Langmuir 1991, 7, 250-253
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Lecithin Microemulsion Gels: A NMR Study of Molecular Mobility Based on Line Widths Donatella Capitani, Anna Laura Segre,* and Roberto Sparapani Zstituto d i Strutturistica Chimica “Giordano Giacomello”, C.N.R. Area della Ricerca di Roma, C.P. 10-00016, Monterotondo Stazione, Roma, Italy
Mauro Giustini, Roger Scartazzini, and Pier Luigi Luisi Znstitut f u r Polymere, ETH-Zentrum, Zurich, Switzerland Received March 12, 1990. In Final Form: July 2, 1990 Microemulsion gels obtained from solutions of lecithin in organic solvents (in particular isooctane) in the presence of a small amount of water are studied by NMR line width measurements. The ‘H, 13C,and slP resonances of the lecithin molecule are investigated as a function of added water w, (w, = [HzO]/ [LEC]). The line width of the 31P resonance increases sharply at the w, value that induces the maximal viscosity (w,= 3 ) , and similar to the viscosity, the 1H resonance of the -N+(CH& polar head, as well as the l3Cresonances of the -CH2CH2N+group, shows a maximum in their line width (i.e. a minimal molecular mobility) around w, = 3. Conversely, at this w, the mobility of water does not appear to be particularly restricted; actually an effect is noticed only by the addition of the first molecule, which induces a significant increase of mobility both in the polar head of the phospholipid moiety and in the water itself. More in general, the molecular details obtained by NMR data are compared with the data obtained by other chemical physical techniques that are only sensitive to the macroscopic molecular structure. The bulk of data indicates that (i) the highly flexible, transient network constituting the gel consists of entangled polymer rodlike lecithin reverse micelles, (ii) such a network structure is formed continuously upon addition of water rather than in an all-or-nothing transition, (iii) solvent and water mobility do not correlate with macroscopic gel viscosity, whereas there is a clear correlation between viscosity and stiffening of the phosphorus atom, (iv) during water addition and gel formation significant change in mobility of specific lecithin groups occur, and (v) in particular, the first added water molecule induces a conformational change of the lecithin polar head.
Introduction It has been recently shown that solutions of surfactants in organic solvents in the presence of a small amount of water can be transformed into gels displaying a very high viscosity (of the order of 10 000 P). Both gelatine’ and lecithin “microemulsion gelsw2have been described and the former have been partly characterized by physical chemical methods.3-8 Their structure is still under investigation. The question of structure is of course more challenging in the case of lecithin gels, because no polymer is present in the system. Recent rheological,9 quasielastic light scattering (QELS) and small angle neutron scattering (SANS) measurementslO clarify some basic structural features; under our conditions, the addition of water to lecithin reverse micellar solution induces one-dimensional growth of the aggregates into long cylindrical reverse micelles. At a threshold lecithin volume fraction, these cylindrical reverse micelles interact with each other forming a dynamic network that breaks and builds up very rapidly in a time long enough to result in a large
* To whom correspondence should be addressed.
(1) Haring, G.; Luisi, P. L. J. Phys. Chem. 1986, 90, 5892.
(2) Scartazzini, R.; Luisi, P. L. J. Phys. Chem. 1988, 92, 829. (3) Capitani, D.; Segre, A. L.; Haring, G.; Luisi, P. L. J. Phys. Chem.
1988, 92, 3500. (4) Petit, C.; Pileni, M. P. J. Phys. Chem. 1988, 92, 2282. (5) Quellet, C.; Eike, H. F. Chimia 1986, 40, 233. (6) Daum, U.; Wei, G.; Luisi, P. L. ColloidPolym. Sci. 1988, 266,657. ( 7 ) Haring, G.; Schurtenberger, P.; Luisi, P. L., work in preparation. (8) Luisi, P. L.; Scartazzini, R.; Haring, G.;Schurtenberger,P. Colloid Polym. Sci. 1990, 268, 356. (9) Schurtenberger, P.; Scartazzini, R.; Luisi, P. L. Rheol. Acta 1989, 28. 372. (10) Schurtenberger, P.; Scartazzini, R.; Magid, L. J.; Leser, M.E.; Luisi, P. L. J. Phys. Chem. 1990, 94, 3695. ~~
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macroscopic viscosity. Recent findings in the investigation of aqueous micellar solution a t high ionic strength support the validity of the proposed m~del.~JO The situation is similar to one described by Candau et al.11J2 and Hoffmann et aI.;l3J4 in particular Candau was able to show that the results from static and dynamic light scattering and rheological measurements performed on viscoelastic surfactant solution could be successfully interpreted in terms of theories used to describe the behavior of semidilute polymers. Recent theoretical work for microemulsion systems by Safran et al.15 predicts the existence of polymer-like phases, associated with the formation of long and flexible cylindrical microemulsions. In the case of lecithin gels, scattering and rheological techniques are certainly the most efficient for characterizing the macromolecular characteristic of the gel structure. However, they are intrinsically incapable of describing the molecular details. Particularly when questions relative to the involvement of different chemical groups in the structure, or questions relative to the interaction between atomic groups and solvent arise, the method of choice is rather NMR. For these reasons, a detailed NMR study of the lecithin gels has been undertaken, which includes both pulse low resolution proton NMR (measurements of T1 and T2) as ~
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(11) Candau, S. J.; Hirsch, E.; Zana, R.; Adam, A. J. ColloidInterface Sci. 1988, 122, 430. (12) Candau, S. J.; Hirsch, E.; Zana, R. J.Colloid Interface Sci. 1985, 105, 521. (13) Rehage, H.; Hoffmann, H. Faraday Discuss. Chem. Sot. 1983,76, 363. (14) Hoffmann, H.; Rehage, H. Surf. Sci. SOC.1988, 22, 209. (15) Safran, S.; Turkevitch, L. A.; Pincus, P. J. Phys. Lett. 1984,45, L-29.
0 1991 American Chemical Society
Lecithin Microemulsion Gels
Langmuir, Vol. 7, No. 2, 1991 251
well as high resolution NMR measurements, 31P (line width, relaxation t i m e ~ ' ~and J ~ NOE), 'H, and I3C. In this paper we will present line width data performed on I H , 31P,and 13CNMR resonances for a family of lecithin gels from isooctane. As it is well known, the line width is the most immediate criterium for molecular mobility; i.e. an enlargement of the line width in general corresponds to an increase of rigidity. As previously reported,2 the addition of water to solutions of lecithin in isooctane, up to w, = 3, induces the formation of a transparent gel with a high value of the viscosity. In this work, we present data from various NMR investigations as a function of added water. This permits us to follow structural changes in a continuous way. For this purpose we have prepared and investigated samples at a constant lecithin concentration (200 mM), in the w, range 1-5, which corresponds to a water content of 0.361.8C(, (v/v). The comparison with w o = 0 has also been performed.
Experimental Section Lecithin gels were prepared as previously described.2 Dynamic shear viscosity measurements were performed as already reported.10 For NMR measurements, the gels were gently heated a t 308313 K, in order to reduce the viscosity,g and rapidly introduced in 5-mm standard NMR tubes (all the samples were allowed to recover their thermal equilibrium before performing any measurement). The outmost attention was paid to the volume of samples. The height of all samples was kept well within the coil of each instrument (1 cm for measurements a t 0.7 T and 3 cm for measurements at 4.7 T). Samples were sealed without degassing, since freezing and thawing processes prevent the formation of gels. All NMR measurements were carried out a t 298 f 0.5 K. In low-resolution IH NMRspectra, diode detection, a t least 48 different delays were used for the inversion recovery experiment used for TI determination; T2 was measured by the Carr-Purcell-Meiboom-Gill technique using 512 echoes.18 All low-resolution NMR spectra were taken a t 30 MHz on a commercial spectrometer "Spinmaster" from Stelar-Mede (PV) Italy. All experimental data were treated with the program "Fit",ls based on a simplex algorithm.20 The line widths of high-resolution NMR spectra (lH, W , and were measured directly a t the half height of the experimental peaks. This value was used as an input in a program for line shape simulation which was performed in order to get reliable line widthz1 (Figure 2). High-resolution NMR measurements were performed in 5-mm tubes, with or without external lock, on a Bruker AC-200 spectrometer (IH, 13C, 31P) or on a Bruker AM 300 WB (IH). All deuterated solvents used were from Dr. Glaser AG-Basel, with a percentage of deuteration always higher than 99.9% (except isooctane-d18 which was 98.0%).
Results and Discussion As previously reported, lecithin gels can be made in a variety of organic solvents such as linear and cyclic alkanes, fatty acid esters, and amines (for more details see Table I in ref 2). One of the first question to take here into account, is whether the two classes of solvents, "gelforming" and "non-gel-forming", induce any differences in the lecithin NMR spectra and in particular in the 'H line width of the surfactant resonances. Figure 1 shows the 'H NMR (300.13 MHz) spectra of (16) Milburn, H. P.; Jeffrey, K. R. Biophys. J . 1987, 52, 791. (17) Milburn, H. P.; Jeffrey, K. R. Biophys. J. 1989, 56, 543. (18)Fukushima, E.; Roeder, S. B. W. Experimental Pulse NMR; Addison- Wesley: Reading, MA, 1981. (19) Program Fit by Sikora, S., Stelar-Mede (PV), Italy. (20) Nelder, J. A.; Mead, R. Comput. J. 1964, 7, 308. (21) Segre, A. L.;Delfini, M.; Paci, M.;Raspolli-Galletti, A. M.; Solaro, R. Macromolecules 1985, 18, 44.
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lecithin solutions (no added water)22in isooctane-dls and cyclooctaneZ3("gel-forming"), compared with a chloroform-d and benzene-& solutions, where lecithin gels cannot be formed (at least under our conditions). The optimal resolution observed in the chloroform solution (Figure 1A) is partially lost in benzene (Figure 1B) and totally in the other solvents (Figure lC,D), where the line widths of most resonances due to lecithin are noticeably broad. This observation suggests that in gel-forming solvents, even at w, values far from the maximal viscosity, some decrease of the molecular mobility of lecithin occurs. This is possibly due to aggregation, which was suggested also by quasielastic light scattering and by small angle neutron scattering (SANS).'O In other words, the formation of the gel is not an all-or-nothingprocess but seems to correspond to a gradual transformation. Also, Figure 1 suggests that NMR spectra can provide a first qualitative indication on whether a solvent may, or may not, lead to a gel structure. Let us now consider the influence of added water on some NMR properties: a first interesting comparison between viscosity and NMR properties is offered in Table I. BothI'? and Tz, measured in bulk for the whole system ('H 30 MHz pulse low resolution; see Experimental Sect,ion),show no dependence on w,. Also the line widths for isooctane (data not shown) is insensitive to w, both on measurements performed on 'H NMR (200.13 MHz) as well as on 13CNMR (50.33 MHz). These results indicate that the molecular mobility of the bulk solvent, as judged by NMR, does not correlate (22) I t is important to point out that the lecithin used contains approximately 0.7 mol of HpO/mol of lecithin as calculated from Fourier transform IR and near-IR spectroscopy and Karl-Fischer measurements. (23) The spectrum of lecithin in cyclooctane was recorded by saturating the strong signal due to the solvent. This was performed by irradiating the cyclooctane singlet a t 1.52 ppm during all the NMR experiment.
Capitani et al.
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252
Table I. Longitudinal and Transversal Proton Relaxation Time (*HMeasured at 30 MHz) and the Zero Shear Viscosity ( q 8 )as a Function of the Added Water to a Lecithin/Jsooctane Solution 200 mM 0 1 2
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0.047 0.47 1.8 5.6 4373 5127 9148 4415 1199 741 28 0.005
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a t all with viscosity data. A similar behavior has been previously observed for gelatine microemulsion gels3 and also for gelatine aqueous gels.24 The NMR spectrum of a gel ([lecithin] = 200 mM, w, = 2) is shown in Figure 2. In the same figure, the full assignment is also reported, based also on literature data.25p26 Figures 3 and 4 show the line width of a series of NMR signals as a function of w,. In particular the behavior of the 'H resonance due to the water (Figure 3) is compared with that of the resonances due to groups inside the polar head
As clearly shown in Figure 3, water mobility increases in the initial w o range from w, = 1 to w, = 2. From this value up to w, = 5 no apparent change in its mobility occurs, as shown by the line width which is independent ~~
(24) Maquet, J.; Theveneau, H.; Djabourow, M.; Leblond, J.; Papon, P. Polvmer 1986. 27. 1103. (25j Wehrly, F. W:; Wirthlin,T. InterpretationofNMR Spectra; Heyden: London, 1980. (26) Burns, R. A., Jr.; Roberts, M. F. Biochemistry 1980, 19, 3100.
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of w,. A similar behavior has been previously found for the 4.8 ppm water resonance in lecithin reverse micelles.*' The lines of Figure 4 show a more complex pattern. Note the sharp decrease of the line width that follows the addition of the first water molecule (this partly parallels the behavior of the water signal); and the onset of a secondary maximum a t w o = 3 (note that this is approximately the w, at which the maximal gel viscosity is observed; see Table I). Figure 5 shows the line width of the 31PNMR signal and the zero shear viscosity (qS)lo as function of added water to lecithin/isooctane solution. With respect to the data of Figure 4, a striking difference appears together with a significant analogy. The difference consists in the fact that addition of the first water molecule does not induce an initial decrease of the line width; the analogy instead is given by the presence of a maximum at about w, = 3. The maximum is extraordinarly pronounced for the 31P signal and it clearly follows the trend of the viscosity. Let us try now to draw some structural information from all these NMR data. The most likely interpretation for the initial sharpening of the line width in the w, region between 0 and 2, as evidenced in Figure 4,has to be sought in a conformational change of the polar head of lecithin; (27) Walter, W. V.; Hayes, R. G. Biochim. Biophys. Acta 1971, 249, 528.
Langmuir, Vol. 7, No. 2, 1991 253
Lecithin Microemulsion Gels
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Figure 6. Proposed water-induced conformational change: the addition of the first water molecule causes a change in the orientation of the phosphocholine moiety of lecithin. The polar head, which is perpendicular to the fatty acid in absence of water (A), orients itself almost parallel to the long axis of the molecule (B).
the polar head, which is perpendicular to the fatty chains in the absence of water, would orient itself almost parallel to the long axis of the fatty chains, gaining in this way a larger mobility as a result of the larger conformational freedom (Figure 6). In this regard, it is important to recall that a conformational transition leading to a large mobility variation has been predicted on the basis of minimal energy calculations.28929 A similar transition was recently observed by Hauser et al.3'J Apparently, the above-mentioned conformational transition, while decreasing the rigidity of the whole lecithin (28) Brosio, E.; Conti, F.; Di Nola, A.; Kovacs, A. L. J . Theor. Biol. 1977,67, 319.
(29) Brosio, E.;Conti, F.; Di Nola, A,; Napolitano, G.; Kovacs, A. L. Chem. Phvs. LiDids 1980.27.127. (30) Hauser, H.; Pascher, I.; Sundell, S. Biochemistry 1988,27,9166.
polar head, does not affect the local mobility of the phosphorus atom. In the adjacent w oregion, from w o = 2 to w o = 5, several signals show a maximum in their line width, which corresponds either to an absolute maximum, as shown in Figure 5, or to a secondary maximum as shown in Figure 4. In any case, this maximum indicates a decrease of molecular mobility. The maxima strictly corresponds to the maximum reported in Table I and in Figure 5 for the viscosity. Thus, an important correlation between a macroscopic property (e.g. viscosity) and a molecular feature is established: the overall increase of viscosity of the sample is attended by a very remarkable stiffening on the phosphorus atom and by a decrease of mobility inside the lecithin polar head. Although these effects are caused by the addition of water, no maximum can be observed in the water signal itself. Thus the overall increase of viscosity does not seem to affect the structure of water, which only a t very low concentration seems to be tightly bound to the lecithin polar head (data not shown). On the basis of the data presented here, it appears that neither changes of the molecular mobility of isooctane, nor of water, correlate with the viscosity changes of the material. Instead, it appears that lecithin molecules, in parallel to a local conformational change induced by the binding of the first water molecule, must engage in a macromolecular,. probably entangled, noncovalent, highly flexible matrix. The details of this novel structure, which may well have some relevance for lipid structures built in vivo, are still unknown. The change in free energy associated with the conformational change of the lecithin molecule may be responsible for the variation in the micellar size distribution upon addition of water to lecithin reverse micelles and, thus, for the gelation phenomenon. Therefore, we hope to eventually be able to quantitatively understand, with the help of the recently published theory of living polymers,3l~~~ the correlation between the NMR results and the rheological properties of the lecithin organogels. Further NMR studies and complementary physical studies are in progress to better clarify the details of the formation and of the structure of the lecithin organogels.
Acknowledgment. We thank the NMR service of CNR in Montelibretti (Rome), especially Mr. Enrico Rossi, who was never too tired to help us. We gratefully acknowledge the support of Mr. F. Bangerter and Dr. P. Skrabal at the NMR service of ETH. We wish also to acknowledge Dr. P. Schurtenberger for his useful comments on this paper. This work was partly supported by the Swiss National Science Foundation (NF-24). (31) Cates, M.E.J. Phys. 1988,49, 1593. (32) We thank one of the referees for suggesting the introduction of ref 31.
