometer equipped with a thermostatically controlled cell at 25 °C. At fixed concentration of pyrene (1.2 × 10-6 M in the final solution), no excimer band was observed. All samples were excited at 335 nm, and the emission spectra of pyrene showed vibronic peaks at λ1 ) 372 nm (intensity I1) and λ3 ) 382 nm (intensity I3), with a slit width equal to 0.5 mm (∆λ1/2 ) 2 nm). As far as the rotor was concerned (concentration 5 × 10-6 M in the final solution), all samples were excited at 511 nm and the emission spectra were recorded in the range 530-650 nm (slit width ) 1.5 mm, ∆λ1/2 ) 6 nm). In all cases, the excitation generated one single fluorescence peak, the position of which was medium-dependent.
Results and Discussion Fluorescence Spectroscopy. Among the techniques encountered in the literature, fluorescence spectroscopy has often been used in the case of amphiphilic polymers in aqueous media to provide information about the change of the macromolecule conformation (e.g. formation of hydrophobic microdomains). The illumination of a chromophore by a radiation of proper wavelength results in (18) Yalpani, M.; Hall, L. D. Can. J. Chem. 1981, 59, 3105.
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Figure 3. Schematic representation of amphiphilic polymers (a) PCnSx prepared in a homogeneous medium (DMSO) and (b) PCnFx prepared in a heterogeneous medium (DMF): n, number of carbon atoms in the alkyl chain; x, substitution ratio in mol % alkyl chain per saccharide unit.
the excitation of the molecule. To return to the fundamental state, the latter emits a photon, the wavelength of which is superior to that of the exciting photon, since desexcitation also gives rise to nonradiative decays. Fluorescence phenomena consist of this photonic emission. In spectroscopic experiments, the chromophore is used as a probe. As a matter of fact, its fluorescence characteristics depend on the medium properties, such as polarity, local cohesion, and so forth. Because of both its long excited lifetime and its sensitivity to the microenvironment, pyrene has largely been used so far. Its monomer emission spectrum is composed of five vibronic peaks, the relative intensities of which highly depend on the solvent polarity (more particularly the I1/I3 ratio of the intensities of the first and the third peaks).19 In aqueous solutions of amphiphilic polymers, pyrene, as a hydrophobic probe, is likely to be found almost selectively in hydrophobic microdomains. Molecular rotors represent another type of fluorescent probe. This time, desexcitation occurs by at least two pathways: radiative decays, such as fluorescence, and nonradiative decays, such as isomerizations or mainly internal rotations of the molecule. The latter particularly depend on the medium viscosity in the surrounding of the probe: the more microdomains form, the more viscous the microenvironment is, the higher the fluorescence quantum yield ratio. As a matter of fact, fluorescence becomes all the more prevalent as the increase in viscosity in the microenvironment reduces the opportunities for the rotor to dissipate energy by internal rotations. As far as the wavelength is concerned, the more the rotor is surrounded by hydrophobic moieties or incorporated in hydrophobic microdomains, the more apolar its environment is; hence, the wavelength corresponding to the maximum fluorescence emission decreases. The rotor we used in our experiments was 1,1-dicyano-(4′-N,N-dimethylaminophenyl)-1,3-butadiene (DMAC) (Figure 2). Polarity Studies Using Pyrene and DMAC. As shown in Figures 4 and 5, an increase in either the hydrophobic substitution ratio or the chain length, for a constant substitution ratio, causes the I1/I3 value (pyrene) or the wavelength corresponding to the maximum fluorescence emission (rotor) to start diminishing at lower concentrations. As a matter of fact, the proportion of apolar parts rises when hydrophobic chains are longer or more numerous. From each curve λmax or I1/I3 versus polymer concentration, it is possible to determine the critical aggregation (19) Nakajima, A. J. Lumin. 1976, 11, 429.
concentration (CACpolarity) values in terms of polarity change. As a matter of fact, the tangent to the flat upper part of the curve (obtained for low concentrations) and the tangent to the sloping part (for higher polymer concentration) intersect at a concentration value which is commonly regarded as the CAC (yet, this procedure is somewhat controversial, and in various instances the CAC is, as an alternative, taken as the intersection of the sloping part of the curve with the lower plateau at high polymer concentration, when observed). The values obtained with both probes, taking into consideration the intersection of the sloping part with the upper plateau, are in relatively good concordance (Table 1). Yet, the hydrophobic character of both probes being highly dependent on their chemical structure, their respective interaction with the apolar moieties of the polymers is very likely different and the corresponding CAC values determined with both probes have accordingly no reason to be identical. Cohesion Study Using the Molecular Rotor. Owing to the dependence of fluorescence quantum yield Φf/Φf0 (where Φf0 represents the fluorescence quantum yield in water) on viscosity, molecular rotors can be used as cohesion fluorescent probes. Figure 6a shows the variations of the relative efficiency of the rotor Φf/Φf0 as a function of polymer concentration for P, PC12S0.6, PC16S0.6, and PC18S0.6. As expected, for the same substitution ratio, the longer the immobilized alkyl chain, the lower the concentration for which the hydrophobic microdomains and consequently the variation of Φf/Φf0 appear. Parts b and c of Figure 6 illustrate the influence of the substitution ratio in the cases of C12 and C18 chains, respectively. It is observed, as expected, that the formation of microdomains occurs for lower concentrations when the substitution ratio increases. From each curve Φf/Φf0 versus polymer concentration, it is also possible to obtain the critical aggregation concentration (CACcohesion) values in terms of local cohesion (Table 2) (graphically determined as the intersection of the plateau at low polymer concentration with the tangent to the sloping part of the curve). Owing to the absence of hydrophobic moieties in unmodified pectin (except the original methyl groups), one might expect, at first thought, that the variation of Φf/Φf0 should be a flat plateau, whatever the polymer concentration. However, Φf/Φf0 is observed to start increasing at a concentration around 1.5-2 g/L. The overlap concentration (C*) of unmodified pectin, that is the transition between dilute and semidilute regimes, is
Hydrophobically Modified Pectin Derivatives
Figure 4. I1/I3 ratio obtained with pyrene as a function of concentration of various pectin derivatives (plain water, 25 °C, [pyrene] ) 1.2 × 10-6 M).
approximately 5 g/L. The increase of Φf/Φf0 for unmodified pectin may therefore be due to the influence of the macroscopic viscosity of the solution, which is, after certain
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Figure 5. λmax obtained with DMAC as a function of concentration of various pectin derivatives (plain water, 25 °C, [DMAC] ) 5 × 10-6 M) (lines and arrows in part a are only guides to the eye, indicating how the various CAC values are determined, throughout).
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Table 1. Comparison between Pyrene and Molecular Rotor in Terms of Critical Aggregation Concentrations critical aggregation concn (g/L) in terms of polarity polymer
according to pyrene
according to rotor
P PC12S0.6 PC12S1.5 PC12S2.3 PC16S0.6 PC18S0.3 PC18S0.6 PC12F11
0.4 0.085 0.07 0.04 0.045 0.045 0.0025 0.02
0.4 0.1 0.08 0.045 0.035 0.05 0.0015 0.01
concentrations close to C*, very likely to play a significant role on the restriction to free rotation of the molecular rotor. In contrast, the situation is totally different with hydrophobically modified derivatives. If one considers the variation of Φf/Φf0 versus polymer concentration, the concentration at which Φf/Φf0 starts to increase (CACcohesion) is, for all amphiphilic derivatives, at least 1 order of magnitude lower than their corresponding overlap concentration (for the various derivatives, C* in the range 2-5 g/L). The onset of the Φf/Φf0 increase thus occurs in the dilute regime, far from C*, and the effect observed cannot therefore be interpreted in terms of a macroscopic viscosity increase. Of course, at higher polymer concentration, above C*, that is in the 5-10 g/L concentration range, it is very likely that the high Φf/Φf0 values observed are due to both cohesion of the microenvironment and, mainly actually, the macroscopic viscosity of the solution. The CAC value derived from local cohesion measurements (variation of Φf/Φf0) is lower for PC12F11 than for PC12S2.3. However, this value of 0.35 is higher than could be expected from the much higher substitution ratio for the former (11% compared to 2.3%). This phenomenon could be explained by the ionic character of the hydrophobic chain fixation in the case of PC12F11, whereas it is covalent for PC12S2.3. No clear-cut evidence can be given to account for the somewhat too loose microdomains observed in the case of ionic immobilization of the hydrophobic chains. As a possible explanation, however, this effect could be due to a temporary (dynamic) participation of these chains in the microdomains, owing to their ionic character, allowing some reversible disconnection from the polysaccharide backbone. In contrast, covalent immobilization would provide a more permanent (static) microenvironment which could then shelter the hydrophobic fluorescence probe more efficiently. Parts a and b of Figure 7 show the variation of Φf/Φf0 for PC12S2.3 and PC12F11, respectively, both in plain water and in 0.025 M NaCl. Concerning covalently linked C12 alkyl chains (PC12S2.3), the addition of salt results in some increase of the fluorescence quantum yield, indicating the reinforcement of the cohesion of the hydrophobic microenvironment. This is in agreement with general principles predicting the enhancement of hydrophobic associations when the ionic strength is increased.20 In opposition, when alkyl chains are immobilized via ionic interactions, some increase in the ionic strength must result in a partial disruption of the links between pectin COO- groups and NH3+-Cn alkyl chains. Hence, fewer long chain alkylamines are available to participate to the
Figure 6. Φf/Φf0 ratio obtained with DMAC as a function of concentration of various pectin derivatives (plain water, 25 °C, [DMAC] ) 5 × 10-6 M).
cohesion of the hydrophobic microdomains and the quantum yield must accordingly decrease. This is what is observed in Figure 7b in the case of the PC12F11 derivative.
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Table 2. Values of the Different Critical Aggregation Concentrations, as Determined with the Fluorescent Molecular Rotor critical aggregation concn (g/L) polymer
in terms of polarity (variation of λmax)
in terms of local cohesion (variation of Φf/Φf0)
P PS PC12S0.6 PC12S1.5 PC12S2.3 PC16S0.6 PC18S0.3 PC18S0.6 PF PC12F11
0.4 0.25 0.1 0.08 0.045 0.035 0.05 0.0015 0.15 0.01
1.5 1.5 0.8 0.75 0.7 0.6 1 0.07 1.5 0.35
Figure 8. Variation of λmax (a) and of Φf/Φf0 (b) with DMAC, as a function of concentration of native pectin and PC4S10, in 0.025 M NaCl (25 °C, [DMAC] ) 5 × 10-6 M) (in part b, the variation of Φf/Φf0 for PC12S2.3 is reproduced from Figure 7, for comparison purposes).
Figure 7. Φf/Φf0 ratio obtained with DMAC as a function of concentration of various pectin derivatives, in plain water and in 0.025 M NaCl (25 °C, [DMAC] ) 5 × 10-6 M): (a) PC12S2.3; (b) PC12F11.
Table 2 shows that the critical aggregation concentration is the same for P, PS, and PF, in terms of local cohesion, indicating that the reaction conditions exert no noticeable influence on the physicochemical properties of the final derivatives and that the effects observed are definitely
only due to the hydrophobic modification. Yet, lowmolecular-weight contaminants which are present in the original polymer (but which are not involved in microdomain formation) could account for the slight difference in CAC determined in terms of polarity. As a matter of fact, after washings in different solvents (ethanol, acetone, ...), polar byproducts are removed, which causes the polarity to change. It is particularly remarkable that CAC values derived from Φf/Φf0 measurements are much superior to those encountered in the study of polarity (almost 1 order of magnitude). The following explanation might be put forward: even before the probe is incorporated in a hydrophobic microdomain, it is likely to approach some of the apolar parts of the macromolecules, such as methyl groups or long alkyl chains. From then onward, the wavelength corresponding to the maximum fluorescence emission can decrease, since the microenvironment of the probe is becoming less and less polar. However, structured microdomains have not compulsorily appeared yet. Hence,
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the concentration for which their presence is detected in terms of local cohesion is much higher. If one refers to this hypothesis, one may anticipate that the behavior of derivatives substituted with short alkyl chains (in our case butyl groups in PC4S10) should be in close agreement with the following expectations: (1) Owing to the presence of about 10% of butyl chains in its structure, the PC4S10 derivative is substantially more apolar than the unmodified pectin. It may therefore be predicted that the plot λmax versus polymer concentration should start sloping down at an earlier concentration for this derivative than for the parent polysaccharide. (2) At a moderate substitution ratio, no organized hydrophobic microdomains are likely to form with alkyl chains as short as C4. Hence, the fluorescence nonradiative decay of the molecular rotor via internal rotations should not be restrained by the presence of the PC4S10 derivative (except a macroscopic viscosity effect) and the variation of Φf/Φf0 versus polymer concentration should accordingly superimpose rather closely with that of the parent pectin. Parts a and b of Figure 8 show that the results obtained are in close concordance with these expectations, and this experiment with PC4S10 therefore provides a convincing support to our assumption. This is the reason which triggered our interest for this type of probe, which gives us more precise information. Whereas the plot λmax versus polymer concentration (as well as the I1/I3 variation) is influenced by the polarity of (20) Tanford, C. The Hydrophobic Effect, 2nd ed.; J. Wiley: New York, 1980.
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the hydrophobic parts of the macromolecules (and the presence of contaminants of all kinds) even when microdomains are not formed, the plot of Φf/Φf0 versus polymer concentration enables us to focus on a somewhat more actual CAC. Nevertheless, the problem of aggregation is not so easily solved: it is to be noticed that preliminary aggregation phenomena can occur, which will not sufficiently restrain the internal rotations of the rotor embedded in the forming microdomains so as to induce an increase of the fluorescence efficiency. Conclusions The aim of this work was to characterize the microdomains formed by the hydrophobic parts of pectin and some of its derivatives obtained by fixation of long alkyl chains. We have been able to detect those microdomains thanks to the variations of several features involved in fluorescence spectroscopy. Furthermore, although pyrene could enable us to determine a critical aggregation concentration in terms of polarity, another type of probe, that is a molecular rotor, provided us further information about the CAC, in terms of local cohesion. This could be a more representative datum concerning the associative tendencies of polymers in dilute solutions. As a matter of fact, unlike indications dealing with polarity, which are likely to be influenced by apolar moieties in the macromolecules and accompanying contaminants, this concentration value directly witnesses changes in local cohesion and, consequently, microdomain formation. LA971362W
