Disintegration of the Lecithin Lamellar Phase by Cationic Surfactants

Dec 1, 1997 - surfactants disintegrate the lamellar structure in the two closely related systems. Lecithin in ... Sites of positive mean curvature are...
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Langmuir 1997, 13, 6956-6963

Disintegration of the Lecithin Lamellar Phase by Cationic Surfactants Jonas Gustafsson,*,† Greger Ora¨dd,‡ and Mats Almgren† Departments of Physical Chemistry, Uppsala University, Box 532, S-75121 Uppsala, Sweden, and University of Umeå, Umeå, S-90187 Umeå, Sweden Received June 5, 1997. In Final Form: October 6, 1997X The phase behavior of egg lecithin mixed with two cationic surfactants, hexadecyl- and dodecyltrimethylammonium chloride (C16TAC and C12TAC, respectively), in brine (100 mM NaCl) has been studied. Combining results from NMR spectroscopy, small-angle X-ray diffraction, and cryo-transmission electron microscopy, the phase sequence in less solvent rich regions of the phase diagram is linked to the aggregate structures appearing in dilute samples. The focus is set on the lamellar phases and their relationship to neighboring phases of positive curvature. NMR line shape studies on 2H-labeled surfactants are employed to reveal changes in bilayer structure. The results demonstrate large differences in the way that the two surfactants disintegrate the lamellar structure in the two closely related systems. Lecithin in mixture with the surfactant of the longer hydrocarbon tail (C16TAC) forms lamellar phase regions where the bilayers display curvature defects. A corresponding defect formation is not observed in the lamellar phase from mixture of lecithin and the short cationic surfactant (C12TAC). Instead, broad coexistence regions of (defect free) lamellar phase and micellar or hexagonal phases are observed. The role played by the defective lamellar phase in altering the phase behavior is discussed. We also conclude that a local segregation of the amphiphiles appears to be associated with curvature defects in the bilayer structure of C16TAC and lecithin.

Introduction The lyotropic lamellar phase frequently occurs as a structure of compromise in mixed amphiphilic systems in water. For instance, two amphiphiles with preference for opposite curvatures are often found to form lamellar phases in a rather broad composition range.1 The large regions spanned by such lamellar phases may seem somewhat unexpected, considering the fact that the lamellar structure is unable to adjust its monolayer mean curvature by simply expanding or shrinking its unit cell. There are alternative mechanisms, however, by which the mean curvature of the lamellar structure may be altered. Sites of positive mean curvature are created when defects such as pores or channels are introduced into the bilayer structure. Lamellar phases featuring equilibrium curvature defects of this kind are found in both single and mixed amphiphilic systems.2-9 The present study deals with lamellar phases and their disintegration into aggregates of high curvature in mixed lipid/cationic surfactant systems. In a previous contribution,10 a defective lamellar structure was described in the pseudoternary system C16TAC/EPC/100 mM NaCl (C16TAC, hexadecyltrimethylammonium chloride; EPC, egg lecithin). Our aim here is to further shed light on the * To whom correspondence should be addressed. † Uppsala University. ‡ University of Umea ˚. X Abstract published in Advance ACS Abstracts, December 1, 1997. (1) Ekwall, P. Adv. Liq. Cryst. 1975, 1, 1-142. (2) Hoffman, H.; Thunig, C.; Munkert, U.; Meyer, H. W.; Richter, W. Langmuir 1992, 8, 2629-2638. (3) Berger, K.; Hiltorp, K. Colloid Polym. Sci. 1996, 274, 269-278. (4) Boltenhagen, P.; Kleman, M.; Lavrentovich, O. D. J. Phys. II 1994, 4, 1439-1448. (5) Funari, S. F.; Holmes, M. C.; Tiddy, G. J. T. J. Phys. Chem. 1994, 98, 3015-3023. (6) Holmes, M. C.; Leaver, M. S.; Smith, A. M. Langmuir 1995, 11, 356-365. (7) Kekicheff, P.; Cabane, B.; Rawiso, M. J. Phys. Lett. 1984, 45, 813-821. (8) Leaver, M. S.; Holmes, M. C. J. Phys. II 1993, 3, 105-120. (9) Quist, P.-O.; Fontell, K.; Halle, B. Liq. Cryst. 1994, 16, 235-256. (10) Gustafsson, J.; Ora¨dd, G.; Lindblom, G.; Olsson, U.; Almgren, M. Langmuir 1997, 13, 852-860.

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properties of this lamellar phase by investigation of a related system (C12TAC/EPC/100 mM NaCl) (C12TAC, dodecyltrimethylammonium chloride) and by comparison of the results to new and old data from the C16TAC system. Both systems are approached in the same way: results from small-angle x-ray scattering (SAXS), NMR, and cryotransmission electron microscopy (cryo-TEM) are considered together in an attempt to obtain a unified view of how the lamellar structures are transformed into aggregates of positive curvature. The key information in the present study is provided by NMR line shape measurements on 2H-labeled surfactants. Such static NMR measurements are often employed in studies of phase equilibria of amphiphilic molecules, since the degree of motional averaging offered by diffusion in the hydrocarbon/water interface varies for different lyotropic liquid crystals.11,12 This gives rise to characteristic line shapes involving quadrupolar splitting from nuclei with spin quantum numbers g1 (in our case from the deuterons of the labeled surfactant). The quadrupolar splitting is expected to vary from a maximum value in lamellar phases to 0 in isotropic phases, such as cubic and micellar phases.12 Observations of changes in the splitting are often helpful for the characterization of different lyotropic phases, especially in studies where intermediate phases are to be identified.5,12-15 Halle and Quist have shown how the quadrupole splitting from a labeled amphiphile can be used to model both curvature defects and undulations in lamellar phases.9,16 Curvature defects as well as undulations cause a decrease in the splitting. In a defective bilayer, amphiphiles may diffuse over a curved interface and thereby gain an additional motional averaging, while undulations instead reduce the splitting due to the (11) Lindblom, G. In Advances of Lipid Methodology; Oily Press: Dundee, U.K., 1996; pp 133-209. (12) Wennerstro¨m, H.; Lindblom, G.; Lindman, B. Chem. Scr. 1974, 6, 97-103. (13) Boden, N.; Corne, S. A.; Jolley, K. W. J. Phys. Chem. 1987, 91, 4092-4105. (14) Henriksson, U.; Blackmore, E. U.; Tiddy, G. J. T.; So¨derman, O. J. Phys. Chem. 1992, 96, 3894-3902. (15) Kang, C.; So¨derman, O.; Eriksson, P. O.; Holstein, J. S. v. Liq. Crys. 1992, 12, 71-81. (16) Halle, B.; Quist, P.-O. J. Phys. II 1994, 4, 1823-1842.

© 1997 American Chemical Society

Disintegration of the Lecithin Lamellar Phase

fluctuation of the bilayer normal. Also extrinsic defects in the lamellar phase, such as the generation of multilamellar vesicles, have recently been illustrated with the aid of quadropole splittings.17 There are apparently several possible sources which could contribute to a reduced splitting from an amphiphile in a lamellar phase. A progressively decreasing quadrupole splitting from the surfactant inside the lamellar phase, together with a strongly nonideal swelling, was used to illustrate the defect formation in the system of C16TAC and EPC.10 Only a limited number of mixed lipid/surfactant systems have been investigated with respect to their phase diagrams,10,18-20 whereas such mixtures occurs more frequently in vesicle solubilization studies,21-26 where the stability of lamellar structures toward disintegration is addressed from a somewhat different point of view. The important vesicle-micelle coexistence, found in almost every solubilization study, has been analyzed in theoretical approaches,27,28 where focus is set on the stabililty of the bilayer structure compared to that of the cylindrical micelle. The outcome of a solubilization study should be largely determined by the phase equilibrium of the components. Vesicle solubilization studies are usually conducted with a set of methods different from the ones commonly employed for phase characterization. With the combination of methods (SAXS, NMR, cryo-TEM) used in the present study, we are able to establish connections between aggregate structures occurring in the dilute region and the phase equilibrium in less solvent rich parts of the diagram. Experimental Section Materials. Egg lecithin of grade 1 was purchased from Lipid Products, Nutfield, U.K. C16TAC was prepared by ion exchange of cetyltrimethylammonium bromide (C16TAB) (Serva). C12TAC obtained from Sigma was recrystallized in methanol. The deuterated versions of the two surfactants were prepared according to a procedure for exhaustive alkylation.29 The corresponding amine, purchased from Fluka, was alkylated with deuterated methyl iodide, isotopic purity 99.5% (Glaser Laboratories, Go¨teborg, Sweden). DMF was used as a solvent, and PMP (1,2,2,6,6-pentamethylpiperidine, Fluka) as a proton acceptor. The reaction product C12TAI-d9 (C16TAI-d9) was recrystallized and finally lyophilized in 25 wt % ethanol in water, and it was ion exchanged into C12TAC-d9 (C16TAC-d9). Samples were prepared by adding cationic surfactant to lecithin lyophilized in chloroform/methanol. The organic solvent was then removed by drying for several hours under vacuum. After addition of electrolyte solution, the samples were periodically mixed during 1 week. Phase equlilibrium was assumed after an additional week at rest at 25 °C. Long-term storage (months) (17) Auguste, F.; Barois, P.; Fredon, L.; Clin, B.; Duforc, E. J.; Beloocq, A. J. Phys. II 1995, 4, 219-2214. (18) Sadaghiani, A. S.; Khan, A.; Lindman, B. J. Colloid Interface Sci. 1989, 132, 352-361. (19) Klose, G.; Eisenbla¨tter, S.; Ko¨nig, B. J. Colloid Interface Sci. 1995, 172, 438-446. (20) Rydhag, L.; Garbran, T. Chem. Phys. Lipids 1982, 30, 309-324. (21) Edwards, K.; Gustafsson, J.; Karlsson, G.; Almgren, M. J. Colloid Interface Sci. 1993, 161, 299-309. (22) Egelhaaf, S. U.; Pedersen, J. S.; Schurtenberger, P. Prog. Colloid Polym. Sci. 1995, 98, 224-227. (23) Heerklotz, H.; Lantzsch, G.; Binder, H.; Klose, G.; Blume, A. J. Phys. Chem. 1996, 100, 6764-6774. (24) Vinson, P. K.; Talmon, Y.; Walter, A. Biophys. J. 1989, 56, 669681. (25) Walter, A.; Vinson, P. K.; Kaplan, A.; Talmon, Y. Biophys. J. 1991, 60, 1315-1325. (26) Maza, A. d. l.; Parra, J. L. Colloid Polym. Sci. 1996, 274, 253260. (27) Andelman, D.; Kozlov, M. M.; Helfrich, W. Europhys. Lett. 1994, 25, 231-236. (28) Fattal, D. R.; David, A.; Ben Shaul, A. Langmuir 1995, 11, 11541161. (29) Sommer, H. Z.; Hayden, I. L.; Jackson, L. L. J. Org. Chem. 1971, 36, 824-828.

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Figure 1. Solvent corner of the pseudoternary diagram of EPC/ C12TAC/100 mM NaCl. Please note that not all multiphase regions are depicted. LR (LR′) denotes the lamellar phase, while I1 and H1 denote the cubic and the normal hexagonal phase, respectively. of samples at room temperature was avoided to prevent degradation of the lecithin. Cryo-TEM. The electron microscopy investigations were performed with a Zeiss 902 A instrument, operating at 80 kV. Specimens were prepared by a blotting procedure, performed in a chamber with controlled temperature and humidity. A drop of the sample solution was placed onto an EM grid coated with a perforated polymer film.30 Excess solution was then removed with a filter paper, leaving a thin film of the solution on the EM grid. Vitrification of the thin film was achieved by rapid plunging of the grid into liquid ethane held at its freezing point. The vitrified specimen was then transferred in the cold state to the microscope and investigated at 108 K. SAXS. Small-angle X-ray diffractograms were obtained using a Kratky compact small-angle system (at Physical Chemistry 1, Lund University), linearly collimated, and equipped with a position-sensitive detector (OED 50M from Mbraun, Graz, Austria) with 1024 channels of width 51.3 µm. The radiation (Cu KR) was provided by a Seifert IF 300 X-ray generator operating at 50 kV and 40 mA. The camera length was 277 mm. The samples were poured into quartz capillaries mounted to a steel body that was sealed with screw caps. Slit-smeared spectra were desmeared according to a standard procedure. SAXS measurements were also performed at station 8.2 at the Daresbury Laboratory (Cheshire, U.K.) using a monochromatic beam of wavelength 1.5 Å. NMR. NMR experiments were performed on a Bruker ACP250 spectrometer. 2H NMR measurements were made at 38.40 MHz using the quadrupole echo sequence31 with a pulse width of 25 µs and a pulse separation of 60 µs. 31P measurements were made at 101.27 MHz using the Hahn echo sequence32 with a broad-band decoupling of the protons. The π/2 pulse length was 10 µs and the pulse separation 60 µs. The FIDs were zero-filled, care was taken to perform the Fourier transform from the top of the echo, and the spectra were analyzed as described elsewhere.10 The quadrupolar splittings, ∆νq, were obtained as the distance between the 90° peaks in the resulting powder pattern line shapes. The 31P chemical shift anisotropy was measured either from the width of the phosphorus powder pattern line shape or, in oriented samples, as 3 times the separation between the 90° peak and the isotropic chemical shift.

Results C12TAC/EPC/100 mM NaCl. Figure 1 shows the solvent corner of the pseudo-ternary system C12TAC/EPC/ 100 mM NaCl. The phase equilibrium was determined mainly by SAXS studies, supported in the multiphase (30) Fukami, A.; Adachi, K. J. Electron Microsc. 1965, 14, 112-118. (31) Davis, J. H. Biochim. Biophys. Acta 1983, 737, 117-171. (32) Rance, M.; Byrd, R. A. J. Magn. Reson. 1983, 52, 221-240.

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Figure 3. 2H (a,c) and 31P (b,d) NMR line shapes illustrating coexisting lamellar/hexagonal (a,b) and lamellar/isotropic phases (c,d), respectively. The magnifications shown to the right on the 2H line shapes are 5 times (a) and 10 times (c) with respect to the original spectra. Sample compositions and the corresponding quadrupolar splitting from the two samples are indicated by arrows in Figure 4.

Figure 2. Desmeared SAXS profiles from (a) the cubic phase and (b) the lamellar phase coexistence. Sample compositions (C12TAC/EPC/brine) were 40/7/53 and 5.8/34.2/60, respectively.

regions by 2H and 31P NMR line shape studies. Note that not all multiphase regions are outlined in the diagram. Only phase equilibria that were directly suggested by the SAXS or NMR results are included in Figure 1. A typical SAXS scattering profile obtained from the rather extended cubic phase is shown in Figure 2a. The q-values for the three dominant peaks are found to relate as x4:x5:x6, which is in accord with a cubic structure built by discrete micelles arranged in a primitive cubic lattice (Pm3n).33 We assume this designation since it is the structure that forms in the binary system of C12TAC in water.33,34 The low viscosity of the micellar phase next to the cubic phase region is also in line with the presence of a so-called micellar cubic phase. Apparently the micelles stay small, even at high concentrations, despite the incorporation of one-fourth by weight of lecithin into the C12TAC micelles. The hexagonal and lamellar phases were identified by the observation of Bragg reflections in the relative positions 1:x3:x4 and 1:x4, respectively. Near the hexagonal phase boundary the micellar solutions are viscous, indicative of micellar growth, as would be expected, since long rodlike micelles are the building blocks of the hexagonal structure. As in the related C16TAC/ EPC system, a region of coexistence of two lamellar phases appears at low ratios of the cationic surfactant (see diffractogram in Figure 2b). The lamellar phase samples were not clear but varied from translucent to semitransparent. In addition, the multiphase samples of lamellar phase in coexistence with excess solvent did not separate (33) Mariani, P.; Luzzati, V.; Delacroix, H. J. Mol. Biol. 1988, 204, 165-189. (34) Balmbra, R. R.; Clunie, J. S.; Goodman, J. F. Nature 1969, 222, 1159-1160.

macroscopically. The swelling limit of the lamellar phase was therefore estimated from how the repeat distance obtained from SAXS changed upon dilution. The phase boundaries so determined in a pseudoternary system are rather uncertain and, therefore, are marked with dashed lines. The identification of other multiphase regions was guided mainly by the 2H NMR line shape studies using C12TAC-d9. In Figure 3 some typical spectra from samples with coexisting phases are shown. Two powder patterns with different peak separations are obtained from the coexistence of the lamellar and hexagonal phases, while a doublet and a narrow isotropic peak result from the two-phase samples with lamellar and micellar phases. The line shape of the hexagonal resonance signal was often found to deviate from a typical powder pattern, indicative of a spontaneous alignment in the magnetic field. The line shapes obtained from the lamellar structures showed the characteristic form of a powder spectrum. Quadrupole splittings from the deuterated C12TAC (C12TAC-d9) were measured in two series, depicted in Figure 1 as lines c and d. Figure 4a shows the results obtained from the dilution series (line d), which starts off in the lamellar/hexagonal coexistence region. Theoretically it is predicted12 (assuming that the local environment stays the same) that the splitting from amphiphile nuclei in the hexagonal phase should equal half of that from the lamellar phase. For the concentrated samples this relation holds approximately, while it is not valid for the less concentrated samples, due to the decreased splitting values in the hexagonal phase. Notable is the seemingly constant splitting measured from the lamellar phase throughout the multiphase regions. An invariant lamellar splitting of the same magnitude is also found in the series of samples that crosses the diagram at 65 wt % solvent (line c). In Figure 4b it is shown that at least six different phase equilibria occur along this line. It is worth noting that the quadrupole splitting from the hexagonal phase inside the single-phase region is somewhat larger (approaching half of the lamellar splitting) than what is measured from samples where a lamellar phase is also present. The magnitude of the splittings may be directly compared to the results of Kang et al.,15 who investigated intermediate phases in the binary system C12TAC-d9/H2O. The comparison shows that the

Disintegration of the Lecithin Lamellar Phase

Figure 4. 2H-Quadrupolar splittings from the deuterons of C12TAC-d9 measured for sample series (a) along the dilution line indicated as d in Figure 1 and (b) at constant solvent content along the line denoted as c. Arrows denote the samples for which line shapes are shown in Figure 3. The splitting given by C12TAC-d9 in the lamellar structure is nearly constant throughout both sample series.

splitting values of the hexagonal phase are somewhat lower in the present system, while the values for the lamellar state are quite similar. A fragmentation of the hexagonal structure could be a probable cause for the low values. Such a fragmentation, which can be considered to reflect the near presence of the micellar phase, would also explain the observed tendency of the hexagonal structure to align in the magnetic field. Cryo-TEM investigations were made on dilute samples, with solvent contents above 99 wt %. Three distinct regions appear as a function of the ratio C12TAC to EPC in the dilute area: a broad region with micellar structures, a region of coexistence of micelles and vesicles (lamellae), and a lamellar region with vesicles only. Due to the high critical micelle concentration (cmc) value of C12TAC, a direct quantitative comparison cannot be made between the composition ranges where the different regions occur in the dilute samples and the locations of the phase sequence at high concentrations. At high ratios of C12TAC to lecithin the micelles remain small, as seen in Figure 5a. When more C12TAC is replaced by lecithin, the micelles change appearance. Rather flexible threadlike micelles

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are observed in Figure 5b, and apparently, the incorporation of lecithin into small C12TAC micelles leads, at some point, to micellar growth. As the multiphase region is entered, the threadlike micelles persist, but now in coexistence with vesicles. Figure 5c illustrates such a coexistence. It is worth noting that many vesicles in this region show disruptions or seem to be just partly closed. A further increase in the proportion of lecithin yields samples that contain only vesicle structures (Figure 5d). Vesicles from this region did not show disruptions in their membranes. C16TAC/EPC/100mM NaCl. The solvent corner of the diagram of C16TAC and lecithin in 100 mM NaCl (adapted from ref 10) is shown in Figure 6. We return to this system to address two issues: the defect formation on approach of the hexagonal phase and the swelling of the lamellar phase. In Figure 7 we recall, for the sake of completeness, the appearance of vesicles from the defective lamellar structure in cryo-TEM. In the previous study10 it was suggested that undulation forces (in addition to the electrostatic interactions) may contribute to the swelling of the lamellar phase and that it would possibly be the dominant repulsive interaction in its most swollen state. A more detailed investigation of the dependence of the surfactant quadrupole splitting on the solvent concentration was therefore performed with a new batch of C16TAC-d9. The results shown in Figure 8, for samples pertaining to the center of the narrow lamellar tongue, were analyzed using the model for flexible bilayers developed by Halle and Quist.16 In their model the bilayer bending modulus is obtained directly from the variation of the quadrupole splitting on dilution. It is also assumed, however, that the defect density is 0 or constant, which is not correct in the present system, since we know from the SAXS data of the previous study that the total bilayer area increases with increasing volume fraction of solvent, i.e., the defect density grows with the solvent concentration. The value of kc obtained from Figure 8, 1.8kT, would have been correct for bilayers with 0 or constant defect density; in other words, it gives the flexibility required if undulations alone would have been responsible for the observed reduction of the quadrupolar splitting. It is notable that this value is on a level with results from related systems of flexible bilayers.17 The SAXS profiles exhibit weak and somewhat broadened Bragg reflection(s) from samples with high solvent contents (>80 wt %). We suggested earlier10 that this was related to undulations of the bilayers, since softened Bragg peaks are expected from swollen lamellar phases stabilized by undulations.35 Additional measurements show, however, that increasing C16TAC concentrations have a similar effect on the scattering profile. Evidently, the lamellar region close to the hexagonal phase also gives rather poor diffraction patterns, and similar changes are thus observed in the scattering patterns along both line c and the earlier investigated dilution line (d). We also note that no additional scattering is observed from these samples on the Kratky camera that can be attributed to the intralamellar features of the defective bilayers. This may appear somewhat confusing since such scattering has been demonstrated in several studies concerned with defective lamellar phases.5-7 However, as noted in ref 7, the use (as in our case) of linear collimation and unoriented samples is likely to obscure these diffuse features in the scattering profile. Some diffractograms measured using the synchrotron facilities at Daresbury Laboratories did, however, show a rather broad band at low angles which possibly could originate from intralamellar structures. (35) Safinya, C. R.; Roux, D. Phys. Rev. Lett. 1986, 57, 2718-2721.

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Figure 5. Cryo-TEM micrographs from series of samples with EPC and C12TAC at a total concentration of 0.75 wt % and decreasing molar ratios of surfactant to lipid. The bar equals 100 nm. (a) Molar ratio 8, spherical micelles. (b) Molar ratio 5, threadlike micelles. (c) Molar ratio 3, coexistence of threadlike micelles and vesicles with disruptions. (d) Molar ratio 2, vesicles (without visible disruptions).

Figure 9 shows an example of such a scattering pattern from a sample of the lamellar tongue with 75 wt % of solvent. The resolution at low q-values is better here than that obtained using the Krakty camera. As with C12TAC-d9, quadrupolar splittings from the labeled C16TAC-d9 were also measured in a sample series that crosses the diagram at a solvent concentration of 75 wt % (depicted as line c in Figure 6). In the lamellar

region of this series, C16TAC-d9 shows a continuous decrease in splitting with increasing proportions of surfactant in the bilayers, as seen in Figure 10. Near the hexagonal phase the lamellar structure actually exhibits values that are below those obtained in the neighboring hexagonal structure. As the hexagonal phase is entered, splittings are obtained that approximately equal half of the maximum splitting observed from the lamellar phase

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Figure 6. Solvent corner of the pseudoternary diagram of EPC/ C16TAC/100 mM NaCl. The diagram is adapted from ref 10, where a description of the phase behavior may be found. Figure 8. 2H-Quadrupolar splittings from C16TAC-d9 in the lamellar phase measured as a function of solvent concentration (line d of Figure 6) at a molar ratio of C16TAC to EPC of 2.1. The solid line corresponds to a two-parameter fit of the splittings according to eqs 3.18 and 3.26 in ref 16. A value for δ/a of 4 was used in the fit. The result did not depend critically on the choice of this parameter within the range 2-8. The uncertainty of this parameter is inserted into the error estimations of κ and ∆ν0q. The fit gives the two parameters kc/kT ) 1.8 ( 0.2 and ∆ν0q) 4.3 ( 0.5 kHz.

Figure 7. Cryo-TEM micrograph of the dispersed defective lamellar phase from C16TAC and EPC, molar ratio 2.1. Socalled perforated vesicles are seen along with open bilayer fragments of the same structure. The bar equals 100 nm.

in the dilution series (Figure 8). The 31P chemical shift anisotropy (csa) was also measured for these samples, and the results are included in Figure 10. The ranges of the axes have been chosen so that the two curves coincide for the hexagonal phase. This normalization of the two data sets is made to emphasize the differences in motional averaging for the two amphiphiles in the defective lamellar structure. It is assumed that both molecules move on the same surface in the hexagonal phase so that the averaging then scales similarly for C16TAC and EPC. In the lamellar phase, the curve for C16TAC is substantially below that of EPC, suggesting a larger motional averaging for the cationic surfactant in the defective bilayer. This can be taken as an indication that C16TAC preferentially resides in the curved surface associated with the defects, while EPC accumulates in intervening flat regions. The csa of

Figure 9. X-ray scattering profile from the defective lamellar phase of C16TAC and lecithin obtained at the Daresbury Laboratories station 8.2. Sample composition (C16TAC/EPC/ brine) was 13/12/75.

the 31P nuclei was also utilized for the discrimination between lamellar and hexagonal phases, since it is of opposite sign in the two structures,36 contrary to the 2H powder pattern which shows the same form for both phases. It should be added that no two-phase region of lamellar and hexagonal phases was observed. The lamellar-hexagonal transition is apparently completed within a very narrow region of compositions. The splittings obtained from C16TAC-d9 may also be directly compared to those from C12TAC-d9, since the two surfactant head groups are identical and the local environment is the same. From the bilayers of C16TACd9 and EPC only the most concentrated sample of the d-line series approaches the 4 kHz obtained from the lamellar phase with C12TAC-d9. Since this value may be taken as normalizing for the situation of a rigid and defect(36) Seelig, J. Biochim. Biophys. Acta 1978, 515, 105-140.

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Figure 10. 2H-Quadrupolar splittings from C16TAC-d9 (open circles) and 31P chemical shift anisotropies (open squares) from samples along line c in Figure 6. The molar ratio of C16TAC to EPC is varied at constant solvent concentrations of 75 wt %. A vertical dashed line indicates the transition from the lamellar to the hexagonal phase.

free bilayer, it is clear that already the first sample in the c-line series of Figure 10 deviates from the ideal situation. Discussion The disintegration of the lamellar phase is shown to occur in distinctly different ways in the two systems. We will in the following argue that the defect formation in the lamellar phase of the C16TAC system and its absence in the C12TAC system are the sources of the differences in phase behavior. First some remarks deserve to be made regarding the interpretation of the results. The excess salt in the systems makes it somewhat difficult to deal with the multiphase regions. The phases in equilibrium are likely to be located out of the studied plane, due to an uneven distribution of the electrolyte. To this it should be added that the lecithin, being a biological extract, is not a single component. The result will nevertheless (and in the lack of suggestions to the contrary) be discussed as if the phases of the multiphase regions are of the same properties and structure as the corresponding single phases present in the studied plane. Disintegration of the Lamellar Phases. The nearly constant value of the quadrupole splitting obtained from the lamellar phase in the system C12TAC/EPC/100 mM NaCl suggests that no significant changes occur in the bilayer structure based on C12TAC and EPC. The appearance of a rather broad two-phase region of lamellar and hexagonal phases may be viewed as a consequence of the large difference in microstructure curvature between the ordinary (intact) lamellae and the rods in the hexagonal structure. The opposite scenario is found in the C16TAC/EPC system, where the progressively decreasing splitting in the lamellar phase suggests an increased defect formation on approach of the hexagonal phase, which in turn leads to a very narrow (not observed) two-phase region. There is a straightforward qualitative correlation between the aggregate structures observed by cryo-TEM and phase equilibria in the less solvent rich part of the diagram in the system with C12TAC and EPC. The observed sequence of liquid crystalline structures is more or less parallel to the succession of aggregate structures observed by cryo-TEM in the dilute samples. Furthermore, the miscibility gap in the C12TAC system between

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the lamellar phase and its neighboring phases of high curvature is in the dilute region manifested in a broad region of coexistence of vesicles (dispersed lamellae) and threadlike micelles. Apparently two aggregation geometries, the cylinder and the bilayer, dominate a very large area of the diagram. Comparing the two surfactants, it is evident that the lamellar-micellar and the lamellarhexagonal transitions occur at a lower molar ratio of the cationic component when C12TAC is used instead of C16TAC. This is likely partly due to the shorter tail of C12TAC, which makes it more effective in promoting microstructures with positive curvature. This observation probably also reflects the dissimilarities of the two disintegration processes. The frequent appearance of a micelle/lamella coexistence, especially in mixed amphiphilic systems (as the ones encountered in vesicle solubilization), has made it a subject for theoretical considerations.27,28 We note that a recent model,28 where chain statistics are also included, does not suggest an increased broadening of the two-phase region due to large disparities in tail length. The source of this discrepancy is probably that the model only considered two types of aggregate geometries, the cylinder and the defect-free bilayer. Somewhat disrupted vesicle membranes were observed in the micelle/lamella coexistence region (Figure 5c). Similar vesicle imperfections in the corresponding mixed aggregate region have been demonstrated in several solubilization studies.25,37,38 It should be stressed that this observation is not in contradiction with the conclusion drawn from the quadrupole-splitting measurements, stating that the lamellae are more or less intact in the multiphase regions. The density of edges in the lamellar structure suggested by the disruptions in the vesicles may be too low to affect the quadrupolar splitting. The apparent correlation between phase equilibria results and cryo-TEM results on this point is, however, not entirely obvious. When investigated by cryo-TEM, the multiphase samples are in a dispersed (nonequilibrium) state. Membrane disruptions may be favored when lamellar aggregates are dispersed in a micellar phase, since it involves an exposure of the lamellae to the surfactant monomer concentration of the micellar phase. In the C16TAC system the reduction of the quadrupole splitting inside the lamellar phase region is substantial, both on dilution and with increasing ratio of surfactant. The decrease is stronger than what was found in earlier studies where curvature defects,9 undulations,16 and texture defects17 have been addressed. Quadrupolesplitting values below those obtained in the hexagonal phase have been measured earlier from deuterated surfactants in intermediate phases.14,15 The defective lamellar structure may be viewed as an intermediate structure, and one would therefore expect its microstructure near the hexagonal phase to show some resemblance to rodlike aggregates. Cryo-TEM observations of a perforated bilayer structure point to the presence of a lamellar phase with pore defects in the dilute area. The connection between a perforated bilayer and connected threadlike micelles was demonstrated in the previous study.10 We note that this relationship was considered several years ago by Porte et al.39 However, as the lamellar phase is concentrated, the defect geometry may very well change. In addition to pores, slit defects giving rise to (37) Edwards, K.; Almgren, M.; Bellare, J.; Brown, W. Langmuir 1989, 5, 473-478. (38) Edwards, K.; Almgren, M. J. Colloid Interface Sci. 1991, 147, 1-21. (39) Porte, G.; Gomati, R.; Haitamy, O. E.; Apell, J.; Marignan, J. J. Phys. Chem 1986, 90, 5746-5751.

Disintegration of the Lecithin Lamellar Phase

ribbonlike aggregates have also been considered as probable defect structures in lamellar phases.5,8,9 Unfortunately, a discrimination between various defect geometries cannot be made from the present set of data. The results obtained provide only indirect information, since the intralamellar features are not revealed in the SAXS profiles, nor do they alter the shape of the NMR signals. Both observations indicate a disordered arrangement of defects in the bilayer plane. The high ability of C16TAC, as compared to C12TAC, to produce defects in the EPC-based bilayer structure may tentatively be rationalized in the following way. The defective lamellar structure is, irrespective of defect geometry, bound to be inhomogeneous in its Gaussian curvature, and probably also in mean curvature. The formation of curvature defects is probably facilitated in a mixed amphiphilic system, where a local segregation of the two components (having different geometries) may account for the inhomogeneity in curvature in a simple way. The different degrees of motional averaging evident from Figure 10 suggest such a local segregation in the defective lamellae of the C16TAC system. However, when there is a large disparity in chain length, segregation may become more difficult to achieve, due to packing mismatches. The chain length difference between C12TAC and EPC, which is important in promoting the early destabilization of the lamellar phase, may therefore at the same time be the reason why a defective lamellar stucture fails to appear. We note that Berger et al.3 came to a similar conclusion concerning chain length differences in their studies on defective lamellar phases formed by sodium dodecyl sulfate and various alcohols. In this connection it also deserves to be mentioned that S. Gustafsson et al.40 were recently able to quantify local segregation for the case of two surfactants in a rectangular phase. Swelling of the Lamellar Phases. The difference in degree of lamellar phase swelling between the two systems may seem puzzling, considering the identity of the surfactant head groups. A broadening of the Bragg peaks from the lamellar phase of C16TAC and EPC was observed in samples from both the highly swollen region and the region close to the hexagonal phase. Aware of only the former effect in the previous study, we suggested that the line broadening in the dilute region may be caused by undulations of bilayers, and further that the undulation force was the reason behind the extensive swelling. Two observations in the present work now point to the role played by an increased defect density: the previously mentioned softening of the Bragg peaks on approach of the hexagonal phase along the c-line and the strong reduction of the quadrupole splitting in the same sample (40) Gustafsson, S.; Quist, P.-O.; Halle, B. Liq. Cryst. 1995, 18, 545553.

Langmuir, Vol. 13, No. 26, 1997 6963

series. Since we do not expect strong undulations at the concentration of the c-line, it appears as if the increased defect density could be the source of both observations, which earlier (when observed along the dilution line) were attributed to undulations.41 Unfortunately, it appears difficult to separate the effect of possible undulations in the reduction of the quadrupole splitting seen in Figure 8 from the contribution caused by motional averaging in the bilayer defects. The early appearance of a micellar phase in the C12TAC system is probably an important reason for the reduced swelling. In coexistence with a micellar phase, the lamellar swelling will be determined by the competition for water between the two phases. A shift in swelling may therefore be a question of a shift in the phase equilibria rather than a matter of interbilayer forces.42 An important question in this connection is how to describe the interbilayer forces between bilayers such as in the present C16TAC/EPC system, which contains up to 30% defects. Another important feature of the lamellar swelling is that, at low ratios of the cationic surfactant, both systems contain coexisting lamellar phases. Jo¨nsson et al.43 have theoretically considered swelling of lamellar phases in systems of two amphiphiles where one is charged. Their study predicts the coexistence of two lamellar phases with different solvent contents at low molar ratios of the charged component. When electrolyte is added to the solvent, this coexistence region is shifted toward an increased fraction of ionic amphiphile. It is likely that the coexistence of two lamellar phases found in the present systems is well described by the theory of Jo¨nnson et al. An observation in accordance with their calculations is that the less swollen lamellar phase, in both systems, shows a repeat distance that is comparable to that of pure lecithin in water. Acknowledgment. This work was economically supported by the Swedish Technical Research Council and by a generous donation from Knut and Alice Wallenbergs Stiftelse. We are grateful to P.-O. Quist for providing the nonlinear least squares fitting procedure used to fit the data in Figure 8. P. Westerby is also acknowledged for his assistance in the use of Matematica for the fitting procedure. LA9705874 (41) An interesting question concerning possible undulations of the CTAC/EPC bilayers is the origin of the bilayer flexibility. When a cosurfactant introduces flexibility to a lipid bilayer, it is usually explained by an overall thinning of the bilayers. If this were the reason in our case, then C12TAC would be more efficient in softening the lecithin bilayers. Once again the defects may play an important role, this time by affecting the bending properties of the bilayers. (42) Dubois, M.; Zemb, T. Langmuir 1991, 7, 1352-1360. (43) Jo¨nsson, B.; Persson, P. K. T. J. Colloid Interface Sci. 1987, 115, 507-512.