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Langmuir 1997, 13, 852-860
A Defective Swelling Lamellar Phase Jonas Gustafsson,*,† Greger Ora¨dd,‡ Go¨ran Lindblom,‡ Ulf Olsson,§ and Mats Almgren† Department of Physical Chemistry, Uppsala University, Box 532, S-75121 Uppsala, Sweden, Department of Physical Chemistry, University of Umeå, S-90187 Umeå, Sweden, and Physical Chemistry 1, Lund University, Chemical Centre, P.O. Box 124, S-22100 Lund, Sweden Received June 3, 1996X The phase equilibria in mixtures of egg lecithin and cetyltrimethylammonium chloride in brine (100 mM sodium chloride) were studied with particular emphasis on the behavior of the lamellar phase. The solvent corner of this pseudoternary system features an extensive lamellar phase which we have characterized by means of cryo-transmission electron microscopy (cryo-TEM), small-angle X-ray diffraction, and 2H and 31P NMR. The cryo-TEM micrographs illustrate a smooth transition in aggregate microstructure between lamellar and micellar structures with a perforated lamellar structure as an intermediate state. The findings from X-ray diffraction and NMR spectroscopy also indicate a deviation from ordinary bilayer structures in the solvent rich region of the lamellar phase. This is concluded from strongly nonideal swelling and progressively decreasing 2H NMR quadrupole splittings of the deuterated cetyltrimethylammonium chloride inside the lamellar region. The correlation between the observed aggregate structures and the phase behavior is discussed in terms of a continuous transition from the lamellar to the micellar phase.
Introduction The lamellar phase is a common form of self-assembly for amphiphilic molecules. In the classical picture it consists of an alternating stack of planar, undisruptured bilayers and intervening layers of solvent. During the last decade the classical picture has been somewhat revised, and today the existence of both strongly undulating bilayers and bilayers with solvent-filled defects are generally accepted. In recent years especially the defective, or perforated, lamellar structure has attracted a growing interest.1-7 The formation of solvent-filled intralamellar defects in lamellar phases may be regarded as related to the formation of so-called intermediate phases. Both types of structures originate from the strive for microstructure curvatures lying between those given in ordinary bilayers and the one offered in the cylinders of the normal hexagonal phase. Detailed X-ray and neutron scattering studies on the structure of intermediate phases have led to the establishment of a number of mesophases with microstructure curvatures between that in the lamellar and the normal hexagonal phase.5,8-17 Some years ago Ke´kicheff et al.10,11 reported four separate intermediate * To whom correspondence should be addressed. E-mail: titus@ fki.uu.se. † Uppsala University. ‡ University of Umea ˚. § Lund University. X Abstract published in Advance ACS Abstracts, January 15, 1997. (1) Kekicheff.; Cabane, B. J. Phys. Lett. 1984, 45, 813-821. (2) Rancon, Y.; Charvolin, J. J. Phys. Chem. 1988, 92, 6339-6344. (3) Leaver, M. S.; Holmes, M. C. J. Phys. Fr. II 1993, 3, 105-120. (4) Hoffmann, H.; Munkert, U.; Thunig, C.; Valiente, M. J. Colloid Interface Sci. 1994, 163, 217-228. (5) Holmes, M. C.; Leaver, S. L.; Smith, A. M. Langmuir 1995, 11, 356-365. (6) Funari, S. S.; Holmes, M. C.; Tiddy, G. J. T. J. Phys. Chem. 1994, 98, 3015-3023. (7) Quist, P.-Q.; Fontell, K.; Halle, B. Liq. Cryst. 1994, 16, 235-256. (8) Holmes, M. C.; Charvolin, J. J. Phys. Chem. 1984, 88, 810-818. (9) Kekicheff, P.; Cabane, B. J. Phys. 1987, 48, 1571-1583. (10) Kekicheff, P.; Grabielle-Madelmont, C.; Ollivon, M. J. Colloid Interface Sci. 1988, 131, 112-132. (11) Kekicheff, P. J. Colloid Interface Sci. 1988, 131, 133-152. (12) Kekicheff, P.; Cabane, B.; Rawiso, M. J. Phys. Lett. 1984, 45, 813-821. (13) Rancon, Y.; Charvolin, J. J. Phys. Chem. 1988, 92, 6339-6344.
S0743-7463(96)00536-7 CCC: $14.00
phases in a narrow region of concentration from the binary system SDS/water. In the same system also a correlation was found between the arrangements of defects in the lamellar phase and the tetragonal lattice structure of the neighboring intermediate phase.12 Defective lamellar phases comprising more or less regular disruptions in the bilayers have been reported from both nonionic and ionic systems,1-7 and are sometimes treated as a separate mesophase denoted LRh.5,6 While several techniques may be employed to reveal lamellar defects, it appears more difficult to determine their topology, and consequently few certain statements on the topology of defects are reported. Contrary to intermediate phases, which are found mostly within narrow fields in the concentrated regions of the phase diagrams, examples of rather extensive defective lamellar phases have been identified in more solvent rich regions.6 In the water corner of a phase diagram the neighboring phase to the lamellar phase may be a micellar solution, and a bilayer to rod transformation in aggregate geometry then corresponds to a LR-L1 phase transition. In the dilute range an additional mechanism for the creation of sites with increased curvature may come into play, namely, the fragmentation of infinite bilayers. This may be expected since the edges, formed when the bilayer is broken, can take on a microstructure similar to the cylindrical micelles. From the micellar point of view, it was suggested by Porte et al.19 that the perforated lamellar phase may form via the growth of branched and multiconnected threadlike micelles. Interconnection of threadlike micelles reduces the overall curvature of the monolayer making up the micelles, and thereby reflects a preference for microstruc(14) Kekicheff, P.; Tiddy, G. J. T. J. Phys. Chem. 1989, 93, 25202526. (15) Henriksson, U.; Blackmore, E. S.; Tiddy, G. J. T.; So¨derman, O. J. Phys. Chem. 1992, 96, 3894-3902. (16) Funari, S. S.; Holmes, M. C.; Tiddy, G. J. T. J. Phys. Chem. 1992, 96, 11029-11038. (17) Burgoyne, J.; Holmes, M.; Tiddy, G. J. T. J. Phys. Chem. 1995, 99, 6054-6063. (18) Blackburn, J. C.; Kilpatrick, P. K. J. Colloid Interface Sci. 1993, 157, 88-99. (19) Porte, G.; Gomati, R.; El Haitamy, O.; Apell, J.; Marignan, J. J. Phys. Chem. 1986, 90, 5746-5751.
© 1997 American Chemical Society
Defective Swelling Lamellar Phase
tures of decreasing curvatures. Other studies20 have, on theoretical grounds, identified conditions for the formation of perforated bilayers or so-called mesh structures. Using a crude model with two parameters, geometry and volume fraction of amphiphile, Hyde demonstrated that mesh structures could be preferred in regions with both higher and lower curvature than the lamellar phase.20 It has been14-16 argued that in the transitions from planar bilayers to rods, the formation of intermediate structures is preferred by long chained amphiphiles, whereas analogues with short chains, which give more flexible monolayers, rather build cubic arrangements. This behavior was demonstrated in the binary systems of various poly(ethylene oxide) surfactants6,16 and further corroborated by the appearance of intermediate structures in systems of the less flexible fluorocarbon amphiphiles.3,5 The present contribution reports on the phase behavior of a mixed amphiphile system of egg lecithin (EPC) and cetyltrimethylammonium chloride (CTAC). The interest in this particular system originates from an earlier cyrotransmission electron microscopy (cryo-TEM) study,21 where perforated vesicles were shown to appear at an intermediate stage in the solubilization of lecithin vesicles by CTAC. In an attempt to relate the aggregate structures observed by cryo-TEM to the phase behavior of the system, we now present the solvent corner of the phase diagram, supported by small-angle X-ray scattering (SAXS), NMR (2H, 31P), and further cryo-TEM studies. Experimental Section Materials. Egg lecithin (EPC) of grade 1 was purchased from Lipid Products, Nutfield, U.K. Cetyltrimethylammonium chloride (CTAC) was prepared by ion exchange (Dowex 1 × 8) from CTAB (Serva). The deuturated version of CTAC (CTAC-d9) was synthesized according to a method described in ref 22. Hexadecylamine (Fluka) lyophilized in DMF was alkylated with deuturated methyl iodide, isotopic purity 99.5%, from Glaser Laboraties, Go¨teborg. The reaction was carried out in the presence of PMP (1,2,2,6,6-pentamethylpiperidine). The proton acceptor PMP was purchased from Fluka. The reaction product CTAI-d9 was washed and recrystallized in methanol/acetone and eventually ion exchanged in 25 wt % ethanol to CTAC. Deuterated water of 99.9% purity was obtained from Sigma. Samples were prepared in 10 or 5 mm tubes of Pyrex glass with screw caps. The preparations were performed by adding CTAC to lecithin lyophilized in chloroform/methanol solution. The solvent was removed by evaporation under a stream of nitrogen followed by an overnight drying under vacum; 100 mM NaCl in deuterium oxide was then added to the dry mixture of amphiphiles. Phase equilibria were assumed to be attained after 1 week of periodical mixing at 25-40 °C and 1 week of rest at 25 °C. The equlibration process was to some extent in competition with the chemical degradation of the lecithin. Prolonged storage of samples was therefore avoided. Centrifugation was in some cases employed for the mixing process and to facilitate the separation of coexisting phases. Samples for the NMR measurements were prepared as described and thereafter transferred to appropriate NMR tubes. All measurements were made on a Bruker ACP-250 NMR spectrometer. The temperature was controlled by a heated air flow and monitored by means of a thermocouple close to the sample. All measurements were performed at 25 °C. SAXS. Small-angle X-ray scattering measurements were performed on a Kratky compact small-angle system, linear collimated and equipped with a position sensitive detector (OED 50M from MBraun, Graz) containing 1024 channels. Nickelfiltered Cu KR radiation (1.542 Å) was provided by a Seifert ID-3000 X-ray generator, operating at 50 kV and 40 mA. Samples (20) Hyde, S. T. Colloq. Phys. 1990, C7, 209-227. (21) Edwards, K.; Gustafsson, J.; Almgren, M.; Karlsson, G. J. Colloid Interface Sci. 1993, 161, 299-309. (22) Sommer, H. Z.; Hayden, I. L.; Jackson L. L. J. Org. Chem. 1971, 36, 824-828.
Langmuir, Vol. 13, No. 4, 1997 853 were filled into a quartz capillary, glued to an invar steel body. The slit-smeared spectra were desmeared using a method of beam height correction. Some 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-measurements have been performed preferentially on samples from the solvent rich region. The phase behavior was studied by 2H NMR on the deuterated water and on CTAC with the methyl headgroups deuterated (CTAC-d9), and finally also by 31P NMR on EPC. 2H NMR measurements were performed at 38.40 MHz on a wide-band probe (Cryomagnet System Inc., Indianapolis, IN) on samples containing either CTAC/EPC/2H2O or CTAC-d9/EPC/ H2O. The signals of 2H2O were acquired with the CYCLOPS sequence, while those of CTAC-d9 were obtained, since the line shapes are much broader, by the quadrupole echo sequence23 with 15 µs pulse width and a pulse separation of 60 µs. The FIDs were zero-filled, and care was taken to perform the Fourier transformation from the top of the echo. 31P NMR measurements were made at 101.27 MHz using the Hahn echo sequence,24 with broad-band decoupling of the protons. Here the π/2 pulse width was 10 µs and the pulse separation was 60 µs. Analysis of NMR Spectra. 2H NMR. For a lamellar arrangement of lipids, the 2H NMR signal of a C-2H group consists of a doublet at resonance frequencies (νq. For most cases the amphiphiles experience an effectively cylindrical symmetry around their long axis. In this case the residual anisotropy is described by a single-order parameter, SCD, and the quadrupolar splitting, ∆νq, is given by:25
[
]
3 3 cos2 θ - 1 ∆νq ) χ |SCD| 2 2
(1)
where χ is the quadrupole coupling constant for the C-2H bond and θ is the angle between the director of the lamellae and the main magnetic field. The order parameter, SCD, is given by
1 SCD ) 2
(2)
where β is the angle between the C-2H bond and the director. For a sample consisting of a random distribution of orientations of liquid crystalline microdomains, the spectrum will adopt a so-called “powder pattern”, where the intensities at each θ is scaled by the probability density, p(θ) ) 1/2 sin θ. A characteristic 2H NMR spectrum with “peaks” (θ ) 90°) and “shoulders” (θ ) 0°) is therefore observed. In the case of macroscopically oriented samples, a doublet will be seen with the quadroupole splitting given by eq 1. Equations 1 and 2 are valid for the water deuterons as well, with the appropriate value of χ for an O-2H bond. The quadrupole splitting obtained from deuterated water is usually much smaller than from deuterated lipids. It also depends much stronger on the water content than the splitting of lipids and surfactants. Quadrupole splittings of water in anisotropic lipid phases can be described by a simple two-site model, where the two sites are comprised of one with water bound to the surfactant aggregates and one with “free” water in an isotropic environment.26 Fast exchange between the two sites is assumed, and the observed quadrupole splitting will be a weighted average of the splittings in the two sites. In water-swollen lamellar phases the average is heavily weighted toward the isotropic line shape, giving small splittings that can be difficult to measure accurately. 31P NMR. The line shapes of 31P NMR spectra in an anisotropic liquid crystalline phase is governed by the chemical shift anisotropy (csa). Again, cylindrical symmetry implies a singleorder parameter, and the chemical shift will be orientation dependent according to:27 (23) Davis, J. H. Biochim. Biophys. Acta 1983, 737, 117-171. (24) Rance, M.; Byrd, R. A. J. Magn. Reson. 1983, 52, 221-240. (25) Wennerstro¨m, H.; Lindblom, G.; Lindman B. Chem. Scripta. 1974, 6, 97-103. (26) Persson, N. O.; Lindman, B. J. Phys. Chem. 1975, 79, 14101418. (27) McLaughlin, A. C.; Cullis, P. R.; Berden, J. A.; Richards, R. E. J. Magn. Reson. 1975, 20, 146-165.
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Langmuir, Vol. 13, No. 4, 1997 σ ) σ0 +
csa (3 cos2 θ - 1) 3
Gustafsson et al. (3)
where σ0 is the isotropic chemical shift and the order parameter is included in the csa. θ is the angle between the director and the main magnetic field. Cryo-TEM. The specimen preparation for the cryo-TEM investigations was carried out using an improved home-built version of the CEVS (controlled environmental vitrifications system), described elsewhere.28 A thin film of the sample solution was prepared by using a filter paper and blotting a small drop of the sample solution placed on an EM-grid. The used grids were of copper (200 mesh) and coated with a perforated polymer film of cellulose acetate butyrate.29 The coating polymer film was in some cases made more hydrophilic through treatment by glow discharge.30 Vitrification was achieved by plunching the grid into liquid ethane held at its freezing point. The vitrified specimens were examined in a Zeiss EM 902 A instrument, operating at 80 kV. Images were recorded at underfocus settings of about 2-3 µm.
Results Phase Equilibria and SAXS. Figures 1 and 2 show the phase behavior in the solvent corner of the pseudo ternary system EPC/CTAC/100 mM NaCl, determined by means of visual inspection, SAXS, and NMR. The swelling limit of the pure lecithin was taken from the literature31 and not measured explicitly. The swelling limit of the lecithin rich lamellar phase was evaluated by studying the evolution of the Bragg peak position on dilution. The region of coexistence of two lamellar phases was inferred by the appearance of two pairs of Bragg peaks, each pair in the positional ratio 1:2. Figure 3 shows such a typical diffractogram from a sample pertaining to the two-phase region of coexisting lamellar phases. Further support for the coexistence of lamellar phases and the overall phase behavior was obtained from the NMR line shape studies presented in the following section. No attempt to construct proper tie lines has been made. It is important to note that the tie lines do not necessarily have to lie in the plane of this pseudoternary diagram, since there may be an uneven distribution of the electrolyte. It was not possible to macroscopically separate the phases of the multiphase samples containing the only partly swelling lecithin rich lamellar phase. On the contrary, a relatively quick (hours) separation was obtained from the two-phase samples comprising lamellar phase from the dilute tongue and excess solvent phase. No distinct Bragg reflections were obtained from the most solvent rich region (Φ < 0.1) of the lamellar tongue. The swelling boundary of the tongue was therefore estimated from the volumes of the coexisting phases. On the CTAC rich side an extensive normal hexagonal phase is observed, as inferred by the diffractograms showing Bragg peaks in the typical ratio 1:x3:x4. From the position of the first peak and the sample composition the radius of the hydrocarbon core may be calculated,32 giving values from about 18 to almost 21 Å for the most lecithin rich sample. The region where the hexagonal and lamellar phase meet has not been studied in detail. Let us instead turn to the enlargement of the solvent corner given in Figure 2. The samples belonging to the (28) Bellare, J. R.; Davis, H. T.; Scriven, L. E. and Talmon, Y. J. Electron Microsc. Tech. 1988, 10, 87-111. (29) Fukami, A.; Adachi, K. J. Electron Microsc. 1965 14, 112-118. (30) Dubochet, J.; Groom, M.; Meuller-Neuteboom, S. Adv. Opt. EM 1982, 8, 107-135. (31) LeNeveu, D. M.; Parsegian, V. A.; Gingell, D. Biophys. J. 1977, 18, 209-230. (32) Luzzati, V. In Biological Membranes; Chapman, D., Ed.; Academic Press: New York, 1968.
Figure 1. Solvent corner of the pseudoternary diagram EPC/ CTAC/100 mM NaCl in water. Please note that not all multiphase areas are depicted.
Figure 2. Enlargement of the solvent corner of Figure 1. Please note that not all multiphase areas are depicted.
Figure 3. Diffraction profile of the sample EPC/CTAC/100 mM NaCl, 47/13/40. Two pairs of Bragg peaks are found in the typical ratio of 1:2, inferring a coexistence of two lamellar phases.
tongue are all birefringent, relatively fluid, and clear. The birefringency is weakenend in the outermost region of the tongue, where also some turbidity may appear. In a small region, denoted L1*, of the lower left corner of the tongue the stationary birefringency is lost, and birefringency is observed only on flow. The micellar region close
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Langmuir, Vol. 13, No. 4, 1997 855
Figure 4. Observed repeat distance (b, the position of the first Bragg peak) as a function of the inverse volume fraction of amphiphiles (1/Φ) for a dilution series with nCTAC/nEPC) 2.10, corresponding to the center of the lamellar tongue. The line connecting the small squares corresponds to the theoretical ideal swelling, as calculated from the apparent bilayer thickness from the most concentrated sample.
to the lamellar phase shows flow birefringency together with a rather high viscosity, but samples from L1* have the same fluidity as the samples from the neighboring stationary birefringent region. The lack of birefringency from the L1* region actually leads to two different twophase regions. One two-phase (LR + W) system of a lower excess solvent phase and an upper birefringent phase is obtained in the region next to the large three-phase area, while two-phase samples (L1* + W) where the upper phase is merely flow birefringent result from the region close to the micellar phase. The parts of the large micellar phase (L1) shown in Figure 2 have low viscosity except for the region closest to the neighboring phases LR and L1*. The dilute samples from this viscous region show viscoelasticity. The LR/L1 phase boundary is thus indicated by a loss of stationary birefringency and an increase in viscosity. Noteworthy is that no two-phase region was found. For symmetry reasons, such a region of coexistence should be present. We therefore conclude that it is narrow and below the used resolution. In the diffraction patterns from samples in the lamellar tongue, two distinct Bragg reflexes usually appeared for solvent concentrations less than 85 wt %. From more dilute (>85 wt %) samples, the scattering profiles are more diffuse: the second Bragg peak is lost, while the first appears to be blurred by an increased low-angle scattering. Unfortunately, a set-up with a larger sample to detector distance and a higher resolution in the low-angle region is needed to fully clarify the diffraction from these samples. In a series the repeat distance (d) was followed with increasing volume fraction of the solvent. This was measured in order to investigate how the swelling in the lamellar tongue relates to the ideal one-dimensional swelling behavior. Equation 4 gives the repeat distance, i.e., the position of the first Bragg peak, for the ideal case:32
d)
2vs δ ) asΦ Φ
(4)
where vs is the (average) molecular volume of the amphiphiles and as is the corresponding value for the (average) area in the bilayer. In the second equality, δ corresponds to an effective bilayer thickness. Figure 4 shows the deviation from eq 4 upon dilution made in the center of the tongue (nCTAC/nEPC ) 2.09). The volume
Figure 5. 31P NMR line shapes from the samples a-d (sample compositions are depicted in Figure 2).
fraction of amphiphiles was estimated by approximating the density of the EPC/CTAC mixture to 0.92 g/cm3. Typical values for the separate compounds are 0.89 for CTAC15 and 0.94 for lecithin.33 The straight line corresponds to the ideal swelling behavior, as calculated from the bilayer thickness of the most concentrated sample. The obtained negative deviation may be interpretated as an increase in the bilayer area. A slight negative deviation may be expected due to increased hydration and expansion of the individual headgroup areas.7 The negative deviation seen in Figure 4 is quite strong and suggests an area increase of almost 30% on dilution from Φ ) 0.49 to Φ ) 0.15. In order to explain such an apparent bilayer thinning, water-filled defects may be considered, since a transfer of solvent into the bilayer region will act to increase the total area of bilayers. NMR. 31P NMR. 31P NMR line shape studies were made on samples pertaining to the dilute regime, as indicated in Figure 2. Samples a-c all had a total content of EPC and CTAC-d9 equivalent to 260 mM. Sample d has the same molar ratio of the amphiphiles as b (nCTAC/ nEPC ) 2.09) but an increased amount of solvent. Figure 5a shows the spectrum of sample a taken from a region where a coexistence of two lamellar phases was predicted from SAXS measurements. The line shape has the characteristic of a uniaxial powder pattern superimposed by a narrow peak of rather high intensity. Inside the lamellar tongue (sample b, figure 5b) a shifted narrow signal is obtained, which corresponds to an oriented sample. This peak appears on the low-frequency side, indicating that the normal to the bilayer is oriented perpendicular to the main magnetic field.34 The chemical shift of this oriented signal is the same as that of the narrow signal from sample a, suggesting that sample a may contain two lamellar phases. Narrow 31P NMR signals are typically observed for samples belonging to the lamellar tongue. In the spectrum from sample d further out in the tongue, however, the NMR peak is broadened and shifted toward an isotropic position (sample d, Figure 5d). Only a very weak anisotropic shift can be traced from sample d. From sample c in the micellar region a narrow isotropic peak is recorded (sample c, Figure 5c). (33) Small, D. M. Handbook of Lipid Research; Plenum Press: New York, 1986. (34) Seelig, J.; Borle, F.; Cross, T. A. Biochim. Biophys. Acta 1985, 814, 195-198.
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Figure 6. Corresponding line shapes of the deuterated CTACd9 from samples a-d.
Figure 7. Quadrupolar splittings of CTAC-d9 as a function of the inverse volume fraction of CTAC-d9 and EPC. The molar ratio of the amphiphiles is fixed at 2.10. 2H NMR. Following the same sample series as in the previous section it is seen in Figure 6a that the line shape of CTAC-d9 from sample a exhibits a powder pattern. Since it is expected to represent a superposition of signals, from two lamellar phases we may conclude that the value of the quadrupolar splitting for the deuterated surfactant must be approximately the same in both the lamellar phases. When the tongue is entered the powder pattern is replaced by a doublet (sample b, Figure 6b). The doublet represents an oriented 2H NMR signal, where the peaks have grown at the expense of the shoulders, indicative again of an orientation with the director perpendicular to the main magnetic field.34 A further increase in the CTACd9 concentration leads to a single peak (sample c, Figure 6c), which reflects the entrance to the micellar phase. In the outermost region of the tongue, the value of the quadrupole splitting is reduced and the lines are somewhat broadened (sample d, Figure 6d). The dependence of the quadrupole spittings from CTAC-d9 on the volume fraction of solvent was investigated on a dilution line including the sample d and more concentrated samples with the molar ratio 2.09 of CTAC to EPC. The result presented in Figure 7 shows a rather strong increase in the values of the quadrupole splitting with increasing concentration. The water 2H NMR spectra of single-phase samples from the lamellar tongue showed doublets from more or less oriented bilayers. As expected the values of the splittings increased with decreasing solvent concentrations. They were also found to decrease with increasing concentrations of CTAC, i.e., for samples in the neighborhood of the micellar phase. To investigate the latter behavior the quadrupole splittings for a sample series with constant water content were measured. Figure 8 shows the progressive decrease in the splitting at a constant solvent
Gustafsson et al.
Figure 8. Quadrupole splittings of the water deuterons when the molar ratio of CTAC to EPC is varied at constant hydration (nD2O/(nCTAC + nEPC) ) 130), corresponding to a solvent weight fraction of about 0.15.
weight fraction of about 0.85. The ratio of CTAC to EPC is varied from 2 to 3, while the molar ratio of water is fixed to 130. Rather rapidly decreasing quadrupolar splittings are observed upon approach of the more CTAC rich micellar phase. Cryo-TEM. Cryo-TEM permits investigation only of dilute samples. The micrographs in Figure 9 are all from samples of concentrations less than 1 wt % of surfactant and lipid. The sequence of structures in this particular system was earlier recognized in a cryo-TEM study on the solubilization of lecithin vesicles by CTAC.21 We now present further micrographs from samples prepared by dilution of the samples used in the determination of the phase diagram. Starting from the micellar region, bearing in mind that CTAC itself forms spherical micelles,35 it is seen in Figure 9a that globular micelles are formed of CTAC and EPC at a molar ratio of 4.0 (nCTAC/nEPC). A growth into threadlike micelles is observed when the amount of lecithin in the aggregates is increased. In Figure 9b it is evident that the threadlike micelles at a molar ratio of 2.6 have a preference for the formation of intermicellar connections. It is particularly interesting that the formation of micellar connections appears to lead to discrete disordered networks of threadlike micelles. When the ratio of the two amphiphiles approaches the value of the L1/L1* phase boundary, the morphology of the aggregates is changed. The elongated micelles are now replaced by sheetlike aggregates (Figure 9c, molar ratio 2.4). The twodimensional character of these aggregates is evident from the presence of edge-on projections, appearing as dense lines. Note also that the aggregates exhibit an internal lacelike structure, which shows some resemblance to the conformations formed by the threadlike micelles in Figure 9b. It is important to note that the way in which the threadlike micelles are connected in this system (Figure 9b) clearly contrasts to the formation of three dimensional network structures, recently visualized in cryo-TEM.36 In this latter study it was found that interconnection of micellar threads in the bile salt/CTAB system resulted in the formation of aggregates that displayed very little internal structure in the cryo-TEM projections. It should also be emphasized that an accumulation, and therefore an increase in concentration of aggregates, during the (35) Imae, T.; Ikeda, S. Colloid Polym. Sci. 1987, 265, 1090. (36) Swanson Vethamuthu, M.; Almgren, M.; Brown, W.; Mukhtar., E. J. Colloid Interface Sci. 1995, 174, 461.
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film-thinning process is an almost unavoidable effect of specimen preparation in cryo-TEM. Such processes may induce the formation of connection and apparent entanglements between micelles. In order to facilitate the interpretation of the micrographs it is therefore of importance to keep a low and homogeneous concentration of aggregates within the film. We have therefore deliberately chosen micrographs where few overlapping micelles are seen and where the arrangement of the aggregates is seemingly not influenced by the presence of the supporting polymer film. At molar ratios (CTAC/EPC) below 2.3 perforated vesicles are observed, sometimes coexisting with the open forms of the same structure. This was documented already in the previous cryo-TEM study of this system.21 Figure 9d shows a stack of continuous layers captured in the thin vitreous film. The protruding single layer gives a clearer view of the perforated lamellarsor if preferredsthe mesh structure. Interestingly, the vesicle form of this structure, i.e., perforated vesicles, dominates at molar ratios between 2.3 and 1.9, which correspond to compositions where the lamellar tongue coexists with excess solvent phase. A further decrease of the CTAC ratio leads to the appearence of ordinary but somewhat ruptured bilayers, in coexistence with perforated vesicles. In Figure 9e both these structures are seen, as well as the open form, which indeed is reminiscent of the micellar structures shown in Figure 9c. Below amphiphile ratios of 1, the samples are completely dominated by ordinary bilayer structures, i.e., ordinary vesicles of various forms. Discussion The original aim of this study was to investigate whether the perforated vesicles observed in cryo-TEM could be correlated to the equilibrium structure and phase behavior at higher concentration of amphiphiles. In the following we shall consider how well the present observations of the phase behavior in general, and on the lamellar phase in particular, may serve this purpose. The Swelling Lamellar Phase. The most striking feature in the appearence of the phase diagram is the narrow tongue of lamellar phase extending far into the solvent corner. In order to explain the swelling of the lamellar phase we must first clarify the dominant interactions at play. The Debye length (κ-1) due to the electrolyte in the solvent is about 10 Å. In this limit the strong interbilayer pressure resulting from the electrical double layers is suppressed for large separations. Instead another repulsive interaction acting at long distances may be considered, namely, the undulation force caused by fluctuations of flexible bilayers. Using approximate descriptions of these interactions, a simple comparision of their magnitude can be made. For a given bilayer bending modulus, kc, the force per unit area between two membranes undulating, under no external constraints, may be expressed as:37
P)
3π2(kT)2 3
64kcD
(5)
where D is the interbilayer distance. This pressure should be compared to the osmotic pressure arising from the double layers of the accumulated electrolyte between two (37) Helfrich, W. Z. Naturforsch. 1978, 33a, 305-311.
charged bilayers. The latter can be estimated in the weak overlap approximation to38
P ) 1.59 × 108[NaCl]Γ2 e-κD
(6)
Where [NaCl] is the molar concentration of salt. In the center of the tongue where there is about two CTAC for every EPC, the factor Γ2, which is a function of the surface potential, is close to unity. If values of a few kT are used for kc, which is reasonable if strong undulations are expected, it is clear that the electrostatic interaction will dominate at small and intermediate separations. For D > 100 Å, the balance will be switched and the pressure from the undulations will now exceed the double-layer interactions, which decreases rapidly at longer distances. The assumption of flexible bilayers implies that the electrostatic contribution to the bending elasticity must be low. Several contributions in recent years have theoretically estimated the electrostatic increment in kc (see ref 39 for review). For situations, as in the present case, where the interbilayer separation is much larger than the Debye length, it is concluded that electrostatic interactions should not substantially suppress undulations.39 Swollen lamellar phases stabilized by fluctuations of flexible bilayers are known from several nonionic systems, where interbilayer distances of several hundred nanometers have been reported.40-42 Earlier studies reporting swollen phases in ternary systems have emphasized the softening effect on lipid bilayers brought about on addition of a cosurfactant.42 Increasing the lamellar flexibility means an increase in the repulsive steric interaction, since it scales with the inverse of the elastic bending modulus of curvature, kc. A related ternary system that exhibits an electrostatically stabilized swollen lamellar phase is the one of CTAB and DMPC determined by Rydhag et al.43 In comparison to the latter diagram it is clear that the swelling in the present system is suppressed for low ratios of the ionic surfactant. Most likely this is due to the presence of salt. As mentioned earlier, fluctuations of bilayers affect the long range order in the lamellar phase. The effect of a reduced positional order of the bilayers may give some explanations to both the X-ray and the NMR data obtained from the swollen lamellar phase in the present system. Recently Halle and Quist44 showed how the quadrupole splitting of a deuturated surfactant in a bilayer decreases whith dilution in a lamellar phase stabilized by the undulation force. In the same study they also used the coupling between the lamellar director fluctuation and the quadrupole splitting to estimate the bilayer rigidity. It is clear from Figure 7 that the values of the quadrupole splitting experience a similar reduction on dilution also in the present system. The observed splitting decreases from 2.88 kHz at Φ ) 0.49 to 1.10 kHz at Φ ) 0.07, along the dilution line in the center of the tongue at a molar ratio of 2.09. However, since we do not expect undulations alone to be responsible for this phenomenon, we have not analyzed the quadrupolar splittings quantitatively. (38) Israelachvili, J. N. Intermolecular & Surface Forces, 2nd ed.; Academic Press, Inc.: San Diego, CA, 1991. (39) Andelman, D. In Handbook of Phys. of Biol. Syst.; Lipowsky, R., Ed.; Elsevier: New York, 1991. (40) Larche, F. C.; Apell, J.; Porte, G.; Bassereau, P.; Marignan, J. Phys. Rev. Lett. 1986, 56, 1700-1703. (41) Strey, R.; Schoma¨cker, R.; Roux, D.; Nallet, F.; Olsson, U. J. Chem. Soc., Faraday Trans. 1990, 86, 2253-2261. (42) Safinya, C. R.; Sirota, E. B.; Roux, D.; Smith, G. S. Phys. Rev. Lett. 1989, 62, 1134-1137. (43) Rydhag, L.; Gabrian, T. Chem. Phys. Lett. 1982, 30, 309. (44) Halle, B.; Quist, P.-Q. J. Phys. II Fr. 1994, 4, 1823-1842.
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Figure 9. Cryo-TEM micrographs from dilute samples (>99 wt % solvent) of various molar ratios of CTAC to lecithin. Bar equals 100 nm (reproduced at 50% of original size): (a) nCTAC/nEPC ) 4.0, small micelles; (b) 2.6, threadlike micellar structures with interconnections; (c) 2.4, sheetlike aggregates with internal structure; (d) 2.3, layers of perforated bilayers; (e) 1.1, mostly ordinary and somewhat disruptured vesicles but also some perforated vesicles (indicated with an arrow).
The alteration of the Bragg peak line shape with the volume fraction of bilayer in a fluctuating lamellar system has been the subject of a number of experimental studies,42,45-47 where SANS, synchrotron X-ray, and light scattering were used. Typical for the scattering profile from samples at high dilution appears to be a broadening of the line shapes and an increase in the low-angle scattering, which eventually blurs the first Bragg singularity. The measurements in our case are of lower resolution, but it is likely that the same effect may account for the less well defined diffraction patterns from the dilute lamellar tongue of the present system. An increased central scattering was recorded from the samples with solvent concentrations greater than 90 wt % solvent, and consequently no distinct first-order peaks appeared. On the other hand, the diffraction profiles from more concentrated samples with the same amphiphilic composition did exhibit two sharp Bragg signals. For a lamellar phase stabilized by electrostatics this is the expected profile, since undulations then should be suppressed. Apparently, the evolution of the diffraction patterns on dilution is in line with a switch in the balance of interbilayer forces somewhere above separations of 100 Å. The most important observation from the SAXS studies is, however, the strong deviation from ideal swelling and the strong increase in bilayer area. In order to explain these observations it is near at hand, referring to the (45) Porte, G.; Marignan, J.; Bassereau, P.; May, R. Europhys. Lett. 1988, 7, 713-717. (46) Nallet, N.; Roux, D.; Prost, J. Phys. Rev. Lett. 1989, 62, 276279. (47) Nallet, F.; Roux, D.; Milner, S. T. J. Phys. Fr. 1990, 51, 23332346.
cryo-TEM observations, to consider water-filled defects in the bilayers. Defects in the Bilayers. We first recall the reason for the occurrence of a defective lamellar structure: to offer an aggregate microstructure with an increasing positive curvature, i.e., more like the one given in the micellar aggregates. Following such a line of reasoning it is important to note the very limited extension of the two-phase area between the lamellar and the micellar phase. Such a narrow two-phase region suggests similarities in aggregate microstructure between the two opposing phases. No direct structural information on any intralamellar structure may be extracted from the present collection of NMR and SAXS data, except that a deviation from the classical bilayer structure occurs. The SAXS data showed a strong nonideal swelling of the lamellar tongue. Heavily undulating bilayers may cause a nonideal swelling, through a reduction of the total projected bilayer. Such a reduction would, however, imply a slight increase in the apparent bilayer thickness41 in contrast to the recorded negative one. If instead water-filled defects in the bilayers are considered, which by no means rule out the presence of undulations, the effect will be to increase the amount of bilayer, and the observed deviation in a plot of d against 1/Φ is obtained (as in Figure 4). Noteworthy is that a similar magnitude of the increase in bilayer area, almost 30%, has been reported from other defective lamellar systems.5,7 Further information on the nature and the topology of the defects could probably be gained from studies in the more concentrated region and in particular from the structure of a possible intermediate phase.
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A reduction of the quadrupolar splitting from the deuterated surfactant may be expected if there are intralamellar defects present, due to the increased motional averaging over the curved surface offered by the defects.7 Since we suspect that undulations are active only in the very dilute regime, it is likely that it is the defect formation alone that accounts for the decrease in the splitting values observed for the concentrated samples. Apparently there is a progressive healing of the defective lamellar structure on increasing concentration of amphiphiles. The same observation was recently made in another mixed amphiphile system,7 and was qualitatively explained in terms of a suppressing of the defect formation by an increased interbilayer repulsion at low solvent concentrations. This occurs since the defect formation effectively leads to a reduction of the interlamellar distance, through an increase of the total area of bilayers. The observed reduction of the splitting from the water deuterons at constant water content (Figure 8) on approach of the micellar phase is consistent with a progressive defect formation near the micellar phase boundary (under the assumption that the degree of hydration of CTAC does not differ greatly from that of EPC). As with CTAC-d9 in the dilution series discussed above, the reduction of the splitting is quite significant. For a comparison, one expects (under the assumption that only the geometry of the amphiphilic aggregate is changed) that the normal hexagonal phase should give quadrupole splittings that correspond to one-half of what is given in the lamellar phase.25 Cryo-TEM and Phase Behavior. The sequence of microstructures observed by cryo-TEM may be summarized as (starting from the CTAC rich side): small micellar structures, elongated and interconnected micelles, a perforated layered structure, coexistence of the latter and ordinary bilayer structure, and eventually only ordinary bilayers. Clearly the cryo-TEM observations support the idea of a smooth transition between the lamellar and micellar structures. No region of coexistence of ordinary bilayers and micelles is found. Instead the perforated structure appears as an intermediate structure and a link between micelles and ordinary bilayers. It appears also very likely that the perforated structure observed by cryo-TEM originates from the lamellar tongue. In that case a nice correlation is found, in both the sequence and the location of structures, between phase equilibria and the microstructures observed by cryo-TEM. The close relationship between a liquid crystalline phase and its neighboring micellar phase near their common phase boundary was recently emphasized by Monduzzi et al.48 In the same way as three-dimensional networks of micelles may exist close to the normal cubic phase, it
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appears as if also a connection of thread micelles, leading to a growth into a sheetlike structure, may occur near a (defective) lamellar phase. Through this growth process the overall curvature of the micelles is reduced, and the micellar structures are made more “lamellar-like”. On the other side of the phase boundary the defect formation in the lamellar phase acts to increase the microstructure curvature in the bilayers. In combination these mechanisms give rise to a very smooth transition between bilayer and micellar structures. Following the above reasoning we may also find suggestions to the structure of the micellar aggregates in the small isotropic region denoted L1*. The remaining possible intermediate structure between a perforated bilayer and interconnected micellar structures is a fragmentized version of the perforated bilayer. One may speculate that the content of the L1* is a stable dispersion of highly defective bilayer fragments, a suggestion that is in accordance with the low viscosity and the flow birefringency observed from the samples of this region. The cryo-TEM observations also supports this scenario. The amphiphile molar ratio of the sample shown in Figure 9c is close to the one found for the L1* region, and perhaps the aggregates of Figure 9c illustrate the content of the L1*, which in that case well fits the description of a stable dispersion of fragmentized perforated bilayers. As a concluding remark we note that several related ternary systems comprising ionic surfactant and cosurfactant (alcohol) also feature a swollen lamellar phase that in the dilute region transfroms into a micellar phase.49 Interestingly, in some of these systems, such as the brinebased pseudoternary system studied by Filali et al.,50 the defective lamellar structure appear as a probable candidate for the aggregate morphology close to the micellar boundary. It should also be noted that a sheetlike structure, of great resemblance to the perforated layer in Figure 9c, has been found in concentrated polymer blends and visualized by means of a different type of transmission electron microscopy method.51 Acknowledgment. This work was financially supported by the Swedish Technical Research Council and by a generous donation from Knut and Alice Wallenbergs Stiftelse. We thank Dr. Patrick Williams for technical assistance and valuable discussions during our measurements at the Darebury Laboratory. LA960536R (48) Monduzzi, M.; Olsson, U.; So¨derman, O. Langmuir 1993, 9, 2914-2920. (49) Ekwall, P. Adv. Liq. Cryst. 1995, 1, 1-142. (50) Filali, M.; Apell, J.; Porte, G. J. Phys. II Fr. 1995, 5, 657-664. (51) Hashimoto, T.; Koizumi, S.; Hasegawa, H.; Izumitani, T.; Hyde, S. T. Macromolecules 1993, 25, 1433-1439.
