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Gel–sol transition in κ-carrageenan systems: microviscosity of hydrophobic microdomains, dynamic rheology and molecular conformation
International Journal of Biological Macromolecules 26 (1999) 69 – 76 www.elsevier.com/locate/ijbiomac
Gel – sol transition in k-carrageenan systems: microviscosity of hydrophobic microdomains, dynamic rheology and molecular conformation Andreas Hugerth, Stefan Nilsson, Lars-Olof Sundelo¨f * Physical Pharmaceutical Chemistry, Faculty of Pharmacy, Uppsala Uni6ersity, Uppsala Biomedical Center, Box 574, S-751 23 Uppsala, Sweden Accepted 8 March 1999
Abstract The effect of gel–sol transition in k-carrageenan systems on the microviscosity of hydrophobic microdomains, as well as its relation to macroscopic rheology and molecular conformation, was studied in k-carrageenan systems. The microdomains were probed by 1,3-di(-1-pyrenyl)propane (P3P) for which the excimer intensity (Ie) provides relative measures of the microviscosity in the immediate probe surroundings. In particular the applicability of P3P to monitor the gel – sol transition was proved, the results showing a dramatic decrease in microviscosity in the vicinity of the transition point. The corresponding changes in rheological properties and carrageenan conformation were investigated by dynamic viscometry (DV) and optical rotation (OR), respectively. The temperature of onset of the transition as indicated by the microviscosity data (T0) was found to correlate well with the OR and DV-results. The application of microviscosity and OR-measurements allowed an estimation of the helical content at T0 to be determined. P3P-data indicate a microenvironment viscosity for the probe sites in the k-carrageenan system comparable to that found in SDS micelles. © 1999 Elsevier Science B.V. All rights reserved. Keywords: k-Carrageenan; Microviscosity; Gel–sol transition; Fluorescent probe; Dynamic viscosity; Optical rotation
1. Introduction Many of the naturally occurring, both charged and uncharged, water soluble macromolecules show a tendency to associate and form gels under specific conditions —high concentration and/or added interacting compounds. Such gels play an important role in many applications, i.e. in pharmaceutical preparations and in food industry. From a more fundamental point of view the more or less complex processes leading to the formation of gels constitute an important field of study in the biophysical sciences. Topologically, a gel is built up by a three dimensional network of chains interconnected by tie points, the space between being filled up by a solvent. For a polyelectrolyte gel this medium is a multicomponent system where the nature of counterions and added * Corresponding author. Tel.: + 46-18-4714373; fax: + 46-184714377. E-mail address: [email protected] (L.-O. Sundelo¨f)
electrolytes, as well as the interaction of these species with the polymer chains making up the network, regulate the gel structure and its properties. Frequently gel formation will take place only within a limited composition interval of this medium, which is the case for the negatively charged polysaccharide k-carrageenan. Considerable efforts have been invested to elucidate the structural and physicochemical properties of carrageenan systems the main part of which have addressed electrostatic aspects of the helix-coil and gel–sol transition. The present work attempts to address the relationship between the tie points, a crucial element of the gel structure, and the chain conformation, by studying the microviscosity of the hydrophobic microdomains, the dynamic rheology and the molecular conformation in the melting process of gel-like systems containing k-carrageenan. This was performed by means of a fluorescent intramolecular excimer forming probe, dynamic viscometry (DV) and optical rotation (OV), respectively. Since the excimer probe has not been used previously in systems containing charged
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polysaccharides, this work also aims at evaluating the applicability of such a probe. It was assumed that a sensitive spectroscopic method like that of a fluorescent intramolecular excimer forming probe, in conjunction with OR and DV, could provide further insight into the molecular interpretation of polymer-polymer association and gelation. For the hydrophobic probe 1,3-di(-1pyrenyl)propane (P3P), the ratio of monomer to excimer intensity (Im/Ie) [1] or the excimer intensity (Ie) alone provides a qualitative measure of the viscosity in the microenvironment of the probe [2]. An intramolecular excimer forming probe is preferable compared to one forming intermolecular complexes, since a much lower probe concentration is needed, thus minimising possible perturbations of the system. P3P has previously been successfully employed in studies of microviscosity of micelles [1], polymer-surfactant systems [3], phase transitions in phospholipids [4,5] and biological membranes [6]. The essential features of the k-carrageenan system, as they are known so far, can be outlined as follows [7–19]: An ordered (helical) conformation is considered necessary for aggregation and gelation to occur. Formation of a helical structure is promoted by adding an appropriate electrolyte to a sufficient ionic strength and/or by lowering the temperature. In addition, the type of counter- and co-ions present has a profound influence on the k-carrageenan conformation and aggregation behaviour. In terms of their promoting efficiency for helix formation and helix-helix association in an aqueous environment, the monovalent counterions can be divided into two main categories, i.e. the ‘non-specific’ counterions [Li + , Na + , and (CH3)4N + ] and the ‘specific’ counterions [NH4+ , K + , Cs + and Rb + ]. The former act primarily by long range Coulomb forces and the latter bind specifically to the carrageenan chain [13]. With respect to anions, k-carrageenan shows specific interaction only with iodide ions [20]. Smidsrød et al. [17] have demonstrated that the iodide ions induce an ordered conformation at the same time as they reduce the tendency for aggregation. Apart from the specific action of certain ions, the polymer concentration plays a decisive role for the formation of a three-dimensional network, as well as for its properties. In the present investigation all experiments were carried out in the semi-dilute concentration regime, close to c*, varying the system with respect to type and amount of added electrolyte as well as temperature, thus providing samples encompassing a number of the main features of the k-carrageenan system outlined above.
2. Experimental section
2.1. Materials k-Carrageenan, (Genugel type X-8944 batch no. 44807920) was a gift from Copenhagen Pectin A/S,
Denmark. The pure potassium and sodium form of k-carrageenan were prepared as described previously [21]. If not otherwise stated, the k-carrageenan concentration used was 10 mM disaccharide (which corresponds to 0.42 % w/w). For the resulting Na + -k-carrageenan a value of [h]= 540 ml g − 1 in 100 mM NaCl was found. Test samples for the experiments were prepared by diluting a stock solution with an appropriate electrolyte solution. All solutions were prepared as moles per kg solvent, but since all solutions can be considered as comparatively dilute the molal and molar scales coincide within the experimental error. Electrolyte concentrations given in the text are, as a rule, not corrected for contributions from the polyelectrolyte counterions. 1,3Bis(1-pyrenyl)propane (P3P), obtained from Molecular probes (Eugene, OR, USA), was used as supplied. The test samples were prepared by adding 5 ml of a saturated P3P acetone solution to a well defined amount of polymer solution (5.00 g) and allowing the solution to equilibrate for 6 days at 25°C to minimise the problem of microcrystals [22]. The final probe concentration was approximately 1× 10 − 7 M. Pyrene (98+%), purchased from Acros Chimica, Belgium, was twice recrystallized from absolute ethanol. Filtered pyren-saturated aqueous solutions (B 106 M) was used to prepare sample solutions. All other chemicals used were of analytical grade.
2.2. Fluorescence measurements The fluorescence measurements were recorded on a SPEX Fluorolog 2 Model FL1T2 steady state spectrofluorometer in the ‘s’ mode with 0.5-mm excitation and emission slits. The temperature of the waterjacketed cell holder was controlled by a circulating water bath. A thermocouple immersed in the sample was used to measure the temperature. In each experiment the temperature was increased stepwise. The sample was allowed to equilibrate at every temperature for a minimum time of 15 min. Emission spectra of P3P were obtained by excitation of the sample at l= 348 nm. The monomer intensity at 420 nm varied only within the limits of experimental error, thus indicating that the solubility of the probe did not change significantly during the experiment. Therefore, the variations in excimer intensity alone can provide information on changes of the microviscosity. The values of Ie and Im/Ie as shown in the figures and the text are averages of at least four consecutive measurements. The monomerto-excimer ratio, Im/Ie, was taken as the ratio of the intensity of the monomer emission (l=3789 1.0 nm) to the excimer emission (l= 48091.5 nm). The monomerto-excimer intensity ratio is essentially a function of the rate constants of excimer formation, dissociation and deactivation and the excimer lifetime [23]. All fluorescence experiments were commenced in the low temperature region following a number of excitations until steady readings were obtained. Due to the
A. Hugerth et al. / International Journal of Biological Macromolecules 26 (1999) 69–76
low levels of intensity (in case of the K + -k-carrageenan solutions at low temperature), the possibility that there were no microcrystals present (a well known problem in P3P and dipyme studies [22]) could not be entirely ruled out. To test for the possible presence of microcrystals and the effects of repetitive excitation to eliminate the effect of microcrystals, a 20 mM sodium dodecyl sulphate (SDS) solution (representing a well known amphiphilic system) was prepared and P3P was added. This solution was thereafter immediately tested with respect to presence of microcrystals, as outlined by Winnik and co-workers [22]. The resulting excitation spectra were not overlapping, as Fig. 1a shows, thus indicating a poorly equilibrated solution with microcrystals present. The initial emission spectra supported this conclusion by exhibiting an extraordinary large excimer maximum around 480 nm, although no shoulder to the red was readily identifiable. The solution was then subjected to excitation at 378 nm every 120th s for 7200 s. Subsequently produced excitation spectra displayed practically no displacement with respect to one another (Fig. 1b) thereby suggesting the attainment of minimisation of the microcrystal effect. In addition, the levels of emission intensity of the test sample decreased relative to the initially-recorded emission intensities. If the UV-radiation should have pro-
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duced an appreciable amount of the photoproduct 1-ethylpyrene [24] the monomer intensity recorded would hardly have decreased. The emission intensity of a pyrene containing solution also decreased initially as a result of repetitive excitation lex = 334 nm) although the ratio of the first (l= 373 nm) to the third vibronic peak (l=384 nm), I1/I3, was not effected. From the results of the SDS and the pyrene experiments it is reasonable to conclude that even if repetitive excitation of the test solution does not eliminate the problem of microcrystals completely its influence is at least significantly reduced.
2.3. Optical rotation Optical rotation was measured at 546 nm on a Perkin Elmer 241 polarimeter, using a 10-cm jacketed cell and a circulating water bath for accurate temperature control. Care was taken to allow the system to reach conformational equilibrium before final readings were made. To allow for possible hysteresis effects the helix-coil transition was monitored through both heating and cooling runs. The fraction of the sample having helix conformation (b) is then calculated from b= (a− acoil)/(ahelix − acoil,) where a equals the measured OR, acoil is determined from the plateau value at high temperatures and ahelix is estimated from the highest observed value of a. In cases were plateau values could not be attained in the low temperature region due to instrument limitations, values for ahelix were obtained by graphical extrapolation. In one case, K + k-carrageenan in 30 mM KC1, the test sample was flushed with Argon in order to avoid the formation of air bubbles during the OR experiment.
2.4. Rheology measurements
Fig. 1. Excitation spectra of 1,3-di(-pyrenyl)propane in an aqueous solution of 20 mM SDS at ambient temperature (a) prior to repetitive excitation and (b) after excitation at 378 nm every 120th s for 7200 s.
Rheological measurements were carried out on a Bohlin VOR rheometer with a temperature controlling unit, using the C14 (concentric cylinders) measuring system and a 0.0246-mNm torsion bar. k-Carrageenan test solutions were heated to a temperature approximately 15°C above the gel–sol endpoint, as estimated from OR measurements, during ample time to make sure that the sample had adopted an all-coil conformation. The sample was then allowed to cool in the measuring system. Each sample was measured at least in duplicate at pre-set temperatures with 15 min of equilibration at every temperature. Test samples were covered with a layer of low viscosity silicon oil (Kebo Lab, DC 200) to prevent evaporation of the sample. Dynamic rheological (oscillatory) measurements (0.01– 20 Hz) were all performed in the linear viscoelastic region at constant amplitude, as estimated from strainsweep measurements. Data given represent values obtained at 1 Hz, unless otherwise specified. The storage
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Fig. 2. Uncorrected fluorescence emission spectra of 1,3-di(-pyrenyl)propane in 10 mM k-carrageenan with 20 mM KCl as a function of temperature.
modulus (G%) and the loss modulus (G¦ data are given as measures of the stress in phase and 90° out of phase with the strain divided by the strain, respectively. Alternatively stated G% expresses the amount of energy stored and recovered per cycle, and G¦ the amount of energy dissipated or lost per cycle [25]. The loss tangent is a function of the phase angle (d) and defined as tan d= G¦/G%.
3. Results and discussion
3.1. Hydrophobic domains In all applications of fluorescent probes, the question of probe localisation is crucial for the interpretation of experimental observations. The ratio of the first to the third vibronic peak in the emission spectrum of pyrene is a sensitive measure of the polarity of the surrounding media (I1/I3 =hydrophobic index) [26]. For the k-carrageenan samples at hand pyrene gives a hydrophobic index of 1.6 – 1.7. This value corresponds to that of the environment found in aqueous solutions of many non-ionic cellulose derivatives [27]. It is thus concluded that hydrophobic sites exist to which P3P-molecules can distribute themselves. Since P3P is composed mainly of two pyrene moieties it can be anticipated that the locus of P3P-molecules will not differ much from that of pyrene.
3.2. Micro6iscosity data 3.2.1. Fluorescence emission spectra Characteristic uncorrected fluorescence emission spectra of P3P for different temperatures are shown in Fig. 2 for a sample containing 10 mM disaccharide
K + -k-carrageenan and 20 mM KCl, thus, providing conditions promoting helix–helix aggregation and gel formation at low temperatures. The shape of the fluorescence emission spectrum is significantly altered around 40°C (for this specific sample). This marked change in spectral properties coincides well with the change in phase angle observed in DV, (Fig. 5a below), which latter is determined by the gel–sol transition. In the low temperature regime, where k-carrageenan is present in the gel state, there is a noticeable contribution to the emission spectrum from the background over a broad wavelength interval.
3.2.2. Effect of temperature and electrolyte Ie as a function of temperature and of KCl and KI concentration, respectively, are shown in Fig. 3a, b. At a certain temperature there is a sharp increase in Ie for all experimental conditions studied. This conspicuous change indicates a reduction of the microviscosity of the system as sensed by the P3P probe. The onset temperature of the marked decrease in microviscosity (designated T0) clearly increases with increasing concentration of K + -ions (see also Table 1). It is a well known fact that an increase in the concentration of K + -ions shifts the gel–sol transition to higher temperatures. A relative measure of the microviscosity, taken as the monomer-to-excimer intensity ratio (Im/Ie), in the K + k-carrageenan systems at T0 was 1.4 9 0.1. This is of the same order of magnitude as in phosphatidylcholine bilayers [5] and in SDS-micelles [3] but one order of magnitude less than that found in clusters of ethyl hydroxyethyl cellulose and SDS [3]. The observation of a comparatively low microviscosity found in k-carrageenan systems is also supported by pulse radiolysis experiments performed by Wedlock et al. [28]. Impeding the gel formation by introducing iodide ions in
A. Hugerth et al. / International Journal of Biological Macromolecules 26 (1999) 69–76
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Fig. 3. The excimer intensity (Ie) as a function of temperature for 10 mM kcarrageenan with (a) ( +) 10; () 15 and () 20 mM KCl and (b) () 30 mM KCl and ( ) 30 mM KI, respectively.
exchange for chloride ions produced a shift in T0 to a lower temperature as is evident in the microviscosity data presented in Fig. 3b. The excimer intensities at temperatures less then T0 are low, with but minor deviations between the systems studied. However, their interpretation are beyond the scope of this paper. In the high temperature region, where k-carrageenan is present in an all-coil state, the microviscosity is low for all systems studied and the observed differences in microviscosity are hardly of any significance.
3.2.3. Effect of polymer concentration The dependence of the microviscosity on K + -k-carrageenan concentration was investigated in the concentration interval 2.5 – 15 mM disaccharide in 15 mM KC1. An increase in polymer concentration from 10 to 15 mM disaccharide resulted in essentially identical features to those in Fig. 3a, although with a minimal increase in recorded Im/Ie ratios. There was also a slight increase in the transition temperature due to the increase in counterion concentration. Solutions containing 2.5 and 5.0 mM disaccharide produced ratios below 0.9. Only in the 5 mM case could a feature indicative of a transition be observed. Thus, as the physical nature of the test sample in the ordered state at temperatures well below the onset of transition transcended from predominately gel-like to a viscous liquid as the polymer concentration decreased, the detectability of the order-disorder transition was reduced. The collected results support the view that the excimer formation of the P3P-probe is preferentially sensitive to the association of polymer segments in the solution.
3.3. Dynamic 6iscometric measurements of the gel–sol transition: correlation to micro6iscosity data 3.3.1. Rheological characterisation of -k-carragenan in the gel state The rheological characteristics of the K + -k-carragenan samples containing KC1 or KI in the ordered state can be evaluated from the observed frequency dependence of the storage modulus (G%) and the loss modulus (G¦) (Fig. 4a, b) determined at temperatures well below the onset of the order–disorder transition, where the dynamic viscosity shows only a minute temperature dependence. In the case of the K + -k-carragenan sample with 10 mM KC1 (Fig. 4a) G% and G¦ are parallel and of the same order of magnitude. Furthermore G% and G¦ show frequency dependence, thereby indicating the presence of a system exhibiting properties intermediate between a very weak gel and a viscoelastic solution. Increasing the concentration from 10 to 15 mM KCl (Fig. 4b) produced a considerable change in the rheological properties and G% became at least one order of magnitude higher in the 15 mM KC1 case. An even Table 1 Helical fraction (b) of k-carrageenan conformation determined by optical rotation measurements at the onset of the order-disorder transition (T0). The value of T0 was obtained from the P3P-probe data Type of electrolyte
Concentration (mM)
T0 (°C)
b(%)
KC1 KCl KCl KCl KI
10 15 20 30 30
31.0 35.0 40.0 47.0 44.5
60 56 42 15 31
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Fig. 4. The frequency dependence of the storage modulus G% (filled symbols) and loss modulus G¦ (unfilled symbols) at temperatures well below the order-disorder transition for k-carrageenan solutions with (a) ()()10 mM KCl and ( )( ) 30 mM KI and (b) ( )(×)15; ( )( ) 20; ()() 30 mM KCl, respectively.
stronger gel is formed in 30 mM KC1 where G% is approximately 100 times higher than G¦, and G% does not exhibit any frequency dependence in the frequency interval investigated. The small variation observed in G¦ is due to equipment limitations. Thus the K + -k-carragenan solutions with 15, 20 and 30 mM KCl formed a set of systems clearly exhibiting gel-like properties, encompassing the range of weak-to-strong gels. The recorded G% values increased with increasing KCl-concentration and, as could be anticipated, the frequency dependence of G% decreased with increasing K + concentration.
3.3.2. Effect of temperature and electrolyte The dependence of the phase angle and of G% on temperature are shown in Fig. 5a, b. The mid-point of transition (TmDV), defined as the temperature where G% = G¦ at 1 Hz, increases with increasing concentration of KC1. (By definition possible frequency dependence is disregarded.) This is in agreement with previously published reports [11,29,30] on the K + -k-carrageenan system as well as with the OR data presented below. Fig. 6 shows the dependence of the melting temperature on electrolyte concentration as obtained from DV, OR, and microviscosity measurements. The very close agreement between 1/TmDV and 1/T0 is evident. This indicates that the P3P-probe responds to an increased mobility in the hydrophobic domains at the gel–sol
Fig. 5. The phase angle (a) and storage modulus G% (b) as a function of temperature for 10 mM k-carrageenan with ( ) 10; ( ) 15; () 20; () 30 mM KCl and ( ) 30 mM KI, respectively.
transition, similarly to DV. The 10-mM KCl case can be characterised as a state intermediate between a weak gel and a viscoelastic solution. This follows from Fig. 6 where for this case the point from the excimer probe technique still follows the linear relationship from the
Fig. 6. The dependence of transition temperature on the total concentration of free counterions as determined by different methods ( ) P3P (1/T0); () DV (1/TmDV); () OR (1/TmOR) and OR ( ) 1/Ton).
A. Hugerth et al. / International Journal of Biological Macromolecules 26 (1999) 69–76
other compositions whereas the point obtained from DV deviates. In addition, compared to the DV measurements the onset of transition is easier to identify by the Ie − data. Exchanging the chloride ion for the iodide ion (30 mM KI) produced a weak gel as indicated by Figs 4a and 5a,band the gel–sol transition is shifted to a lower temperature. This result agrees with the microviscosity data presented.
3.4. Optical rotation measurements of the gel– sol transition: correlation with micro6iscosity data. 3.4.1. Effect of temperature and electrolyte Optical rotation has, in many investigations, been proven as a dependable tool in the study of conformational transitions in carrageenan systems. Results from such studies are therefore well suited for a comparison with data obtained by an alternative method such as the use of a bichromophoric excimer forming fluorescent probe sensitive to the microviscosity. The inverse of the melting temperature (TmOR, defined as the inflection point of optical rotation curves) for the k-carrageenan system depends linearly on the logarithm of the total concentration of free counterions (log CT) [11,15], as predicted by Manning’s limiting law [31]. 2.3R 1 1 = − j−1 log CT +E. 2 DHm Tm
(1)
In the calculation of CT the contribution from the polymer counterions and the dependence of the average activity coefficient on the helical content at T0, TmDV, TmOR, and Ton, respectively, were taken into account [15]. (Ton is the temperature of onset of the ordered conformation as obtained form OR cooling runs). DHm denotes the enthalpy change per mole disaccharide in the transition (assuming one charged group per disaccharide) and j denotes a dimensionless structural parameter proportional to the charge density. OR-data from both heating and cooling runs gave linear plots of 1/TmOR and 1/Ton, respectively, as a function of log CT (Fig. 6). When plotted together with the data obtained earlier by Rochas and Rinaudo [15] almost no difference could be discerned which ensures the similarity of our system to theirs. The data obtained by the P3P-probe gave a linear relationship between 1/T0 and log CT (1/T0 = − 3.93× 10 − 4 log CT +2.55×10 − 3, R =0.995), thus supporting the view that the onset of transition, as detected by the P3P-probe, reflects a common feature of the gel–sol transition.
3.4.2. Enthalpy of transition The enthalpy of transition, (DH0), corresponding to the ‘melting’ of the hydrophobic microdomains was estimated by a procedure outlined by Rochas et al. [32] and utilising the P3P-data of this study a value DH0 =
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14.7 kJ mol − 1 was obtained. This estimation agrees quite well with values attained by calorimetric measurements for gelling carrageenan systems under comparable conditions: 15.0 kJ mol − 1 in 33 mM KNO3 [33] and 17.6 kJ mol − 1 in 20 mM CsCl and 80 mM NaCl [19]. All these values of DH are larger than the enthalpy of transition (12.1 kJ mol − 1) for a 0.4 g l − 1 K + -kcarrageenan solution obtained in the absence of added electrolyte [33]. This is reasonable since in the latter case there should be no contributions to the enthalpy of transition from intermolecular crosslinks.
3.4.3. Helical content at T0 From the optical rotation measurements the helical content of the k-carrageenan conformation present at the onset of the order-disorder transition (T0) could be estimated (Table 1). All test solutions containing KC1 gave the same values of amax and amin within the limit of experimental error. Considering the variation in superhelical structure produced by a variation in KCl-concentration any contribution to the OR originating from a three-dimensional network thus appears to be so small that it falls within the experimental error. Consequently, the OR measurements provides a fair basis of quantification of the helical content. However, the data should be interpreted with some caution since amax for the 30 mM KI case is higher than for the corresponding KC1 case, thus indicating that a higher degree of order is reached as pointed out by Ciancia et al. [7] It is evident from Table 1 that the fraction in helical form at T0 decreases as the KCl-concentration increases. There is even a direct relation between the potassium ion concentration and the helical content present at T0. The data of Table 1 could be fitted to a polynomial, B(log Ci )= −253(log Ci )2 − 963(log Ci )− 855, with a coefficient of correlation of 0.999. Electron microscopy results obtained by a mica-technique indicated the formation of a coarser three-dimensional network structure as the KCl-concentration increased [34]. Considering this and the DV-measurements presented it is reasonable to assume that the fraction in helical form at T0 will reflect the conformation of the network structure of k-carrageenan in the gel state. It was shown above that substituting iodide for chloride as coion resulted in the formation of a weaker gel. Also, from the higher helical content at T0 in this case the reduced tendency of helix–helix aggregation is apparent.
4. Concluding remarks Since the excimer probe is preferentially redistributed to the hydrophobic domains of k-carrageenan, the data presented clearly indicate a considerable decrease in microviscosity of these domains of k-carrageenan at the gel–sol transition temperature. Thus, the results em-
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phasise the importance of these domains in building the gel structure and illustrate the delicate balance between the charged and the hydrophobic domains on the polyelectrolyte skeleton of k-carrageenan. The change in excimer intensity of the fluorescent microviscosity-probe P3P correlates well with changes in conformation and rheology as studied by optical rotation and dynamic viscometric measurements, these latter being in good agreement with previous findings. Combining the microviscosity and OR-results the helical content of k-carrageenan at T0, the onset temperature of the marked decrease in microviscosity, could be evaluated. Furthermore, the sensitivity of T0 to the gel – sol transition is supported by the corresponding dynamic viscometric measurements which indicate a close correlation between TmDV and T0. Estimations of the enthalpy change, (DH0), for the order–disorder transition under aggregating conditions as obtained from the P3P-probe studies, (DH0 =14.7 kJ mol − 1), compares well with data from other investigations on similar systems where different methods have been used. This gives further strong support for the validity of the excimer probe technique. In conclusion, the results support the applicability and versatility of the fluorescent probe technique as a sensitive tool to monitor changes of the gel tie points and thus the structural stability of the network. As such the results presented do encourage the pursuit of similar studies by other probes preferably using both steady state and time-resolved fluorescence.
Acknowledgements Financial support from the Swedish Natural Science Research Council (NFR) and the Swedish Council for Engineering Sciences (TFR) is gratefully acknowledged. We thank Dr Katarina Edsman and the Department of Pharmaceutics, Faculty of Pharmacy, for helpful discussions and the use of a Bohlin VOR rheometer. We also express our thanks to Dr Ninus Caram-Lelham for valuable contributions to the development of this project and Copenhagen Pectin A/S, Denmark, for providing k-carrageenan.
.
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Gel – sol transition in k-carrageenan systems: microviscosity of hydrophobic microdomains, dynamic rheology and molecular conformation Andreas Hugerth, Stefan Nilsson, Lars-Olof Sundelo¨f * Physical Pharmaceutical Chemistry, Faculty of Pharmacy, Uppsala Uni6ersity, Uppsala Biomedical Center, Box 574, S-751 23 Uppsala, Sweden Accepted 8 March 1999
Abstract The effect of gel–sol transition in k-carrageenan systems on the microviscosity of hydrophobic microdomains, as well as its relation to macroscopic rheology and molecular conformation, was studied in k-carrageenan systems. The microdomains were probed by 1,3-di(-1-pyrenyl)propane (P3P) for which the excimer intensity (Ie) provides relative measures of the microviscosity in the immediate probe surroundings. In particular the applicability of P3P to monitor the gel – sol transition was proved, the results showing a dramatic decrease in microviscosity in the vicinity of the transition point. The corresponding changes in rheological properties and carrageenan conformation were investigated by dynamic viscometry (DV) and optical rotation (OR), respectively. The temperature of onset of the transition as indicated by the microviscosity data (T0) was found to correlate well with the OR and DV-results. The application of microviscosity and OR-measurements allowed an estimation of the helical content at T0 to be determined. P3P-data indicate a microenvironment viscosity for the probe sites in the k-carrageenan system comparable to that found in SDS micelles. © 1999 Elsevier Science B.V. All rights reserved. Keywords: k-Carrageenan; Microviscosity; Gel–sol transition; Fluorescent probe; Dynamic viscosity; Optical rotation
1. Introduction Many of the naturally occurring, both charged and uncharged, water soluble macromolecules show a tendency to associate and form gels under specific conditions —high concentration and/or added interacting compounds. Such gels play an important role in many applications, i.e. in pharmaceutical preparations and in food industry. From a more fundamental point of view the more or less complex processes leading to the formation of gels constitute an important field of study in the biophysical sciences. Topologically, a gel is built up by a three dimensional network of chains interconnected by tie points, the space between being filled up by a solvent. For a polyelectrolyte gel this medium is a multicomponent system where the nature of counterions and added * Corresponding author. Tel.: + 46-18-4714373; fax: + 46-184714377. E-mail address: [email protected] (L.-O. Sundelo¨f)
electrolytes, as well as the interaction of these species with the polymer chains making up the network, regulate the gel structure and its properties. Frequently gel formation will take place only within a limited composition interval of this medium, which is the case for the negatively charged polysaccharide k-carrageenan. Considerable efforts have been invested to elucidate the structural and physicochemical properties of carrageenan systems the main part of which have addressed electrostatic aspects of the helix-coil and gel–sol transition. The present work attempts to address the relationship between the tie points, a crucial element of the gel structure, and the chain conformation, by studying the microviscosity of the hydrophobic microdomains, the dynamic rheology and the molecular conformation in the melting process of gel-like systems containing k-carrageenan. This was performed by means of a fluorescent intramolecular excimer forming probe, dynamic viscometry (DV) and optical rotation (OV), respectively. Since the excimer probe has not been used previously in systems containing charged
0141-8130/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 4 1 - 8 1 3 0 ( 9 9 ) 0 0 0 6 5 - 3
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polysaccharides, this work also aims at evaluating the applicability of such a probe. It was assumed that a sensitive spectroscopic method like that of a fluorescent intramolecular excimer forming probe, in conjunction with OR and DV, could provide further insight into the molecular interpretation of polymer-polymer association and gelation. For the hydrophobic probe 1,3-di(-1pyrenyl)propane (P3P), the ratio of monomer to excimer intensity (Im/Ie) [1] or the excimer intensity (Ie) alone provides a qualitative measure of the viscosity in the microenvironment of the probe [2]. An intramolecular excimer forming probe is preferable compared to one forming intermolecular complexes, since a much lower probe concentration is needed, thus minimising possible perturbations of the system. P3P has previously been successfully employed in studies of microviscosity of micelles [1], polymer-surfactant systems [3], phase transitions in phospholipids [4,5] and biological membranes [6]. The essential features of the k-carrageenan system, as they are known so far, can be outlined as follows [7–19]: An ordered (helical) conformation is considered necessary for aggregation and gelation to occur. Formation of a helical structure is promoted by adding an appropriate electrolyte to a sufficient ionic strength and/or by lowering the temperature. In addition, the type of counter- and co-ions present has a profound influence on the k-carrageenan conformation and aggregation behaviour. In terms of their promoting efficiency for helix formation and helix-helix association in an aqueous environment, the monovalent counterions can be divided into two main categories, i.e. the ‘non-specific’ counterions [Li + , Na + , and (CH3)4N + ] and the ‘specific’ counterions [NH4+ , K + , Cs + and Rb + ]. The former act primarily by long range Coulomb forces and the latter bind specifically to the carrageenan chain [13]. With respect to anions, k-carrageenan shows specific interaction only with iodide ions [20]. Smidsrød et al. [17] have demonstrated that the iodide ions induce an ordered conformation at the same time as they reduce the tendency for aggregation. Apart from the specific action of certain ions, the polymer concentration plays a decisive role for the formation of a three-dimensional network, as well as for its properties. In the present investigation all experiments were carried out in the semi-dilute concentration regime, close to c*, varying the system with respect to type and amount of added electrolyte as well as temperature, thus providing samples encompassing a number of the main features of the k-carrageenan system outlined above.
2. Experimental section
2.1. Materials k-Carrageenan, (Genugel type X-8944 batch no. 44807920) was a gift from Copenhagen Pectin A/S,
Denmark. The pure potassium and sodium form of k-carrageenan were prepared as described previously [21]. If not otherwise stated, the k-carrageenan concentration used was 10 mM disaccharide (which corresponds to 0.42 % w/w). For the resulting Na + -k-carrageenan a value of [h]= 540 ml g − 1 in 100 mM NaCl was found. Test samples for the experiments were prepared by diluting a stock solution with an appropriate electrolyte solution. All solutions were prepared as moles per kg solvent, but since all solutions can be considered as comparatively dilute the molal and molar scales coincide within the experimental error. Electrolyte concentrations given in the text are, as a rule, not corrected for contributions from the polyelectrolyte counterions. 1,3Bis(1-pyrenyl)propane (P3P), obtained from Molecular probes (Eugene, OR, USA), was used as supplied. The test samples were prepared by adding 5 ml of a saturated P3P acetone solution to a well defined amount of polymer solution (5.00 g) and allowing the solution to equilibrate for 6 days at 25°C to minimise the problem of microcrystals [22]. The final probe concentration was approximately 1× 10 − 7 M. Pyrene (98+%), purchased from Acros Chimica, Belgium, was twice recrystallized from absolute ethanol. Filtered pyren-saturated aqueous solutions (B 106 M) was used to prepare sample solutions. All other chemicals used were of analytical grade.
2.2. Fluorescence measurements The fluorescence measurements were recorded on a SPEX Fluorolog 2 Model FL1T2 steady state spectrofluorometer in the ‘s’ mode with 0.5-mm excitation and emission slits. The temperature of the waterjacketed cell holder was controlled by a circulating water bath. A thermocouple immersed in the sample was used to measure the temperature. In each experiment the temperature was increased stepwise. The sample was allowed to equilibrate at every temperature for a minimum time of 15 min. Emission spectra of P3P were obtained by excitation of the sample at l= 348 nm. The monomer intensity at 420 nm varied only within the limits of experimental error, thus indicating that the solubility of the probe did not change significantly during the experiment. Therefore, the variations in excimer intensity alone can provide information on changes of the microviscosity. The values of Ie and Im/Ie as shown in the figures and the text are averages of at least four consecutive measurements. The monomerto-excimer ratio, Im/Ie, was taken as the ratio of the intensity of the monomer emission (l=3789 1.0 nm) to the excimer emission (l= 48091.5 nm). The monomerto-excimer intensity ratio is essentially a function of the rate constants of excimer formation, dissociation and deactivation and the excimer lifetime [23]. All fluorescence experiments were commenced in the low temperature region following a number of excitations until steady readings were obtained. Due to the
A. Hugerth et al. / International Journal of Biological Macromolecules 26 (1999) 69–76
low levels of intensity (in case of the K + -k-carrageenan solutions at low temperature), the possibility that there were no microcrystals present (a well known problem in P3P and dipyme studies [22]) could not be entirely ruled out. To test for the possible presence of microcrystals and the effects of repetitive excitation to eliminate the effect of microcrystals, a 20 mM sodium dodecyl sulphate (SDS) solution (representing a well known amphiphilic system) was prepared and P3P was added. This solution was thereafter immediately tested with respect to presence of microcrystals, as outlined by Winnik and co-workers [22]. The resulting excitation spectra were not overlapping, as Fig. 1a shows, thus indicating a poorly equilibrated solution with microcrystals present. The initial emission spectra supported this conclusion by exhibiting an extraordinary large excimer maximum around 480 nm, although no shoulder to the red was readily identifiable. The solution was then subjected to excitation at 378 nm every 120th s for 7200 s. Subsequently produced excitation spectra displayed practically no displacement with respect to one another (Fig. 1b) thereby suggesting the attainment of minimisation of the microcrystal effect. In addition, the levels of emission intensity of the test sample decreased relative to the initially-recorded emission intensities. If the UV-radiation should have pro-
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duced an appreciable amount of the photoproduct 1-ethylpyrene [24] the monomer intensity recorded would hardly have decreased. The emission intensity of a pyrene containing solution also decreased initially as a result of repetitive excitation lex = 334 nm) although the ratio of the first (l= 373 nm) to the third vibronic peak (l=384 nm), I1/I3, was not effected. From the results of the SDS and the pyrene experiments it is reasonable to conclude that even if repetitive excitation of the test solution does not eliminate the problem of microcrystals completely its influence is at least significantly reduced.
2.3. Optical rotation Optical rotation was measured at 546 nm on a Perkin Elmer 241 polarimeter, using a 10-cm jacketed cell and a circulating water bath for accurate temperature control. Care was taken to allow the system to reach conformational equilibrium before final readings were made. To allow for possible hysteresis effects the helix-coil transition was monitored through both heating and cooling runs. The fraction of the sample having helix conformation (b) is then calculated from b= (a− acoil)/(ahelix − acoil,) where a equals the measured OR, acoil is determined from the plateau value at high temperatures and ahelix is estimated from the highest observed value of a. In cases were plateau values could not be attained in the low temperature region due to instrument limitations, values for ahelix were obtained by graphical extrapolation. In one case, K + k-carrageenan in 30 mM KC1, the test sample was flushed with Argon in order to avoid the formation of air bubbles during the OR experiment.
2.4. Rheology measurements
Fig. 1. Excitation spectra of 1,3-di(-pyrenyl)propane in an aqueous solution of 20 mM SDS at ambient temperature (a) prior to repetitive excitation and (b) after excitation at 378 nm every 120th s for 7200 s.
Rheological measurements were carried out on a Bohlin VOR rheometer with a temperature controlling unit, using the C14 (concentric cylinders) measuring system and a 0.0246-mNm torsion bar. k-Carrageenan test solutions were heated to a temperature approximately 15°C above the gel–sol endpoint, as estimated from OR measurements, during ample time to make sure that the sample had adopted an all-coil conformation. The sample was then allowed to cool in the measuring system. Each sample was measured at least in duplicate at pre-set temperatures with 15 min of equilibration at every temperature. Test samples were covered with a layer of low viscosity silicon oil (Kebo Lab, DC 200) to prevent evaporation of the sample. Dynamic rheological (oscillatory) measurements (0.01– 20 Hz) were all performed in the linear viscoelastic region at constant amplitude, as estimated from strainsweep measurements. Data given represent values obtained at 1 Hz, unless otherwise specified. The storage
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Fig. 2. Uncorrected fluorescence emission spectra of 1,3-di(-pyrenyl)propane in 10 mM k-carrageenan with 20 mM KCl as a function of temperature.
modulus (G%) and the loss modulus (G¦ data are given as measures of the stress in phase and 90° out of phase with the strain divided by the strain, respectively. Alternatively stated G% expresses the amount of energy stored and recovered per cycle, and G¦ the amount of energy dissipated or lost per cycle [25]. The loss tangent is a function of the phase angle (d) and defined as tan d= G¦/G%.
3. Results and discussion
3.1. Hydrophobic domains In all applications of fluorescent probes, the question of probe localisation is crucial for the interpretation of experimental observations. The ratio of the first to the third vibronic peak in the emission spectrum of pyrene is a sensitive measure of the polarity of the surrounding media (I1/I3 =hydrophobic index) [26]. For the k-carrageenan samples at hand pyrene gives a hydrophobic index of 1.6 – 1.7. This value corresponds to that of the environment found in aqueous solutions of many non-ionic cellulose derivatives [27]. It is thus concluded that hydrophobic sites exist to which P3P-molecules can distribute themselves. Since P3P is composed mainly of two pyrene moieties it can be anticipated that the locus of P3P-molecules will not differ much from that of pyrene.
3.2. Micro6iscosity data 3.2.1. Fluorescence emission spectra Characteristic uncorrected fluorescence emission spectra of P3P for different temperatures are shown in Fig. 2 for a sample containing 10 mM disaccharide
K + -k-carrageenan and 20 mM KCl, thus, providing conditions promoting helix–helix aggregation and gel formation at low temperatures. The shape of the fluorescence emission spectrum is significantly altered around 40°C (for this specific sample). This marked change in spectral properties coincides well with the change in phase angle observed in DV, (Fig. 5a below), which latter is determined by the gel–sol transition. In the low temperature regime, where k-carrageenan is present in the gel state, there is a noticeable contribution to the emission spectrum from the background over a broad wavelength interval.
3.2.2. Effect of temperature and electrolyte Ie as a function of temperature and of KCl and KI concentration, respectively, are shown in Fig. 3a, b. At a certain temperature there is a sharp increase in Ie for all experimental conditions studied. This conspicuous change indicates a reduction of the microviscosity of the system as sensed by the P3P probe. The onset temperature of the marked decrease in microviscosity (designated T0) clearly increases with increasing concentration of K + -ions (see also Table 1). It is a well known fact that an increase in the concentration of K + -ions shifts the gel–sol transition to higher temperatures. A relative measure of the microviscosity, taken as the monomer-to-excimer intensity ratio (Im/Ie), in the K + k-carrageenan systems at T0 was 1.4 9 0.1. This is of the same order of magnitude as in phosphatidylcholine bilayers [5] and in SDS-micelles [3] but one order of magnitude less than that found in clusters of ethyl hydroxyethyl cellulose and SDS [3]. The observation of a comparatively low microviscosity found in k-carrageenan systems is also supported by pulse radiolysis experiments performed by Wedlock et al. [28]. Impeding the gel formation by introducing iodide ions in
A. Hugerth et al. / International Journal of Biological Macromolecules 26 (1999) 69–76
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Fig. 3. The excimer intensity (Ie) as a function of temperature for 10 mM kcarrageenan with (a) ( +) 10; () 15 and () 20 mM KCl and (b) () 30 mM KCl and ( ) 30 mM KI, respectively.
exchange for chloride ions produced a shift in T0 to a lower temperature as is evident in the microviscosity data presented in Fig. 3b. The excimer intensities at temperatures less then T0 are low, with but minor deviations between the systems studied. However, their interpretation are beyond the scope of this paper. In the high temperature region, where k-carrageenan is present in an all-coil state, the microviscosity is low for all systems studied and the observed differences in microviscosity are hardly of any significance.
3.2.3. Effect of polymer concentration The dependence of the microviscosity on K + -k-carrageenan concentration was investigated in the concentration interval 2.5 – 15 mM disaccharide in 15 mM KC1. An increase in polymer concentration from 10 to 15 mM disaccharide resulted in essentially identical features to those in Fig. 3a, although with a minimal increase in recorded Im/Ie ratios. There was also a slight increase in the transition temperature due to the increase in counterion concentration. Solutions containing 2.5 and 5.0 mM disaccharide produced ratios below 0.9. Only in the 5 mM case could a feature indicative of a transition be observed. Thus, as the physical nature of the test sample in the ordered state at temperatures well below the onset of transition transcended from predominately gel-like to a viscous liquid as the polymer concentration decreased, the detectability of the order-disorder transition was reduced. The collected results support the view that the excimer formation of the P3P-probe is preferentially sensitive to the association of polymer segments in the solution.
3.3. Dynamic 6iscometric measurements of the gel–sol transition: correlation to micro6iscosity data 3.3.1. Rheological characterisation of -k-carragenan in the gel state The rheological characteristics of the K + -k-carragenan samples containing KC1 or KI in the ordered state can be evaluated from the observed frequency dependence of the storage modulus (G%) and the loss modulus (G¦) (Fig. 4a, b) determined at temperatures well below the onset of the order–disorder transition, where the dynamic viscosity shows only a minute temperature dependence. In the case of the K + -k-carragenan sample with 10 mM KC1 (Fig. 4a) G% and G¦ are parallel and of the same order of magnitude. Furthermore G% and G¦ show frequency dependence, thereby indicating the presence of a system exhibiting properties intermediate between a very weak gel and a viscoelastic solution. Increasing the concentration from 10 to 15 mM KCl (Fig. 4b) produced a considerable change in the rheological properties and G% became at least one order of magnitude higher in the 15 mM KC1 case. An even Table 1 Helical fraction (b) of k-carrageenan conformation determined by optical rotation measurements at the onset of the order-disorder transition (T0). The value of T0 was obtained from the P3P-probe data Type of electrolyte
Concentration (mM)
T0 (°C)
b(%)
KC1 KCl KCl KCl KI
10 15 20 30 30
31.0 35.0 40.0 47.0 44.5
60 56 42 15 31
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Fig. 4. The frequency dependence of the storage modulus G% (filled symbols) and loss modulus G¦ (unfilled symbols) at temperatures well below the order-disorder transition for k-carrageenan solutions with (a) ()()10 mM KCl and ( )( ) 30 mM KI and (b) ( )(×)15; ( )( ) 20; ()() 30 mM KCl, respectively.
stronger gel is formed in 30 mM KC1 where G% is approximately 100 times higher than G¦, and G% does not exhibit any frequency dependence in the frequency interval investigated. The small variation observed in G¦ is due to equipment limitations. Thus the K + -k-carragenan solutions with 15, 20 and 30 mM KCl formed a set of systems clearly exhibiting gel-like properties, encompassing the range of weak-to-strong gels. The recorded G% values increased with increasing KCl-concentration and, as could be anticipated, the frequency dependence of G% decreased with increasing K + concentration.
3.3.2. Effect of temperature and electrolyte The dependence of the phase angle and of G% on temperature are shown in Fig. 5a, b. The mid-point of transition (TmDV), defined as the temperature where G% = G¦ at 1 Hz, increases with increasing concentration of KC1. (By definition possible frequency dependence is disregarded.) This is in agreement with previously published reports [11,29,30] on the K + -k-carrageenan system as well as with the OR data presented below. Fig. 6 shows the dependence of the melting temperature on electrolyte concentration as obtained from DV, OR, and microviscosity measurements. The very close agreement between 1/TmDV and 1/T0 is evident. This indicates that the P3P-probe responds to an increased mobility in the hydrophobic domains at the gel–sol
Fig. 5. The phase angle (a) and storage modulus G% (b) as a function of temperature for 10 mM k-carrageenan with ( ) 10; ( ) 15; () 20; () 30 mM KCl and ( ) 30 mM KI, respectively.
transition, similarly to DV. The 10-mM KCl case can be characterised as a state intermediate between a weak gel and a viscoelastic solution. This follows from Fig. 6 where for this case the point from the excimer probe technique still follows the linear relationship from the
Fig. 6. The dependence of transition temperature on the total concentration of free counterions as determined by different methods ( ) P3P (1/T0); () DV (1/TmDV); () OR (1/TmOR) and OR ( ) 1/Ton).
A. Hugerth et al. / International Journal of Biological Macromolecules 26 (1999) 69–76
other compositions whereas the point obtained from DV deviates. In addition, compared to the DV measurements the onset of transition is easier to identify by the Ie − data. Exchanging the chloride ion for the iodide ion (30 mM KI) produced a weak gel as indicated by Figs 4a and 5a,band the gel–sol transition is shifted to a lower temperature. This result agrees with the microviscosity data presented.
3.4. Optical rotation measurements of the gel– sol transition: correlation with micro6iscosity data. 3.4.1. Effect of temperature and electrolyte Optical rotation has, in many investigations, been proven as a dependable tool in the study of conformational transitions in carrageenan systems. Results from such studies are therefore well suited for a comparison with data obtained by an alternative method such as the use of a bichromophoric excimer forming fluorescent probe sensitive to the microviscosity. The inverse of the melting temperature (TmOR, defined as the inflection point of optical rotation curves) for the k-carrageenan system depends linearly on the logarithm of the total concentration of free counterions (log CT) [11,15], as predicted by Manning’s limiting law [31]. 2.3R 1 1 = − j−1 log CT +E. 2 DHm Tm
(1)
In the calculation of CT the contribution from the polymer counterions and the dependence of the average activity coefficient on the helical content at T0, TmDV, TmOR, and Ton, respectively, were taken into account [15]. (Ton is the temperature of onset of the ordered conformation as obtained form OR cooling runs). DHm denotes the enthalpy change per mole disaccharide in the transition (assuming one charged group per disaccharide) and j denotes a dimensionless structural parameter proportional to the charge density. OR-data from both heating and cooling runs gave linear plots of 1/TmOR and 1/Ton, respectively, as a function of log CT (Fig. 6). When plotted together with the data obtained earlier by Rochas and Rinaudo [15] almost no difference could be discerned which ensures the similarity of our system to theirs. The data obtained by the P3P-probe gave a linear relationship between 1/T0 and log CT (1/T0 = − 3.93× 10 − 4 log CT +2.55×10 − 3, R =0.995), thus supporting the view that the onset of transition, as detected by the P3P-probe, reflects a common feature of the gel–sol transition.
3.4.2. Enthalpy of transition The enthalpy of transition, (DH0), corresponding to the ‘melting’ of the hydrophobic microdomains was estimated by a procedure outlined by Rochas et al. [32] and utilising the P3P-data of this study a value DH0 =
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14.7 kJ mol − 1 was obtained. This estimation agrees quite well with values attained by calorimetric measurements for gelling carrageenan systems under comparable conditions: 15.0 kJ mol − 1 in 33 mM KNO3 [33] and 17.6 kJ mol − 1 in 20 mM CsCl and 80 mM NaCl [19]. All these values of DH are larger than the enthalpy of transition (12.1 kJ mol − 1) for a 0.4 g l − 1 K + -kcarrageenan solution obtained in the absence of added electrolyte [33]. This is reasonable since in the latter case there should be no contributions to the enthalpy of transition from intermolecular crosslinks.
3.4.3. Helical content at T0 From the optical rotation measurements the helical content of the k-carrageenan conformation present at the onset of the order-disorder transition (T0) could be estimated (Table 1). All test solutions containing KC1 gave the same values of amax and amin within the limit of experimental error. Considering the variation in superhelical structure produced by a variation in KCl-concentration any contribution to the OR originating from a three-dimensional network thus appears to be so small that it falls within the experimental error. Consequently, the OR measurements provides a fair basis of quantification of the helical content. However, the data should be interpreted with some caution since amax for the 30 mM KI case is higher than for the corresponding KC1 case, thus indicating that a higher degree of order is reached as pointed out by Ciancia et al. [7] It is evident from Table 1 that the fraction in helical form at T0 decreases as the KCl-concentration increases. There is even a direct relation between the potassium ion concentration and the helical content present at T0. The data of Table 1 could be fitted to a polynomial, B(log Ci )= −253(log Ci )2 − 963(log Ci )− 855, with a coefficient of correlation of 0.999. Electron microscopy results obtained by a mica-technique indicated the formation of a coarser three-dimensional network structure as the KCl-concentration increased [34]. Considering this and the DV-measurements presented it is reasonable to assume that the fraction in helical form at T0 will reflect the conformation of the network structure of k-carrageenan in the gel state. It was shown above that substituting iodide for chloride as coion resulted in the formation of a weaker gel. Also, from the higher helical content at T0 in this case the reduced tendency of helix–helix aggregation is apparent.
4. Concluding remarks Since the excimer probe is preferentially redistributed to the hydrophobic domains of k-carrageenan, the data presented clearly indicate a considerable decrease in microviscosity of these domains of k-carrageenan at the gel–sol transition temperature. Thus, the results em-
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phasise the importance of these domains in building the gel structure and illustrate the delicate balance between the charged and the hydrophobic domains on the polyelectrolyte skeleton of k-carrageenan. The change in excimer intensity of the fluorescent microviscosity-probe P3P correlates well with changes in conformation and rheology as studied by optical rotation and dynamic viscometric measurements, these latter being in good agreement with previous findings. Combining the microviscosity and OR-results the helical content of k-carrageenan at T0, the onset temperature of the marked decrease in microviscosity, could be evaluated. Furthermore, the sensitivity of T0 to the gel – sol transition is supported by the corresponding dynamic viscometric measurements which indicate a close correlation between TmDV and T0. Estimations of the enthalpy change, (DH0), for the order–disorder transition under aggregating conditions as obtained from the P3P-probe studies, (DH0 =14.7 kJ mol − 1), compares well with data from other investigations on similar systems where different methods have been used. This gives further strong support for the validity of the excimer probe technique. In conclusion, the results support the applicability and versatility of the fluorescent probe technique as a sensitive tool to monitor changes of the gel tie points and thus the structural stability of the network. As such the results presented do encourage the pursuit of similar studies by other probes preferably using both steady state and time-resolved fluorescence.
Acknowledgements Financial support from the Swedish Natural Science Research Council (NFR) and the Swedish Council for Engineering Sciences (TFR) is gratefully acknowledged. We thank Dr Katarina Edsman and the Department of Pharmaceutics, Faculty of Pharmacy, for helpful discussions and the use of a Bohlin VOR rheometer. We also express our thanks to Dr Ninus Caram-Lelham for valuable contributions to the development of this project and Copenhagen Pectin A/S, Denmark, for providing k-carrageenan.
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