Langmuir 1991, 7, 223-231
223
Structural Study of Microemulsion-Based Gels at the Saturation Point C. Petit,? Th. Zemb,z and M. P. Pileni*ltJ Laboratoire S.R.I. bbtiment de chimie physique, Universit6 P . et M . Curie, 11 Rue P. et M . Curie, 75005 Paris, France, and Centre d'6tudes nucleaires de Saclay, D.Ph.G-S.C.M., 91191 Gif sur Yvette, France Received December 15, 1989. In Final Form: July 2, 1990 The quaternary organic gels (water, AOT, gelatin, isooctane) have been studied by small-angle X-ray and neutron scattering, as well as by conductivity and pulse radiolysis. Among the possible microstructures, we show that only a model of water spheres, interconnected by gelatin rods that are free from surfactant, makes it possible to explain the shape of the scattering spectra near the saturation point. This structure also allows us to calculate the phase diagram.
Introduction
several different models have been proposed to describe the microscopic structure of the gel state (Figure 1): For several years AOT reverse micelles have been (1)A microstructure of aggregated macromolecules in increasingly used to solubilize proteins' or biopolymers.2 a rigid network surrounded by water and AOT.' For the A new field of study has been opened with the solubiliadvocates of this model, the structure of the gelatin in the zation of gelatin in this system, which leads to obtaining water is essentially conserved; the role of the water is to transparent hydrophobic gels based on micro emulsion^.^-^ hydrate the gelatin and these connected hydrated cylinders These transparent gels constitute a dispersive medium are, subsequently, surrounded by a monolayer of AOT, with a viscosity varying from that of the liquid to true the solvent being hydrophobic. This model is consistent physical gels. The new mediaoffer numerous perspectives with the electrical conductivity values of the gel but does from the fundamental point of view for the study and not allow an easy explanation of the demixion phase understanding of gelation mechanisms. This is based on observed in the phase diagram. the competition between the branching network induced (2) A model5 where the gelatin network and the reverse by the strands of gelatin and the spontaneous compartmicelles are independent. In this model, the spherical mentation induced by the surfactant. From the practical AOT micelles are fixed by a gelatin network, protected by point of view, the photochemical applications appear to the surfactant, independently of the position of the AOT be promising, as an optically clear solid organic medium micelles, as these are not connected to the water pools. is made a ~ a i l a b l e .The ~ possibility offered of incorporating The hydrophilic parts of the gelatin are covered by AOT; hydrophilic proteins or d r ~ g s ~ ?suggests " ~ ~ interesting This model allows us to understand the shape of the phase applications in biotechnology. diagram but it does not predict the electrical percolation. However, if studies have been carried out on the (3) A model of spherical droplets containing water and structure of these gel~,3-~ there has not yet been a part of the gelatin. These reverse microemulsions are satisfactory model of the gel phase determined by scattering. Since the first work done by Haering4andQ ~ e l l e t , ~ connected by the gelatin, arranged in helical strands, but not covered by ~urfactant.~*6 For this system of interconnected spheres, the droplet structure, the characteristic + Universitk P. e t M. Curie. of the water/AOT/isooctane system is retained during all f Centre d'ktudes nuclkaires de Saclay. of the gelation process. (1) (a) In Structure and Reactioity in Reoerse Micelles;Pileni, M. P., Each of these structures (Figure 1)can, a priori, exist Ed.; Elsevier Science Publisher: Amsterdam, 1989. (b) In Reverse Micelles;Luisi, P. L., Straubs, B., Ed.s; Plenum Press: New York, 1983. (c) in a part of the phase diagram (see note). The X-ray Nicot, C.; Vasher, M.; Vincent, M.; Gallay, s.; Waks, M. Biochem.Bioscattering spectra (especiallysensitive to gelatin and water) phys. Res. Commun.1986,135,629. (d) Martinek, K.; Levashov, A. V.; and neutron spectra (where only the aqueous pools are Klyachko, N. K.; Berezin, I. V. Biochim. Biophys. Acta 1981,657,277. (e) Martinek, K. Eur. J. Biochem. 1986,155,453. (f) Fletcher, P. D. I.; seen) consistent with these models are qualitatively similar Robinson, B. H.; Freedman, R. B.; Oldfield, C. J. Chem. Soc., Faraday but quantitatively different. For example, the measureTrans. I 1985,81,2667. (9) Hilhorst, R.; Spruijt, R.; Laane, C.; Veeger, ment on an absolute scale of the total specific interfaces C. Eur. J. Biochem.1984, 144,459. (h) Fendler, J. H. J.Phys. Chem. 1982,86, 947. by X-rays and neutrons allows us to measure the total (2) (a) Haering, G.; Luisi, P. L.; Meussdorfer, F. Biochem.Biophys. interfacial area, 2 in A2/A3, and to numerically compare Res. Commun. 1985, 127, 991. (b) Imre, V. E.; Luisi, P. L. Biochem. this with the value predicted by the models. Biophys. Res. Commun. 1982, 107, 538. (c) Zulauf, M.; Eicke, H. F.; J. Phys. Chem. 1979,83,480. (d) Pileni, M. P.; Zemb, T.; Petit, C. Chem. We, thus, present here a study of the structure by smallPhys. Lett. 1985, 118, 414. angle scattering of neutrons or X-rays with the objectives (3) Quellet, C.; Eicke, H. F. Chimia 1986, 40, 233. of discriminating between the different models and (4) Haering, G.; Luisi, P. L. J.Phys. Chem. 1986, 90,5892. (5) Atkinson, P. J.; Grimson, M. J.; Heenan, R. K.; Howe, A. M.; Robproposing a structure consistent with the mechanisms of inson, B. H. J. Chem. Soc., Chem. Commun. 1989, 1807. gel formation. (6) Quellet, C. Thesis, Basel; Ekonom Drdck AG, Basel, 1988. ~~~
(7) Capitani, D.; Segre, A. L.; Haering, G.; Luisi, P. L. J. Phys. Chem.
--.
1988. - - - ,92. 3500. ----
(8) Eicke, H. F.; Quellet, C.; Xu, G. J . Surf. Sci. Technol. 1988,4, 2. (9) Petit, C.; Pileni, M. P. J.Phys. Chem. 1988, 92, 2282. (10) Petit, C. Thesis, Universite P. et M. Curie, Paris, 1985. (11) Luisi, P. L.; Majid, L. J. C.R.C.Crit. Reo. Biochem. 1986,20,409. (12) Terech, P. T h h e d'btat, Grenoble, 1983.
0743-7463/91/2407-0223$02.50/0
Experimental Section Gel Preparation. In aqueous media, the gel is in the form of fibers where the molecular unit is a rod around 3000 A long with a diameter that can vary, depending on the degree of 0 1991 American Chemical Society
Petit et al.
224 Langmuir, Vol. 7, No. 2, 1991
/"
0 0
I
1
IO
20
I/
s
OT
V
I
I
I
0
IO
10
30
Polu Volum
Figure 2. "Phase diagrams" of gelatin-water-AOT-isooctane. This representation shows the different regionsobserved a t room temperature for two different concentrations in AOT. The two phase region is a demixion, the precipitate region is a segregation (there is not enough water to solubilize all the gelatin). The junction of the four regions is called the "saturation point".
Figure 1. Three models of gel (see text): (Model 1)cylindrical model (from ref 7); (Model 2) reverse micelles and cylindrical structure (from ref 5); (Model 3) reverse micelles interconnected by gelatin rods (from ref 3 and our results). hydration, between 11 and 14 A.13 This unit is made up of approximately 1000 amino acid residues and has an average molecular weight of around 200 OOO. We must, nevertheless,stress the very large differences in weight and size of the molecular unit, depending on the its origin and the extraction method, as all these studies were made with the same lot of gelatin. The solubilization of the gelatin in reverse AOT micelles was carried out by using the procedure described by Haerings et al.:4 The gelatin was directly incorporated in powder form (Fluka bloom 250) in the micellar AOT solution (Sigma, used without further purification) of isooctane (Fluka, puriss) and water in the desired amount ( W = [H20]/[AOT]). The solution was immediately heated to 50 "C with rapid stirring until a homogeneous opalescent mixture was obtained. The sample was then cooled to room temperature. The solutions thus obtained are optically clear and their viscosities vary with the initial gelatin concentration. Conductivity. The conductivity measurements were made with a Tacussel CD810 conductivity meter using a GK2401C cell from the same manufacturer. This cell was placed in the sample being studied. The measurements were made a t room temperature once equilibrium was established and were, thus, perfectly reproducible. SAXS Experiments. The small-angle X-ray scattering was done at L.U.R.E., Orsay (France), on the D22 beam line with a detector-sample distance of 1 m and radiation with X = 1.22 A. Additional studies were made on a GDPA30 goniometer with copper Ke radiation (1.54 A). The experimental arrangement used has been described previ0usly.1~ SANS Experiments. The small-angleneutron scattering was carried out with the PACE spectrometer of the Orph6e reactor (13) Djabourov, M.; Leblond, J.; Papon, P. J . Phys. (Paris) 1988,49, 319. (14) (a)Zemb, T.;Charpin, P. J . Phys. (Paris)1986,46,249. (b) Zemb, T.; Rapport CEA R-3501, Service de Documentation du CEN Saclay, 1985.
a t L.L.B. (CENSaclay, France). The wavelength was 5 A and two sample-detector distances were chosen, giving a range of the accessible Q values from Qmin = 5 x to Q- = 0.4 A-1and, thus, a resolution in real space from Dm,, = 200 A to D m i n = 2.5 A.The samples were placed in 1 mm thick quartz cells; only the water was deuterated (99.5 % D20 from the "Service des molbcules marqu6esn, CEN Saclay, France). Quasi-Elastic Light (QELS) Experiments. The QELS experiments were made with a 136-channel Brookhaven 2130 AT correlator. The light source is an argon laser (5145 A). The power is 0.5 W. The measurement were made a t 25 "C; the angle of diffusion is 45O. All the samples are filtered and centrifuged to eliminate dust.
Results Phase Diagram. The different regions observed a t room temperature for two different AOT concentrations as a function of the gelatin concentration in the medium (expressed in weight percent of gelatin in the volume of the solution) and of the polar volume fraction of the medium are shown in Figure 2. The relation between these quantities is Four different regions can be distinguished macroscopically: A liquid region: non-birefringent; optically clear liquid solution. A solid region: optically clear physical gel. This is not exactly a distinct phase, but as observed for gelatin in water, a concentrated region of the sol phase; the transition line is not abrupt but presents a certain extension. As usual, there is no structural variation along the gelation line, but only a strong variation in the rheologic property of the sample. A precipitate zone, where all or part of the gelatin in powder form cannot be solubilized. This means a macroscopic segregation. A two-phase region: there is a demixion zone in the presence of a solid gel below a high viscosity liquid. For the structure studies we will call the junction point of the different regions in the phase diagram (Figure 2) the "saturation point". These points are found a t the gelation threshold with the minimum and maximum of gelatin; the addition of gelatin is impossible (solubility limit). If the amount of gelatin is decreased, the sol region is reached. In the same way, if water is added, the system
Structure of Microemulsion-Based Gels
Langmuir, Val. 7, No. 2, 1991 225
Table 1. Evolution of the Ratio [Gelatin]/[Micelle] at W = 25 for Different Concentration of AOT [gelatin], vi2 (m/v) 6.5 10 14
[gelatin], mol.L-’ 3.25 x 10-4 5 x 10-4 7 x 10-4
[ AOT], mo1.L-l
0.1 0.15 0.25
[micelle], mo1.L-l 3.3 x 10-4 5.1 X 8.5 x lo-*
[gelatin]/ [micelle] 0.96 0.98 0.82
goes over to the gel region. If the water content is decreased, then there are two phases. A t the “saturation point” all the water and all the gelatin are necessary and take part in the structure. It is first essential to determine the structure a t the saturation point, then to study the variation of the structure when one of the components is in excess. There is a unique saturation point for each gelatin concentration and it is remarkable to observe that the ratio, [gelatin]/[micelle] (Table I), is constant; it is, therefore, the number of micelles that appears to influence the gelation and the concentration of surfactants for these different saturation points. This process is difficult to understand on the basis of model 1 or 2. The sol-gel transition is shown by an increase of the viscosity of the medium in arather narrow range (typically, 1w t r o ) of gelatin concentration. In the same way, NMR studies on the broadening of the water signal,’ as well as our results,1° show the continuing increase in the line widths, which is characteristic of an increasing immobilization of part of the water on ge1ati0n.l~By comparison of the gelation regions for several gelatin concentrations (0.15 and 0.25 mo1.L-’) (Figure 2), the following three assertions can be made: 1. Gelation occurs only with significant gelatin concentrations (6-15 50, depending on the experimental conditions) while in aqueous solutions 0.5 % of gelatin (M/ V) is enough togel the medium.13 This difference increases with the AOT concentration. This result is uncompatible with the model 1. Inside the micelles, radius of gyration (R,) of the polar part of gelatin has to be decreased due to the ionic strength (local concentration of Na+ = 3 mo1.L-l). Therefore, the radius of the water pools at W > 25 is always larger than R, of the gelatin. 2. The ratio of the solubilized gelatin concentration on the micellar concentration shows that there is practically one molecule of gelatin per micelle at the saturation point (Table I). 3. For the different surfactant concentrations used, it was never possible to obtain a gel for a water content in the initial microemulsion of less than W = 25. This water content, W ,corresponds to a water-pool radius of the microemulsion equal to 40 These three observations show that in microemulsionbased gels, the micellar structure strongly influences the mechanism of gelation: in bulk water, gelatin molecules stretch due to stiff helixes in the structure, whereas the reverse micelles induce confinement of the polar part of the gelatin inside small regions. This micellar size effect on the gelation process is incompatible with models 1 or 2, in which gelatin, surfactant, and water are redistributed. Electrical Conductivity. The measurement of the electrical conductivity allows us to test the continuity of the aqueous media. The electrical conductivity shows an increase of 3 orders of magnitude between the liquid (insulating) phase and the solid (conducting) phase. The conductivity curves obtained for different samples are shown in Figure 3; they
a.2d
(15) Abragam, A. In Principe du Mag6tisme Nuclgaire; PUF: Paris,
1961.
0
2
4
6
8
10
12
% of Celatin(WN)
Figure 3. (A) Variation of the hydrodynamics radius, obtained by QELS with the gelatin content (in percent of weight per volume) ([AOT] = 0.15 mo1.L-l W = 45). (B)Variation of the conductivity with the gelatin content (in percent of weight per volume). The hatched aera corresponds to the sol-gel transition. The increase in the hydrodynamics radius is strongly correlated with the increase in the conductivity: the transition is a percolative phenomenon.
are small a t low gelatin concentrations, then increase a t the sol-gel transition (hatched area in Figure 3). There is also a strong correlation between the modification of the physical properties of the medium and the change of its macroscopic state; the increase of the electrical conductivity with the gelatin content starts a little before the sol-gel transition and seems to stabilize a t a high value (10pS) that is quite similar for the different samples tested. This abrupt change of the conductivity is a typical manifestation of the passage from a dispersed to a highly interconnected continuous medium.16 This behavior has been observed in other very similar systems.3~5,~This drastic modification of the physical properties of the medium is again found by QELS in the very large and rapid increase of the hydrodynamic radius of the solution shortly before gelation (Figure 3). Below the gelation, solubilization of gelatin in reverse micelles first increases the apparent hydrodynamic radius of the micelle. At a gel content lower than 45’6,the (16)(a) Mitescu, C. D.; Musolf, M. J. Phys. (Paris),Lett. 1983,44, L-679. (b) Eicke, H. F.; Hilficker, R. H.; Thomas, H. Chem. Phys. Lett. 1986,125, 295.
Petit et al.
226 Langmuir, Vol. 7, No. 2, 1991 Table 11. Composition of the Sample Studied by SANS or SAXS and Surface per Polar Head Group Obtained by SANS sample
A
R C
n
[gelatin],
%I
10 8.5 14 14
(m/v)
W
[AOT], mo1.L-*
30 50 35 30
0.15 0.15 0.25 0.25
0,
A2
50
IO 50 45
apparent hydrodynamic radius (obtained by dilution of the sample) is close to an “empty” reverse micelle. The change in the diffusion coefficient observed by dilution shows that even before the gelation occurs, there is intermicellar attraction due to the gelatin solubilized in the sample. It probably forms micellar aggregates that are the precursor of the macroscopic gel. This shows the change of scale of the medium in the course of the process. The report of Quellet6 on gelation under experimental conditions close to these clearly shows the occurrence of this percolation phenomenon on gelation. According to model 3, these conductivity measurements can be explained in term of an electric percolation threshold associated with the gelation. Using model 1or 2, the electrical conductivity should be very high and invariant. Pulse Radiolysis Experiments. The observation of the hydrated electron by pulse radiolysis makes it possible to confirm the presence of free water in the microstructure and here also to distinguish between the models. The irradiation with high-energy electrons of a micellar gelatin solution results in the formation of hydrated electrons, transient species characterized by a strong absorbance band centered a t 720 11m.l’ The fact that the hydrated electron can be observed proves the existence of sufficiently large pockets of free water in the gelled medium.18 The reorganization of the medium in the gel phase, therefore, leaves a place for the presence of “water pockets”. These have a nonnegligible size but one that is still less than that of the initial micelles. It must be noted, however, that the formation yield decreased rapidly with the gelatin concentration and initial water content of the system. There are practically no electrons observed at the saturation points of the phase diagram, which shows the absence of free water which, therefore, is used to hydrate the polar heads of the s ~ r f a c t a n t ’or ~ to hydrate the gelatin’s This is so even if a very fast reaction of the hydrated electrons with the gelatin cannot be excluded, which would indicate a much greater proximity of the species with low water contents. SANS Experiments. The small-angle neutron scattering measurement is sensitive only to the structure of heavy water and it is understood that for model 1 this should be a spectrum for a hollow cylinder,12while models 2 and 3 imply a spectrum for spherical water droplets. The deuteration of samples does not change the phase diagram. All the scattering experiments both with neutrons and X-rays were carried out on four samples with different gelatin and AOT concentrations (Table 11);these correspond to the two saturation points observed in the phase diagram for [AOT] = 0.15 mo1.L-l and [AOT] = 0.25 mo1.L-’ (samples A and D), respectively, as well as for (17) Pileni, M. P.; Hickel, B.; Ferradini, C.; Pucheault, J. Chem. Phys. Lett. 1982, 92, 3087. (18) Petit, C.; Brochette, P.; Pileni, M. P. J. Phys. Chem. 1986, 90,
651 7 .
(19) Wong, M.;Thomas, J. K.; Nowak, T. J. Am. Chem. SOC.1976,98, 2391.
two gels obtained with higher water concentrations (samples B and C). In this experiment only the deuterated water is visible. This involves, therefore, the form of the structures which contain the water in the gelled system: the small-angle neutron scattering spectrum for these samples (Figure 4) shows, in the log (Z(q)), = f(1og ( q ) ) plot, behavior asymptotic to the great wave vector, q ; in q-4 (a constant slope of -4),this is characteristic of an interface.20 The total surface per unit volume, ET,can, therefore, be measured by using Porod’s equation20as well as the surface per AOT head, u, since the AOT concentration is known (Table 11). This value is very close to the value presently accepted in the literature for the surface per polar head of AOT.21 Figure 4 shows that the diffusion is clearly that of spherical droplets. In particular, there is no region in q-l, characteristic of cylinders for qR, C 1, visible in Figure 4. Moreover, for q = 0.1, model 1 (cylinder model) predicts 100 times greater scattering (I(q = 0.1) 10 cm-l) than that actually observed (Z(q = 0.1) 0.1 cm-l). There are two possible interpretations: Strong repulsions between cylinders lead to S(q) C 1, which allows us explain the difference between the theoretical curve for the cylinders and the observed scattering. But the lack of scattering a t q C 1A-1 cannot be explained nor can the physical origin of this repulsion between cylinders. Attractive interactions between spherical droplets explain the excess scattering (S(q = 0) = 10) at small angles. This effect has already been observed for mixtures of micelles and proteins21bs22 and may be attributed to attractive interactions due to the gelatin (protonated, thus, directly invisible to neutrons in this contrast). 5. SAXS Experiments. Small-angle X-ray scattering (SAXS) makes it possible to observe the diffusion of gelatin and water, due to their high electron density ( p = 0.41 e-/A3 for the gelatin). The different plots of the scattered intensity I ( q ) as a function of the wave vector q are given in Figure 5 for the sample A (similar results are obtained for the other samples). Here again, we find an asymptotic behavior of log (Z(q))= f(1og ( q ) )close to -4 a t large values of q. Ths allows us to measure the sum of the oil-gelatin and oilAOT interfaces in the medium. Figure 5 shows that the scattering spectrum obtained for X-rays is very different from that obtained from neutron scattering. At very small wave vectors the behavior observed is no longer that of a spherical structure; the log (qZ(q))= f ( q 2 )plot clearly shows a linear slope. This plot is characteristic of cylinders,23 since for qR < 1, the scattered intensity from a cylindrical structure is given by24 q I ( q ) = d P o 1 V , FAp2 exp(-q2R2/2)
qR C 1 Where V I is the volume per length unit of the cylinder ( s R 2 )and F is the Thompson factor. (20) Porod,G. In Small AngleX-RaysScatterings;Glatter,O., Kretky, O., Eds.; Academic Press: New York, 1982. (21) (a) Cabos, C.; Delord, P. J. Appl. Crystallogr. 1979,12,502. (b) Brochette, P.; Petit, C.; Pileni, M. P. J. Phys. Chem. 1988, 92, 3505. (22) Chen, S. H.; Texeira, J. Phys. Reu. Lett. 1986, 57, 2583. (23) Cabane, B. In Surfactant in Solution, New Method of Inoestigation; Zana, R., Ed.; Marcel Dekker: New York, 1987; p 57. ( 2 4 ) Porte, G.; Marignan, J.; Bassereau, P.; May, R. J. Phys. (Paris) 1988, 49, 511.
Langmuir, Vol. 7, No. 2, 1991 227 4
-
--.
i
2-
-
0-
3 -2
Sample A
-4-
-
-6
I I I I I I I I I -2.3
-1.9
-1.5
-1.1
-0.7
-0.3
-2.3
-1.9
-1.5
-1.1
-0.7
-0.3
log(q)
-4
-2.3
-1.9
-1.5
-1.1 log(q)
-0.7
-0.3
' -2.3
I
I I I I I I I I I -1.9
-1.5
-1.1
-0.7
-0.3
loe(P)
Figure 4. SANS experiments on the four samples studied (for the composition see Table 11). All plots are log ( I ) = f(1og ( 9 ) ) . (- - -1
Simulated diffusion for a cylindrical system (&,.I = 54 A). In this model all the gelatin and water are coated by the surfactant (see text). (-) Simulated diffusion for spherical system. The polar volume corresponds to the total water and 30% of the gelatin volume. I ( q ) = Ssphere(q) Paphere(q) is obtained by the Hayter-Penfold method (see, for example,'ref 14b).
The slope of this linear plot gives the average radius of these cylinders (Figure 5c). The radius of the gelatin cylinders connecting the micelles thus shown is close to 25 A for the samples studied (Table 111). The reference micellar sample used does not give such a linear slope. The determination of the total interface of the diffusing particles also shows an increase of the former, due to the presence of gelatin in the system (Table 111). This demonstrates the direct contact of, a t least, part of the gelatin with the isooctane since the surface per polar head of the surfactant is constant; thus, part of gelatin participates in the interface and is directly exposed to contact with the apolar phase.
Discussion The two scattering techniques clearly show both the structure of 25 8,radius cylindrical strands, seen by smallangle X-ray scattering, and the structure of self-attractive spherical drops, seen by neutron scattering. The scattering studies are, therefore, a priori, consistent with models 2 and 3, for only these studies allow us to affirm the simultaneous occurrence of water droplets and gelatin cylinders. Only the study of the phase diagram allows us to see if connecting links in the network of cylinders correspond or not to the number of drops in going through the sol-gel transition; the observed demixion (two-phase domain) is, for example, difficult to explain in the framework of model 1 or 2. As the surface per polar head of AOT is invariant, the total interface of the system, ZAOT,is, a priori, known for each composition. Knowing the dispersed polar volume, due to the water and the gelatin, the geometry of this quaternary system is known: number of cylinders per unit
volume, number of spheres, length of the cylinders, and the average distance between two adjacent micelles. The gelation threshold is reached when the number of cylinders is of the order of one per micelle (connectivity index, 2 = l . l ) . 2 5 One can, therefore, also calculate, a priori, the position of the gelation threshold. Figure 4 has shown that the scattering of neutrons can be simulated by that of spheres where the geometry is fixed by the experimental conditions where p is the only quantity to be fixed; it is the fraction of the polar volume of the gelatin. The increase in the size of the droplets obtained from neutron scattering in reference to the empty micelles shows that part of the gelatin must be solubilized in the aqueous phase; this fraction is estimated to be 30% of the total gelatin, depending on the geometry of the system. Thus, p is fixed at 0.3; approximately 30% of the gelatin penetrates the water pools and 70% is in direct contact with the isooctane or in the interface. The proportion of gelatin present outside or in contact with the apolar medium may seem large for this hydrophilic protein.'3 But the structure in a helices is known to be more hydrophobic than the random coil structure26since i t results in a greater fraction of the hydrophobic amino acids on the surface. Moreover, our X-ray scattering results show an increase of the total interface of the order of 30% with respect to that obtained from neutron scattering, the available surface per polar head being invariant. This shows that part of the total (25) Zemb, T. N.; Hyde, S. T.; Derian, P. J.; Barnes, I. S.;Ninham, B. W. J. Phys. Chem. 1987, 91, 3814. J . Phys. Chem. 1988, 92, 2286. (26) Lehninger, A. L. Biochemistry; Worth Publisher: New York,1975.
Petit et al.
228 Langmuir, Vol. 7, No. 2, 1991
Table IV. Comparison between the Theoretical Surfaces Needed for a Cylindrical Network and the Real Surface
A B C D
B
0.15 0.15 0.25 0.25
30 50 35 30
3.4 x 10-2 2.9 X 4.8 X 4.8 X
9 x 10-4 7.7 X 12.6 X 12.6 X
5.4 x 10-3 5.4 X 9X 9X
structure of this gel is, therefore, a continuous water structure, as it is conducting, which, nevertheless, keeps the dispersion of the spherical water droplets very close to that of the initial micellar phase. The possibility of producing a significant amount of hydrated electrons in this confirms the presence of this water pocket. Let us examine, for example, the scattering that should be observed in the case of model 1:a network of hydrated gelatin chains covered by a layer of ~ u r f a c t a n t .Since ~ the geometry of the system is fixed, we can calculate the total interface necessary to cover such a cylindrical network 2, = 2nRg1,
C
0,001
0.0026
0,0018
0,003
q2
Figure 5. Small-angleX-rayscatteringobtainedon D22 at LURE (Orsay, France) ([AOTI = 0.15 mol-L-', W = 30, [HzOI = 4.5 mol.L-l, and 10% (w/v) gelatin): (A) I ( q ) = f ( q ) ; (B) log (I) = f(1og ( q ) ) , the slope at lwo q (close to -1) is characteristic of cylinders; (C) log ( I ( q ) q ) versus 92, the slope gives the radius of the cylinders. p = - (R,/2)1/2. Table 111. Radius of the Cylinder and Total Interface, 9, for the Different Samples Studied samDIes
A B C D reP
[gelatin], ?, ( m / v ) 10
8.5 14 14 0
[AOT],
mo1.L-1
W
0.15 0.15 0.25 0.25 0.25
30 50 35 30 35
&I,
A
25 34 28 25 -
ZT, A2/A3
7.5 X 7X 11.5 X 12 X 9 x 10-3
Reverse micelle without gelatin.
interface of the system observed by X-ray scattering is due to the gelatin which is, therefore, in direct contact with the apolar phase. Other studies of the gelatin have shown that it can be easily absorbed on waterloil and water/air interfaces.*' It is, then, probable that the gelatin ensures the junctions between the spherical droplets observed by neutron scattering, as Quellet assumed in his thesis.6 This picture is confirmed by small-angle X-ray scattering which shows the presence of 25-A cylindrical structures (which implies several chains of gelatin per cylinder as the radius of the hydrated gelatin is 7 A, thus, ensuring conducting channels) which would ensure the junctions between the spherical components (which essentially contain the water). The swelling observed for the cylinders in sample B is, then, due to the presence of free water in the connecting cylinders (Table 111). The (27) Wustneck, R.; Warnheim, T. Colloid Polym. Sci. 1988,266,926.
where R , is the average radius of the hydrated gelatin and 1, is the length of the gelatin chains in the medium. Table IV clearly shows, then, that in this model there is not enough interface to cover all of the network; another order of magnitude is required (this is especially true for samples A and D, corresponding to the saturation points of the phase diagram where the water is involved only in hydration of the constituents and where the geometrical data are the most accurate). The same conclusion is also valuable, in our experiments, for model 2. Even if a cylindrical network, where the gelatin chains were tightly coiled in the 25 A radius cylinders, is envisaged, there is still not enough interface to cover the network. Moreover, the neutron spectra are not consistent with the formation of a cylindrical network (Figure 4); the simulation of the neutron scatterings by a network of cylinders is, in all the cases, very different from those actually observed (Figure 4). The simulation was made for infinite cylinders where the radius is fixed by the system geometry Rc
= "pol/
'AOT
where ap0lis the fraction of the polar volume, taking the total of the gelatin and the water into account. The scattering calculated on an absolute scale for all the D2O in the sample if the water was contained in a locally cylindrical structure or dispersed droplets is shown on a logarithmic scale in Figure 4. The theoretical calculation was made by assuming independent entities: S(q) = 1for any value of q. The radius of the cylinders is known (R, = 54 A), since the volumes and the surfaces are known. The scattered intensity is given by the product I ( q ) = P(44 S(q) We assume that S(q) = 1. An excess of observed scattering will, then, be interpreted as an attraction and conversely. The following theoretical equations give the structure factor, P(q)? q P ( q ) = x+p,lV,F Ap2 exp(-q2RC2) if qR,
, )1I ~ Jo(x)is the first Bessel function. Figure 4 clearly shows that the scattering from a cylindrical structure never takes (28) Guinier,A.; Fournet, G. Small Angle Scatterings of X-rays;Wiley: New York, 1955.
Structure of Microemulsion-Based Gels
Langmuir, Vol. 7, No. 2, 1991 229
I(a 1
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01 0 -0 I t
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9
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q
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i
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q
Figure 6. Experimental X-ray diffusion (0) and theoretical diffusion calculated for the model 3. Z(q) = f(q). The lines represent ~ ~ @.pol the theoretical diffusion of our models: spheres (radiusdepends on the polar volume and the total interface, Rs= 3 @ p o l / 2 ~with = @water + O.3*p,ei) and cylinders (R, = 25 A). I ( q ) = S(q)spheres(P(q)sph iP(q),l) (see text).
account of the experimental results, especially for the asymptotic limit a t great wave vectors. Our measurements are, therefore, consistent uniquely with model 3, with 25 A radius cylinders containing approximately 70% of the initial volume of the gelatin. These cylinders connect, between each other, spheres which have a radius determined by the polar content. What, then, are the diffusion predicted by model 3? Simulation of t h e Experimental Results by t h e Model. The radius of the droplets, R,, is given by
R , = 39p,,/2.AOT with
aPol = @water + 0.39ge1
The intermicellar distance, D*, is fixed by the concentration, nmic,of micelles in the medium29
D* = l.22n,ic-1/3 Knowing the length of the gelatin, I,, available in the medium, gives us the connectivity index, 2, of the gel
z=(1- P)~,(R,/R,,~)~
nmicD* withp = 0.3, the gelatin partition coefficient, R, the radius of the hydrated gelatin in the helices (7 A), and Rcyl the radius of the connecting cylinders (25 A). The structure factor, P ( 9 ) ,is, then, approximated by
neglecting the sphere-cylinder cross terms: Z cylinders are associated with each sphere.25 We take here S(q) = S(q)spheres. These expressions allow us to calculate the theoretical scattering on an absolute scale of our model. It is important to specify that it is not a question of adjusting the theory to the experiment, as is generally done, but of the scattering calculated from a quantitative model obtained from the ensemble of our experimental results. Good agreement with the resultsis then observed, since the difference, theory-experiment, is less than 10% for all of the samples (Figure 6). It is also possible to better understand even the gelation process and, in particular, the phase diagram of the system as shown in Figure 2. The results previously published for this process show, as do our results, that it involves a polar media percolation phenomenon, a process clearly explained for this type of “unorthodox The important parameter controlling the gelation is the chain length, l,, of the available gelatin in the medium. This must be sufficient for the percolation of the system; there is, then, an important criterion for the gelatin concentration necessary to provide, at least, a continuous path between the different micelles30 where Zminhas been determined to be 1.1from geometrical
(29) Ninham,
B.W.;Barnes, I. S.; Hyde, S. T.;Derian, P.J.; Zemb,T.
N. Europhys. Lett. 1987,4, 561.
(30)Clarkson, M.T.Phys. Reu. Lett. A 1988, 137, 2079.
Petit et al.
230 Langmuir, Vol. 7, No. 2, 1991 AUT = 0. 15 I11oI.L- I
studies of the percolation between random ~pheres.3~ The length of the cylinders, Lcyl,is derived from the length of the gelatin and the cylinder radius, determined experimentally and verified by the calculation of the scattering intensity
20
15
Lcyl
= 1,(1 - P)(R,/RCYJZ
where R, is the average radius of the hydrated gelatin and 1, is the length of the gelatin chains in the medium (which is easily determined from the average length of the molecular unit, 3000 A).13 Enough water is required to hydrate the system, Le., both the AOT and the gelatin, giving the hydration condition
10
5
-
The gelatin should not have steric constraints that hinder the random coil-helices transition that characterizes the ge1ati0n.l~ In particular, the size of the micelle should be, at least, similar to that of the random coil of the gelatin (in the opposite case, as has been already seen with other biopolymers there is hindering of the renaturation of the proteinza)
0
5
e-”
20
I
10
I5
20
I
I
I
2s
30
’
Rmicelle Rrandom coil (3) The radius of the coil is easily obtained for a random chain of 3000 A consisting of N = 1000 residues, each one contributing a length of b = 3 A to the total length of the chains; the radius is given by
10
(R2) = b2N/6,R = 39 8, which corresponds to micelles with a minimum water content, W = 25; here, we again find a limitation observed for the phase diagram of the system. From these three constraints, which are the minimal constraints operating on the system, it is possible to determine the ensemble of the compositions in the phase diagram for which these three conditions are confirmed. The two gel existence regions thus determined for two different AOT concentrations re shown in Figure 7; it can be seen that the sphere-cylinder model is in good agreement with the experimental results, in particular, for the composition of the different saturation points, although there are no adjustable parameters in this theoretical diagram. It is to be noted that, experimentally, the gel phase is always of lesser extent on going to lower gelatin concentrations; this may be due to a salt effect as there is a nonnegligible concentration in the water pool (counter Na+ ions of the AOT21b). It is, in fact, known that the addition of salt perturbs the random coil-helices transition by screening the charges and thus increasing the concentration required to obtain a gel.32 The purely geometrical aspects of the model do not take into account the effect of the surfactant counterions on the conformation of the gelatin. Similarly, the existence of the twophase region is not predicted by this structural model. It may involve a separation between two media, one a connecting liquid, Z = 1.1,the other a strongly connected solid. The attraction, here, is induced by a molecule but the situation is very analogous to the separation into two phases near critical points for reverse micelles.33 The quantitative parameters used in our model, which are not adjusted, give, thus, a good account of both the phase diagram and the small-angle scattering. (31) De Gennes, P.G . Scalling Concept in Polymer Physics; Cornel1 University Press: New York, 1979. (32) Boedtker, D.; Doty, P. J . Am. Chem.SOC.1954,58,969. (33) Roux, D. Thesis, Universite de Bordeaux I, Bordeaux, 1984.
5
0 0
5
10
I5
Q of
Polar Volume
20
25
30
Figure 7. Simulatedphase diagrams, obtained from our models (seetext). Each AOT micelle which contains a part (30%)of the gelatin molecules and water is connected via one gelatin strand to a neighboring micelle, ensuring that the extent of the connections is large enough to induce a macroscopic gelation of the solution, since the percolation threshold is 1.1cylinder per sphere. The line refers to the experimentaldiagram (see Figure 2), and the theoretical gel region corresponds to the circle region (0).
Conclusion The structure of the gel obtained by solubilization of the gelatin in reverse AOT micelles provides a good description of the process as a mutual gelation of the gelatin and the microemulsion.3 The discontinuous spherical structure characteristic of the reverse AOT micelles is retained during all of the gelation; the radius is always fixed by the ratio of the polar volume to the surface of the available surfactant. These independent drops are interconnected by cylindrical links mainly free from surfactant monolayer and made up of the apolar parts of coiled gelatin strands. Indeed, the precision of the scattering experiments allows us only to evaluate the interfacial area with 1070 precision; the major part of the surfactant is needed to coat the discontinuous spherical droplets. However, a minor part of the surfactant molecules can be adsorbed on the gelatin strains. Partial adsorption of AOT on the gelatin strains can explain the high ionic conductivity which would be due to the Na+ counterions of the surfactant (as observed by Howe et al.5). Gelation occurs when the number of strands per micelle is greater than one. In the case of
Structure of Microemulsion-Based Gels large micelles and in the presence of excess gelatin there is very strong connectivity and demixion is observed. The medium appears to be, then, like a reverse micellar system interconnected by helical chains of gelatin, tightly coiled in joining cylinders with 25-A radii. These joining cylinders are directly exposed to the continuous apolar phase and are ensured by around 70% of the total gelatin concentration. We have, thus, been able to specify model 3 qualitatively and quantitatively and it is in good agreement with the ensemble of the results, while models 1 and 2 are only in partial agreement with our results (although the experimental conditions are quite different, particularly in the case of model 2 (see note)). This model allows, in particular, the prediction of the observed scattering as well as the stability zone of the gel phase in the phase diagram. Finally, each of the models allows the calculation of a composition range for which the proposed structure can be formed. For example, to construct the microstructure 1it would be necessary to have enough surfactant to cover all the cylinders. To construct the microstructure 3, the amount of gelatin necessary is independent of the water content of the microemulsions. The range of composition for which a given microstructure is sterically possible can be calculated by taking the molecular volumes and surfaces into account. Our model allows an estimation of the relative volume contained in the connecting cylinders and spherical droplets for any composition. At the saturation point, a ratio of 30% of the gelatin volume inside the micelles and 70!‘0 in the links gives good agreement with scattering.
Langmuir, Vol. 7,No. 2, 1991 231 However, this partition ratio of 30% to 70% has no reason to be constant in the phase diagram. Measurement of this partition ratio with different proteins and polymers is probably a key issue in the understanding of protein/ reverse micelles composite structures.
Note Added in Proof: Since the redaction of this paper, Atkinson et a1.34 describe experiments supporting model 2, incompatible with our measurements. However, it should be noticed that the compositions of the samples studied by Atkinson (AOT 0.1 M, W = 55, gel = 3.5%)are located very far from the saturation point, deeply inside the gel domain. Therefore, the structure they have found is probably also right because they work in a very different part of the phase diagram. Our study is relative to the “saturation point” because quantitative calculation of the full X-ray and neutron scattering on absolute scale are only possible near the saturation point. In their case, the area per headgroup, directly measured by the Porod asymptotic limit without any structural assumption, is 50 A2/molecule. The total surface of the gelatin network is l o 5 cm2/cm3. The available surfactant film in their sample is 3 x lo5cm2/cm3. Hence, for their composition, model 2 is acceptable because there is enough available surfactant to cover gelatin and independent excess reverse micelles. The model proposed by Atkinson is clearly incompatible in our composition range with the difference seen between X-ray and neutron scattering for our composition. (34) Atkinson, P.J. J. Chem. Soc., Chem. Commun. 1989, 1807.
