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Gelatin Microemulsion-Based Gels with the Cationic Surfactant Cetyltrimethylammonium Bromide: A Self-Diffusion and Conductivity Study Francesco Lopez,*,† Francesco Venditti,† Luigi Ambrosone,† Giuseppe Colafemmina,†,‡ Andrea Ceglie,† and Gerardo Palazzo*,†,‡ Consorzio Interuniversitario per lo sviluppo dei Sistemi a Grande Interfase (CSGI), c/o Department of Food Technology, University of Molise, I-86100 Campobasso, Italy, and Department of Chemistry, University of Bari, I-70126, Bari, Italy Received July 27, 2004. In Final Form: September 14, 2004 In this contribution we demonstrate that gelatin can jellify also water-in-oil microemulsions based on the cationic surfactant cetyltrimethylammonium bromide (CTAB). The partial stability diagram of the system CTAB + 1-pentanol + water + hexane + gelatin was determined. The resulting microemulsionbased gels (MBGs) were characterized by means of conductivity and pulsed gradient spin-echo NMR. For the first time the water self-diffusion coefficient was measured in gelatin MBGs, and the results were successfully analyzed in terms of diffusion in an interconnected network of aqueous channels.
Reverse micelles have been used to solubilize a variety of biopolymers in apolar solvents with a low water content.1-3 In particular, the effect of gelatin solubilization on the rheological behavior of water-in-oil (w/o) microemulsions had a deep impact in several biotechnological processes. Gelatin is a typical gelling agent in water. This polypeptide is obtained by thermal degradation of collagen, a protein widely present in biological connective tissues (about 25% of all the proteins in our body is collagen). The ability of gelatin to jellify organic solvents, when dissolved in reverse micelles, was independently assessed in the 1980s by two Swiss groups.4,5 It was demonstrated that when solid gelatin is dispersed in reverse micelles at high enough water loading, a moderate warming followed by a careful decrease in temperature leads to the jellification of the whole sample, which remains fully transparent but becomes solidlike. These systems, often referred to as microemulsion-based gels or MBGs,6 were of outstanding importance in biotechnology because they can easily trap enzymes, retaining their catalytic activity, in organic solvents.7,8 Evaporation of organic solvent gives rise to gelatin films that efficiently immobilize enzymes also in aqueous environments and has been proposed as a tool for water detoxification.9 Moreover, MBGs can be silica* Corresponding authors. Address: Consorzio Interuniversitario per lo sviluppo dei Sistemi a Grande Interfase (CSGI), c/o Department of Food Technology (DISTAAM), University of Molise, I-86100 Campobasso, Italy (F.L.); Dipartimento di Chimica, via Orabona 4, I-70126, Bari, Italy (G.P.). E-mail:
[email protected] (F.L.);
[email protected] (G.P.). † University of Molise. ‡ University of Bari. (1) Luisi, P. L.; Giomini, M.; Pileni, M. P.; Robinson, B. H. Biochim. Biophys. Acta 1988, 947, 209. (2) Fletcher, P. D. I.; Robinson, B. H. J. Chem. Soc., Faraday Trans. 1985, 81, 2667. (3) Lopez, F.; Cinelli, G.; Ambrosone, L.; Colafemmina, G.; Ceglie, A.; Palazzo, G. Colloids Surf., A 2004, 237, 49. (4) Haering, G.; Luisi, P. L. J. Phys. Chem. 1986, 90, 5892. (5) Quellet, C.; Eicke, H. F. Chimia 1986, 40, 233. (6) Atkinson, P. J.; Grimson, M. J.; Hennan, R. K.; Howe, A. M.; Robinson, B. H. J. Chem. Soc., Chem. Commun. 1989, 1807. (7) Luisi, P. L.; Scartazzini, R.; Haering, G.; Schurtenberger, P. Colloid Polym. Sci. 1990, 268, 356. (8) Hinze, L.; Uemasu, I.; Dai, F.; Braun, M. Curr. Opin. Colloid Interface Sci. 1996, 1, 502. (9) Crecchio, C.; Ruggiero, P.; Pizzigallo, M. D. R. Biotechnol. Bioeng. 1995, 48, 585.
hardened, giving a matrix that is almost temperatureand solvent-insensitive.10 Almost all the reported MBGs were prepared using reverse micelles made of bis-2-ethylhexylsodiumsuccinate (AOT), an anionic surfactant. Few exceptions are represented by microemulsions based on other anionic and nonionic surfactants.11,12 In the present contribution, we demonstrate that MBG can be made also in a w/o microemulsion stabilized by the cationic surfactant cetyltrimethylammonium bromide (CTAB). CTAB, 1-pentanol, hexane and gelatin (type A from porcine skin, bloom 300) were purchased from Sigma. CTAB was three times recrystallized from anhydrous ethanol; other chemicals have been used as received. Water was twice distilled in an all-quartz device. Water, CTAB, pentanol, and hexane form, when mixed in suitable ratios, reverse micelles.13,14 To define the microemulsion composition we use as parameters the molar ratios: water/CTAB (W0), pentanol/CTAB (P0), and the overall concentration of CTAB ([CTAB]). MBGs were prepared by adding known amounts of gelatine and water to a microemulsion at W0 ) 5, [CTAB] ) 0.1 M, and the desired P0. The mixtures were kept at 60 °C for 20 min and then cooled at 35 °C; all the steps were performed under stirring. Finally the sample was left standing at 23 °C without stirring. The final composition is defined by the parameters P0, W0 (evaluated taking into account the overall water content), and the overall gelatin content (cg expressed as grams of gelatin/100 mL of solution). An initial screening suggested that the formation of singlephase MBG requires (in the present system) a minimum amount of water (W0 > 40), and it is hindered by the cosurfactant. Therefore, we have fixed the P0 to the lowest value compatible with microemulsion formation (P0 ) 4.8), and the W0 ranges from 40 to 120. The upper panel of Figure 1 shows the stability diagram of this system in the variables W0 and gelatin concentration. At low gelatin (10) Schuleit, M.; Luisi, P. L. Biotechnol. Bioeng. 2001, 72, 249. (11) Murdan, S.; Gregoriadis, G.; Florence, A. T. Int. J. Pharm. 1999, 180, 211. (12) Kantaria, S.; Rees, G. D.; Lawrence, M. J. J. Controlled Release 1999, 60, 355. (13) Giustini, M.; Palazzo, G.; Colafemmina, G.; Della Monica, M.; Giomini, M.; Ceglie, A. J. Phys. Chem. 1996, 100, 3190. (14) Palazzo, G.; Lopez, F.; Giustini, M.; Colafemmina, G.; Ceglie, A. J. Phys. Chem. B 2003, 107, 1924.
10.1021/la048110x CCC: $27.50 © 2004 American Chemical Society Published on Web 09/30/2004
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Figure 1. (Upper panel) partial stability diagram for MBGs at 23 °C. Circles, triangles, and diamonds represent liquid, gel, and two-phase systems, respectively; open and closed symbols denote isotropic and birefringent samples, respectively. The bold gray curve represents the compositions where the conductivity reaches a plateau ∼100 µS/cm. (Lower panel) phase diagram for the system CTAB/1-pentanol/water/hexane at P0 ) 4.8, [CTAB] ) 0.1 M, and different W0 values. The ordinate represents the relative volumes of different phases. RM denotes reverse micelles, and LC denotes liquid crystals.
loading the samples are low viscous (circles in Figure 1). Samples at W0 < 78 are optically isotropic so they can be considered as ordinary liquids, while at higher W0 values they are birefringent suggesting the presence of long-range anisotropic structures. For high enough gelatin loading, samples are solidlike and do not flow under gravity (triangles in Figure 1). Also at this high gelatin content the W0 ∼ 78 represents a boundary between isotropic and birefringent samples. The gelatin concentration where the sol-gel transition takes place decreases upon water loading. For very high water contents (W0 > 120) the system phase-separates leading to equilibrium between excess hexane and a very stiff gel. This solidlike phase is birefringent in the range 115 < W0 < 145 and isotropic for higher W0 values (in this last case the solid phase is transparent but slightly bluish). Gelatin influences the phase behavior of the system. For the purpose of comparison, the lower panel of Figure 1 shows the phase diagram in the absence of gelatin. We found that the microemulsion exists as a single isotropic phase up to W0 ∼ 48. Diffusion properties are typical of reverse micelles with water and CTAB diffusion mutually close and very low (around 10-11 m2 s-1). For higher water loading, the system phase separates leading to coexistence between reverse micelles and liquid crystals. Moving toward higher W0 results in a growth of the birefringent phase that, for W0 ∼ 80, occupies the whole sample. For water loading higher than W0 ∼ 90 the liquid crystalline phase shrinks expelling almost pure hexane. The formation of MBGs is usually described in terms of a protein-assisted percolation process. This topological
Figure 2. Conductivity behavior of MBGs. (A) Samples at three W0 values and different gelatin loadings. (B) Conductivity upon water dilution for MBGs at a fixed (Cg ) 3%) gelatin content (open diamonds) and for gelatin-free microemulsions (closed triangles); the liquid crystal region is labeled LC. Inset: birefringence texture of MBG at Cg ) 7% and W0 ) 100 observed between two cross-polarizers; the sample was stirred at 60 °C for a time not long enough to dissolve all the water so that the subsequent cooling trapped some macroscopic water droplets.
transition can be suitably monitored by means of conductivity measurements. Here we have measured the specific conductivity κ with a CDM230 conductivity meter (Radiometer Analytical) equipped with a two-pole conductivity cell tailored for small volumes CDC749 (cell constant ) 1.84 cm-1). Introduction of the electrode into the sample before and after gelation did not change the conductivity values after setting. Measurements were done after equilibration times ranging from 0.5 h to 1 day; the constant conductivity readout was used as a criterion of sample equilibrium. Figure 2A shows representative κ versus cg curves obtained for systems at constant W0. The increase of at least 3 orders of magnitude in κ is evident above a critical gelatin content. Such dramatic changes in electrical conductivity have been already reported for MBGs prepared with AOT as the surfactant and were usually interpreted as a consequence of the proteininduced increase in connectivity of aqueous domains.15,16 From Figure 2A it is clear that the higher the W0, the (15) Quellet, C.; Eicke, H. F.; Sager, W. J. Phys. Chem. 1991, 95, 642. (16) Eicke, H. F.; Hilfiker, G. R.; Struis, P. W. J.; Xu, G. J. Phys. Chem. 1992, 96, 5175.
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lower the gelatin content needed to reach the κ plateau. Interestingly, the locus where κ reaches the plateau (gray bold line in Figure 1) successfully divides liquids from jelly-like samples in the phase map. The synergetic effect of water and gelatin content on conductivity is confirmed by the data of Figure 2B, where the influence of water loading at a constant gelatin content is reported. It is clear that, in the presence of protein, water addition induces electrical percolation already before the formation of a birefringent phase. On the contrary, without gelatin the conductivity is always very low, as expected for reverse micelles, and a high κ value is reached only when the sample is birefringent (the small maximum in κ versus W0 plot around W0 ) 20 is a feature of migration of statistically charged droplets).13,17 The birefringence in samples at W0 ) 80 was previously attributed, in gelatinfree microemulsions, to a dilute lamellar phase.18 Likely the lamellar structure is retained in MBG as well. Actually, when macroscopic water droplets are trapped in the gel, a millimeter-size texture similar to a maltese cross is revealed between cross-polarizers (see inset in Figure 2B). To have insight on the connectivity of aqueous domains, a study on the water diffusion was undertaken through pulsed gradient spin-echo (PGSE) NMR experiments19 on a Tesla 587 A spectrometer (1.88 T, operating at 80 MHz for 1H) equipped with a Stelar field gradient probe. Gels were gently warmed and transferred into NMR tubes (5 mm) that were subsequently sealed. At a constant gelatin content (Cg ) 3%) the water selfdiffusion coefficient (DW) grows monotonically upon increase in the molar ratio of water/CTAB (Figure 3A). This is consistent with the increase in connectivity inferred from conductivity data (Figure 2B). However, although the system appears to be electrically percolated already at W0 ) 80, the water traslational mobility grows continuously along the water dilution path independently from the phase transitions encountered. At constant W0, the dependence of DW on the gelatin content is markedly affected from water loading as shown in Figure 3B. At high W0 values (viz., 80 and 65) a maximum in the DW versus Cg plot is clearly discernible, while at low W0 values (viz., 50 and 40) the water diffusion is essentially unaffected by the gelatin content. The most important feature of the NMR self-diffusion approach is the fact that it monitors movements over micrometer distances. In the present system, water traslational motion is affected mainly by two factors. One is the connectivity of aqueous channels. The other is the local self-diffusion coefficient of water inside the aqueous domains; the presence of gelatin is expected to hinder the water diffusion because a fraction of water will be involved in protein hydration. Depending on the compositional path explored, these parameters interplay leading to different diffusional behaviors. Upon water loading, at constant overall gelatin content (Figure 3A), both the factors conspire to increase the water traslational mobility because an increase in W0 corresponds to a more percolated system and a higher water/gelatin ratio. At variance, the samples in Figure 3B describe a path where the connectivity increases (because of protein-assisted percolation) but the local water diffusion is expected to decrease (because the gelatin/water ratio grows). The resultant of these two opposite effects could give rise to a well-defined maximum in a DW versus Cg plot as found in the case of samples at high water content. To discuss this hypothesis on a more quantitative (17) Bumajdad, A.; Eastoe, J. Phys. Chem. Chem. Phys. 2004, 6, 1597. (18) Palazzo, G.; Carbone, L.; Colafemmina, G.; Angelico, R.; Ceglie, A.; Giustini, M. Phys. Chem. Chem. Phys. 2004, 6, 1423. (19) Stilbs, P. Prog. Nucl. Magn. Reson. Spectrosc. 1987, 19, 1.
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Figure 3. Water diffusion in MBGs. (A) DW along the water dilution line for MBGs at Cg ) 3%; LC ) liquid crystals. (B) DW as a function of Cg for different W0 values; the samples at W0 ) 80 are birefringent, and all the others are isotropic.
ground it is useful to examine the water diffusion along a connected network (above the percolation threshold).20 Let us describe the diffusion path in terms of the concepts developed in polymer statistics. The aqueous channels can be described as an N segment freely jointed path with each segment of length Λ (the Kuhn’s length that is twice the persistence length). Let Lb be the mean curvilinear distance between connections; the diffusion time between two branches (τb) depends on the local (curvilinear) selfdiffusion coefficient Dc according to τb ) Lb2/2Dc. The corresponding mean square distance traveled by water molecules in the laboratory frame is 〈Rb2〉 ) LbΛ, where Λ is the Kuhn’s length of the aqueous channel. The selfdiffusion coefficient observed by means of PGSE-NMR gives a time-scale independent laboratory frame selfdiffusion coefficient:21 2 Dc 1 〈Rb 〉 DW ) ) 3 2τbranch 3N
(1)
where N ) Lb/Λ is the mean number of Kuhn’s lengths (20) Ambrosone, L.; Angelico, R.; Ceglie, A.; Olsson, U.; Palazzo, G. Langmuir 2001, 17, 6822 and references therein. (21) Callaghan, P. T. Principles of Nuclear Magnetic Resonance Microscopy; Clarendon Press: Oxford, 1991.
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Figure 4. Water diffusion as a function of the gelatin concentration in the water pool. The DW data are the same as those of Figure 3B; closed symbols denote samples above the electrical percolation threshold (κ > 100 µS/cm) while samples below this threshold are shown as open symbols. Gray symbols refer to the water self-diffusion coefficient obtained on gels made only by gelatin and water (Dc); triangles represent Dc/3 (N ) 1 in eq 1), and hexagons represent Dc/4.5 (N ) 1.5 in eq 1).
self-diffusion coefficient of water inside the aqueous domains is expected to be function of the gelatin/water ratio, and this suggests that the relevant variable for the data of Figure 3B is the gelatin concentration in the water pool. In Figure 4 the DW values have been plotted against the percentage of gelatin evaluated only with respect to water and gelatin. It is clear that all the points above the electrical percolation threshold (closed symbols) follow the same trend. Such a trend reflects a decrease in water diffusion (inside the channels) upon gelatin loading. Actually we found that in hydrogels (only gelatin and water) the water self-diffusion coefficient decreases with gelatin content. In hydrogels the water diffuses freely in three dimensions, while in MBG its motion is locally monodimensional. We can take into account this difference, using as Dc in eq 1 the self-diffusion coefficient measured in hydrogels. In Figure 4 are also shown the data collected in hydrogels expressed either as Dc/3 (N ) 1, i.e., interconnected stiff pipes) or as Dc/4.5 (N ) 1.5). It is clear that in this representation the water diffusion in MBGs and hydrogels share the same order of magnitude and in particular the prediction for N ) 1.5 and the data of percolated MBGs follow the same master curve. This strongly indicates that for interconnected aqueous channels water diffusion depends essentially on the gelatin concentration in the water pool (through Dc in eq 1), while below the percolation threshold DW depends mainly on the degree of connectivity and, thus, on the water and gelatin overall concentration.
between two connections; for N e 1 we have a system of interconnected stiff rods for which DW ) Dc/3. The local
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