Langmuir 2001, 17, 3227-3234
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Intermediate Phases in the System Egg Lecithin/CTAC/ Brine. SAXS and NMR Studies Greger Ora¨dd,*,† Jonas Gustafsson,‡,§ and Mats Almgren‡ Department of Chemistry; Biophys. Chem., Umeå University, S-901 87 Umeå, Sweden, and Department of Phys. Chem., Uppsala University, Box 532, S-751 21 Uppsala, Sweden Received December 19, 2000. In Final Form: March 5, 2001 The phase behavior and phase structures in the pseudo ternary system egg lecithin (EPC)/ cetyltrimethylammonium chloride/brine (100 mM NaCl) have been investigated by 2H and 31P NMR spectroscopy and small-angle X-ray diffraction (SAXS). At solvent contents higher than 55 wt %, micellar, hexagonal, and lamellar phases are formed. At lower solvent content, an intermediate phase occurs consisting of rods with noncircular cross-sections forming a centered rectangular phase of space symmetry c2mm. This phase is connected to both the lamellar phase and the hexagonal phase by two-phase regions. There is a large region of the lamellar phase where the bilayers contain water-filled defects in the form of pores and/or slits. SAXS diffractograms from the defect lamellar phase exhibit interlamellar Bragg reflections as well as a broad reflection originating from the water-filled defects. The variation of the distances obtained as a function of water content and CTAC/EPC ratio is consistent with theoretical predictions on waterfilled defects in lamellae. The rectangular phase is characterized by several sharp SAXS reflections which have been indexed to the space group c2mm. The NMR line shapes are of biaxial symmetry with an asymmetry parameter ranging from 0.6 to 1.0. From an analysis of the SAXS and NMR data, we have found evidence of molecular segregation in that lecithin is enriched in the flat regions of the noncircular cylinders.
Introduction The lamellar phase (LR) of egg lecithin (EPC) in water is stable over a broad temperature range and shows that aggregates with almost no curvature are favored.1 The lamellar phase swells to about 40% in water and salt solution. Addition of a charged component, such as cetyltrimethylammonium chloride (CTAC), a cationic surfactant, results in two effects. First, there is a very pronounced swelling in water, already at very low additions, to about 99% water at a CTAC/EPC molar ratio (X) of about 2.2 Second, addition of surfactant gives a destabilization of the bilayer and ultimately formation of mixed micelles.3 In a salt solution of 0.100 M NaCl, one could expect strongly reduced swelling, because of the screening of the electrostatics. With small ions present also in the aqueous layers of the lamellar phase, the entropic or osmotic repulsion between the bilayers is suppressed. It turns out, however, that a narrow tongue of swollen lamellar phase remains, around a certain surfactant-to-lipid ratio.4 This tongue can take up about 94% 0.100 M NaCl solution and is followed by a small island of a flow birefringent fluid phase, probably a sponge phase. On dispersion of the lamellar phase in excess brine, liposomes with perforated walls are formed. Investigations using NMR and SAXS have shown the lamellar tongue to contain defected bilayers, forming a random mesh phase (LRh).4,5 †
Umeå University. Uppsala University. § Present address: GDPC, Universite Montpellier II, Case 026, 34095 Montpellier Cedex 5, France. ‡
(1) Small, D. M. The physical chemistry of lipids. From alkanes to phospholipids; Plenum Press: New York, 1986. (2) Rydhag, L.; Gabra´n, T. Chem. Phys. Lipids 1982, 30, 309-324. (3) Edwards, K.; Gustafsson, J.; Almgren, M.; Karlsson, G. J. Colloid Interface Sci. 1993, 161, 299-309. (4) Gustafsson, J.; Ora¨dd, G.; Lindblom, G.; Olsson, U.; Almgren, M. Langmuir 1997, 13 (4), 852-860. (5) Gustafsson, J.; Ora¨dd, G.; Almgren, M. Langmuir 1997, 13 (26), 6956-6963.
The repulsive forces responsible for the swelling in this system are probably Helfrich undulation forces,6 as in many systems where cosurfactants have made the bilayers soft and easy to bend.7 In this case, however, it is the perforations and defects that soften the bilayer. A system with C12TAC replacing C16TAC develops no lamellar mesh phase, shows reduced swelling, and exhibits also in other respects a different, more normal phase behavior with clear two-phase regions between LR and micellar solution (L1) or hexagonal (H1) phases.5 The difference between the two systems with respect to the swelling supports the coupling between the swelling and the formation of defects. The reason for the difference in behavior may be the fact that the short surfactant imposes much more curvature in the structure than the long one, so that a breakup of the bilayers is induced at a lower content of surfactant in the bilayer; a surfactantto-lipid ratio Xsat ≈ 2 is required with C16TAC, but Xsat is only ≈1 with C12TAC. This means a lower charge density, but it also means that a possible segregation of lipids and surfactant, with the latter enriched at the rim of the holes, would be counteracted by a larger loss of mixing entropy in the latter case. Which of these effects is most important is not clear. Previously, the phase behavior and phase structure have been studied, mainly in the dilute parts of the pseudo ternary phase diagram.4,5 It is also of interest to investigate the behavior in the more concentrated parts: what happens with the defects at higher concentration of lipids, and how does the transition occur, from the lamellar phase of the lipids to the hexagonal phase of CTAC, on increase of the surfactant concentration? These questions were the motivation of the present study. There have been several studies of systems in which a micelle-forming molecule has been mixed with a lamella(6) Helfrich, W. Z. Naturforsch. 1978, 33, 305-315. (7) Safynia, C. R.; Sirota, E. B.; Roux, D.; Smith, G. S. Phys. Rev. Lett. 1989, 62 (10), 1134-1137.
10.1021/la001772b CCC: $20.00 © 2001 American Chemical Society Published on Web 05/05/2001
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forming molecule.8-11 The phase diagrams of these systems often show a transition from a hexagonal phase to a lamellar phase passing one or several so-called intermediate phases. The intermediate phases, which are anisotropic, can be topologically characterized into three groups: rectangular, lamellar mesh, and bicontinuous three-dimensional structures. Intermediate phases have been found in a multitude of surfactant/water systems12 and similar to the cubic phases can be seen as ways to bridge the gap between the flat lamellae and the highly curved hexagonal phase. The intermediate phases provide a compromise between the packing constraints and the attempt to maintain an average curvature close to the spontaneous curvature of the system. The fact that intermediate phases exhibit nonuniform curvature allows them to change the mean curvature rather smoothly, both within a phase and on transition from one phase to another, by adjusting the relative amounts of highly curved and flat parts of the structure. If two or more amphiphiles are involved, a molecular segregation may occur, where the lamella-forming lipids reside preferentially in the flat regions of the phase while the micelle forming surfactants are enriched in the curved regions.13,14 This work extends the investigation of the ternary phase diagram of CTAC, EPC, and 100 mM NaCl (brine) to the more concentrated region where, apart from the LRh phase, at least one other intermediate phase is also found. This phase, which exists in equilibrium with the LRh and the H1 phase, is formed by noncircular cylinders organized in a centered rectangular phase (R). By a combination of 2H and 31P NMR line shape analysis with small-angle X-ray scattering (SAXS), phase equilibria and phase structures in this region have been determined and the role of the LRh phase as a mediating medium between the lamellar and the micellar and hexagonal phases is discussed. Experimental Section Materials. EPC of grade 1 was purchased from Lipid Products, Nutfield, U.K. CTAC was prepared by ion exchange (Dowex 1 × 8) from cetyltrimethylammonium bromide (Serva). Headgroup deuterated CTAC (CTAC-d9) was prepared as described elsewhere.4 Deuterated water of 99.9% purity was obtained from Sigma. Samples were prepared in 8 mm tubes of Pyrex glass by weighing appropriate amounts of EPC and CTAC into the tubes. To ensure mixing of the two amphiphiles, they were dissolved in chloroform/methanol which was then removed by evaporation under a stream of nitrogen followed by overnight drying under vacuum. NaCl (100 mM) in deuterium oxide was then added to the dry mixture of amphiphiles. Phase equilibria were assumed to be attained after one week of periodic mixing at 25-40 °C and one week of rest in 25 °C. Measurements were completed within five weeks after preparation with the samples stored at -30 °C in periods between measurements. NMR. 2H and 31P measurements were performed on a 10 mm wide-band probe on a Varian CMX-400 Infinity system. An ordinary Hahn echo sequence15 with WALTZ decoupling of the protons was used for phosphorus, and a quadrupole echo sequence16 was used for deuterium measurements. The 90° pulse widths were 22 µs for phosphorus and 30 µs for deuterium, and (8) Almgren, M. Biochim. Biophys. Acta 2000, 1508, 146-163. (9) Hendrikx, Y.; Charvolin, J. Liq. Cryst. 1992, 11 (5), 677-698. (10) Quist, P.-E.; Halle, B. Phys. Rev. E 1993, 47 (5), 3374-3395. (11) Hagsla¨tt, H.; Fontell, K. J. Colloid Interface Sci. 1994, 165, 431444. (12) Holmes, C. H. J. Curr. Opin. Colloid Interface Sci. 1998, 3, 485. (13) Chidichimo, G.; Golemme, A.; Doane, J. W.; Westerman, P. W. J. Chem. Phys. 1985, 82 (1), 536-540. (14) Quist, P.-O.; Halle, B. Mol. Phys. 1988, 65 (3), 547-562. (15) Rance, M.; Byrd, R. A. J. Magn. Reson. 1983, 52, 221-240. (16) Davis, J. H. Biochim. Biophys. Acta 1983, 737, 117-171.
Ora¨ dd et al. the time between the two pulses in both experiments was 100 µs. The temperature was held within 1 °C by a heated air stream passing the sample. All measurements were made at 25 °C. Line Shape Analysis. The deuterium line shape is governed by three parameters, namely, the motionally averaged quadrupole coupling constant, 〈χ〉, the asymmetry parameter, η, and the angular distribution function, f(θ,φ), of the polar and azimuthal angles that specify the orientation of the principal axis system with respect to the main magnetic field. The quadrupolar frequency of the two transitions (m ) 1 f 0 and m ) 0 f -1) is defined as16
3 νq(θ,φ) ) ( 〈χ〉(3 cos2 θ - 1 + η sin2 θ cos 2φ) 8
(1)
Detailed theoretical analyses of the quadrupolar line shapes in liquid crystalline systems can be found in the literature.14,16-18 31P NMR line shapes are governed by the chemical shift anisotropy (csa), and the line shape analysis can be treated in the same way as for the quadrupole interaction with the exception that there is now only one transition given by the frequency
ν(θ,φ) )
csa (3 cos2 θ - 1 + η sin2 θ cos 2φ) 3
(2)
where csa is the motionally averaged chemical skift anisotropy and η is the asymmetry parameter for the chemical shift.19 An oriented sample would thus give two peaks for 2H and one peak for 31P, whereas samples with microcrystallites oriented in many different directions give more complex line shapes where a nonlinear fit of the line shape can give 〈χ〉, csa, and η.20 Note that in contrast to 〈χ〉 from 2H NMR, one can determine the sign of the csa. This is particularly valuable in studies of phase transitions from a lamellar to a hexagonal phase when additional motional averaging due to motion around the cylinders scales down the csa by a factor of -1/2.21 SAXS. Small-angle X-ray diffraction experiments were performed on Station 8.2 of the Daresbury Laboratory, U.K., using a monochromatic beam of wavelength 1.54 Å.22 The samples were transferred to a 1 mm thick sample holder with thin mica windows and mounted on the heating element of a modified THM 600 thermally controlled microscope stage (Linkam, Tadorth, U.K.). The sample temperature was held at 25 °C at all measurements and was monitored by a thermocouple embedded in the sample adjacent to the beam. The camera length was 3.5 m, and the system was calibrated using wet rat-tail collagen (repeat distance 670 Å). Data were achieved as an average of 15 consecutive data sampling periods, each of 20 s length. No changes in the diffraction patterns were observed during the experiments. Some experiments were also done on Station 2.1 (8 m camera length) and on Station 16.1 (1.5 m camera length) with similar experimental setups. The diffraction data were processed using the XOTOKO software (G. Mant, Daresbury Laboratory, U.K.), and the diffraction peaks were indexed to the different space symmetries.23 Calculation of Structural Parameters. The molecular volumes were estimated to 531 Å3 (CTAC) and 1305 Å3 (EPC) by using 1 g/cm3 as the density of both amphiphiles. Only the hexadecyl chain and the oleoyl chains were assigned to the (17) Chidichimo, G.; Vaz, N. A. P.; Yaniv, Z.; Doane, J. W. J. Phys. Rev. Lett. 1982, 49, 1950. (18) Wennerstrom, H.; Lindblom, G.; Lindman, B. Chem. Scr. 1974, 6, 97-103. (19) Seelig, J. Biochim. Biophys. Acta 1978, 515, 105-140. (20) Gustafsson, S.; Quist, P.-O.; Halle, B. Liq. Cryst. 1995, 18 (4), 545-553. (21) Lindblom, G. Nuclear magnetic resonance spectroscopy and lipid phase behaviour and lipid diffusion. In Advances in lipid methodology; Christie, W. W., Ed.; Oily Press: Dundee, Scotland, 1996; pp 133-209. (22) Bras, W.; Derbyshire, G. E.; Ryan, A. J.; Mant, G. R.; Felton, A.; Lewis, R. A.; Hall, C. J.; Greaves, G. N. Nucl. Instrum. Methods Phys. Res. 1993, A326, 587-591. (23) Hahn, T. International tables for crystallography, 4th rev. ed.; Kluwer Academic: Dordrecht, 1996.
Intermediate Phases in Egg Lecithin/CTAC/Brine
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Figure 1. 2H (left) and 31P (middle) NMR line shapes and SAXS diffraction (right) from the four phases determined in the phase diagram. The different phases are, from bottom to top, LR (d ) 64.7 Å), LRh (d ) 69.3 Å), R (a ) 59.6, b ) 141 Å), and H1 (a ) 83.6 Å). The sample compositions are, from bottom to top, given as (X ) nCTAC/nEPC, wt % brine), (1.50, 50.0), (2.14, 65.0), (3.50, 50.0), and (3.00, 65.0). The inset in the SAXS figure shows the same sample with a longer camera length. The SAXS intensity is plotted on a logarithmic scale, whereas the NMR intensity scales are linear. The vertical lines correspond to the expected peak positions. hydrophobic part of the molecules, and the volumes of the chains were 460 and 590 Å3, respectively.24 From these values, the apolar volume fraction, φ, could be calculated for each sample.25 SAXS data from the lamellar phase can be used to calculate the hydrophobic thickness of the bilayer, dapp ) φd, and the molecular area at the polar-apolar interface, A ) 2Vm/dapp. Here, d is the repeat distance between bilayers and V is the apolar volume of the molecule. For the hexagonal phase, A can be calculated as A ) 2V/r, where r is the radius of the hydrocarbon core, given by r ) a0xx3φ/2π, and a0 is the distance between the cylinders obtained from SAXS. These calculations, however, are based on the ideal lamellar and hexagonal structures and should be used with caution in the present system where the investigated samples form less ideal structures (bilayers including defects and noncircular cylinders).
Experimental Results Characterization of the Phases. The combination of SAXS and NMR proves to be well suited for the characterization of the different phases in the system as they provide complementary information which, put together, provides the means for an unambiguous interpretation. Shown in Figure 1 are SAXS diffractograms and 2H and 31P NMR spectra from representative samples in four different phases found in the present investigation. The phases are, from bottom to top, LR, LRh, R, and H1. From left to right is displayed 2H NMR line shapes, 31P NMR line shapes, and SAXS diffractograms. In the SAXS diffractograms, in which the intensities are on a log scale, (24) Tanford, C. The hydrophobic effect. Formation of micelles and biological membranes.; John Wiley and Sons: New York, 1973. (25) Hagsla¨tt, H.; So¨derman, O.; Jo¨nsson, B. Liq. Cryst. 1992, 12 (4), 667-688.
the vertical lines indicate the calculated positions of the Bragg peaks corresponding to each phase according to the parameters listed in the figure legend. The LR phase (bottom row) is characterized by powder Pake line shapes for 2H and 31P, indicating that the microcrystallites are randomly oriented within the sample. The SAXS diffractogram shows two Bragg peaks with distance relations 1:1/2. The LRh phase (second row from bottom) gives NMR line shapes rather similar to those of the LR phase except that the intensities are enhanced for the 90° peaks, because of partial orientation of the director in the 90° direction with regard to the main magnetic field. The SAXS diffractogram shows Bragg peaks characteristic for the lamellar phase, but, in addition, there is also a diffuse scattering at low q-values attributed to the diffraction of the water-filled defects, which show little long-range correlation (hence the broad peak). The inset shows the same sample measured with a camera length of 8 m, thereby focusing on the low q region in order to separate the diffuse peak from possible beam stop effects that could distort its shape. Note that LRh should not strictly be seen as a phase separate from LR. Rather, the symbol LRh is used to emphasize the fact that these lamellae contain a considerable portion of defects. Moving on to the rectangular phase (second row from top), there is a marked change in the appearance of the NMR line shapes. They are now typical biaxial line shapes, reflecting the fact that the asymmetry parameter, η, now deviates from zero. SAXS gives seven diffraction peaks that can be indexed to the first seven peaks for a rectangular phase (c2mm). The indexation of the peaks is shown in Table 1. The centered rectangular phase gave the smallest error of all tested structures.
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Table 1. Indexation of the Centered Rectangular Phase Shown in Figure 1a hk dcalc dexp error a
02
11
13
04
20
22
15
70.5 70.6 -0.1
54.9 54.9 0.0
36.9 36.7 0.2
35.2 35.1 0.1
29.8 29.7 0.1
27.4 27.3 0.1
25.5 25.6 -0.1
Rectangular phase: a ) 59.6 Å, b ) 141 Å.
Figure 2. The tentative phase diagram of the pseudo ternary system of CTAC/EPC/100 mM NaCl at 25 °C: L1, micellar solution; H1, normal hexagonal phase; R, centered rectangular phase; LR, lamellar phase. The shaded area shows the part of the LR phase that contains defects.
The H1 phase (top row) gives NMR line shapes compatible with uniaxial powder patterns with the frequency scaled by approximately -1/2 compared to the lamellar phase. This scaling factor is a consequence of the additional motional averaging due to fast diffusion around the cylinders.21 The SAXS diffractogram from the hexagonal phase shows rather broad lines, and only a few of the allowed reflections can be observed. The positions of the expected peaks with distance ratios 1:1/x3:1/x4:1/x7 are indicated by the lines in the figure. Figure 1 shows the necessity of using complementary techniques when determining the phase structures as none of the methods alone give enough information to unambiguously determine what phases are present. Using only SAXS would have caused problems in indexing the H1 phase, and NMR alone cannot properly distinguish between the ordinary lamellar and the lamellar mesh phases. (It should be noted that the absence of the broad peak in SAXS does not strictly exclude the possibility of defects in the bilayers. If the defects are dilute and/or transient, the effect from them will be too small to detect in the SAXS experiment. We have, however, chosen to distinguish between LRh, where the broad reflection is observed, and LR, which can be defect-free or contain smaller amounts of defects.) However, by combining the information given by both SAXS and NMR a clear picture is obtained of the phases present in the system. As 31P NMR gives both the sign and the magnitude of the csa, whereas 2H NMR gives only the magnitude of the quadrupole splitting, the majority of the samples were prepared using only the natural isotopes. The Phase Diagram. The tentative phase diagram is presented in Figure 2. The dilute region with more than 75 wt % brine was thoroughly investigated in an earlier study4 and will not be discussed here. The main charac-
Figure 3. SAXS and 31P NMR data from the series of samples with constant solvent concentration marked with O in Figure 2. The sample compositions are listed in Table 2 and are numbered from bottom to top as 1-6.
teristic of the phase diagram is the narrow tongue of LRh extending from slightly above 50 wt % brine and far into the solvent corner (the dotted area represents the part of the phase diagram where an intralamellar reflection was observed by SAXS and, at very high dilution, perforated membranes have been observed in cryo-TEM). Contained between X ) 1.8-2.2 and over a large range of solvent concentration, this tongue separates the lecithin-rich part of the diagram, where only lamellar phases are formed, from the CTAC-rich part, in which there is a succession of phases, going from the solvent corner, of L1, H1, and R. There are two-phase regions connecting the hexagonal, rectangular, and lamellar phases. The hexagonal-lamellar two-phase region, however, is very narrow and difficult to observe. Figure 3 shows 31P NMR and SAXS data from six samples with 55 wt % solvent marked in Figure 2 as circles. As X is increased, the phase sequence in the figure is, from bottom to top, LR, LRh, LRh and/or intermediate, LRh + R, R, and R + H1. A summary of the results obtained for these samples is given in Table 2. Apart from the lamellar phase, the SAXS diffractograms are rather diffuse and would be difficult to interpret by themselves. However, the information given by the 31P NMR line shape makes it possible to conclude that the phase sequence is the one given. As an example of a two-phase sample (no. 4), Figure 4a shows how the 31P NMR line shape can be fit to the sum of one uniaxial (LR) and one biaxial (R) line shape. Given this information, it can be seen that the SAXS data, shown in Figure 4b, are compatible with this interpretation although the quality of the diffractogram is not good enough to draw such a conclusion based solely on SAXS.
Intermediate Phases in Egg Lecithin/CTAC/Brine Table 2.
31P
Langmuir, Vol. 17, No. 11, 2001 3231
NMR and SAXS Parameters Obtained from the Samples at Constant Solvent Concentrationa
sample number
wt % brine
X ) nCTAC/nEPC
φ
phase
1 2 3
55 55 55
1.5 2.0 2.5
0.40 0.40 0.40
4
55
3.0
0.40
5 6
55 55
3.5 4.0
0.40 0.40
LR LRh LRh see text LRh R R H1 R
a
31P
csa (ppm)
66.8 57.1 52.2 not det. 49.3 40.6 35.8 31.8 32.7
31P
η
SAXS distances (Å)
0 0 0 not det. 0 0.60 0.90 0 0.97
F
d ) 70.9 d ) 62.5, D ) 102 d ) 61.7 d ) 60.3 a ) 65.2, b ) 153 a ) 64.6, b ) 144 not det. a ) 65.8, b ) 146
1.46 1.37 1.36
The samples are marked with O in Figure 2.
Figure 4. Line shape analysis of the two-phase sample 4. (a) Experimental (solid) and simulated (dotted) 31P NMR line shapes together with the individual simulated line shapes from the rectangular and the lamellar phases. The integrated intensity fractions of the two line shapes are 56% (lamellar) and 44% (rectangular). (b) SAXS data together with the calculated positions of the rectangular phase peaks (bottom lines) and the lamellar phase peaks (top lines).
Sample 3 is somewhat ambiguous. The NMR spectrum of this sample shows no sign of biaxial character, yet the SAXS diffractogram clearly indicates the presence of something different from a lamellar phase. The same feature was observed for several samples at about 50-55 wt % solvent and X ) 2-2.5. This is further discussed in the next section. To investigate the diffraction peaks originating from defect correlations, a number of samples (marked with + in Figure 2 and summarized in Table 3) were prepared in the LRh region. Figure 5 shows the SAXS data of these samples. The SAXS diffractograms feature, in addition to the first- and second-order interlamellar peaks, a broad peak centered at q-values of 0.04-0.06 Å-1. The diffractograms from the three highest solvent contents are grouped two by two so that samples of the same solvent content can be compared in the same figure. (At the two highest concentrations, only one sample was made at each concentration. These two measurements were made with a different camera setup which introduced the artifacts at q < 0.04 Å-1 from the central beam and beam-stop seen in the top two figures.) As the solvent concentration is Table 3.
31P
decreased, the Bragg peaks move to smaller distances as the interlamellar distance is decreased. From the three lower figures, it is also clear that the Bragg peaks move to smaller distances as X is increased at constant solvent content. The broad reflection moves to smaller distances as the solvent concentration is decreased, and it also moves slightly to smaller distances when X is increased at the same solvent content.
NMR and SAXS Parameters Obtained from the Samples in the Lamellar Mesh Phasea
sample number
wt % brine
X ) nCTAC/nEPC
f
7 8 9 10 11 12 13 14
81.2 81.2 74.2 74.2 65.0 65.0 60.0 55.0
1.90 2.14 1.90 2.14 1.80 2.14 2.00 2.00
0.17 0.17 0.23 0.23 0.31 0.31 0.35 0.40
a
Figure 5. SAXS data from the lamellar mesh phase. The sample compositions are listed in Table 3 and are numbered from bottom to top as 7-14. The samples are marked with + in Figure 2.
The samples are marked with + in Figure 2.
31P
csa (ppm) 50.0 44.8 50.1 46.5 58.0 50.9 57.1 54.5
SAXS distances (Å)
apparent hydrocarbon thickness (Å)
d ) 116.1, D ) 145 d ) 103.2, D ) 140 d ) 90.4, D ) 129 d ) 83.6, D ) 126 d ) 76.7, D ) 113 d ) 69.3, D ) 108 d ) 67.7, D ) 109 d ) 62.5, D ) 102
21.8 19.4 23.3 21.6 26.8 24.3 27.1 28.1
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Discussion The Phase Diagram. The phase diagram is divided into two parts by the narrow tongue of the defect lamellar phase. On the EPC-rich part, only lamellar phases are formed,4 and this part of the phase diagram has not been studied in detail. The other part of the diagram, the CTACrich part, shows a succession of phases going from micellar solution over a hexagonal phase into a rectangular phase as solvent concentration is decreased. In the binary system of CTAC/D2O, a similar phase sequence was observed at 45 °C.26 Upon increasing amphiphile concentration, they observed a micellar and a hexagonal phase followed by two intermediate phases of which the first was a twodimensional monoclinic phase consisting of cylinders with noncircular cross sections. The second intermediate phase was tentatively assigned to a three-dimensional network, such as the rhombohedral, orthorhombic, or tetragonal phase. The phase diagram in this study shows a remarkable similarity with a ternary system studied by Hagsla¨tt and Fontell.11 Their system, composed of a divalent, micelleforming surfactant and an uncharged, lamella-forming surfactant, shows many of the features that the system in this study exhibits. The lamellar phase found in their system swells extensively at an average charge per surfactant below 0.7, whereas above this limit a phase succession of micellar, hexagonal, and rectangular phases is found. The authors also mention the possibility of a lamellar mesh phase located close to an average charge per surfactant of +0.7. A direct comparison is difficult to make, but it can be noted that the narrow tongue in our system occurs at an average charge per hydrocarbon chain of 0.7. This similarity in phase equilibria is somewhat unexpected because one would expect the electrostatic contribution to the free energy of the system to be much larger in the system of Hagsla¨tt. The phase equilibria are in both systems governed by the fact that one is mixing a micelle-forming molecule with a lamella-forming molecule and not by the precise nature of the molecular interactions. The general appearance of the phase diagram is well in accord with a theoretically calculated phase diagram of single-chain surfactants with variable average charge.27 This phase diagram was constructed using the so-called hexagon-rod Poisson-Boltzmann cell model, and the transition from micelle-forming to lamella-forming molecules was governed by the charge of the molecules. It was found that, with average charge per surfactant above +0.8, the transformation from a hexagonal phase consisting of circular cylinders to a lamellar phase was achieved by deforming the cylinders slightly into a noncircular shape. It was suggested that the noncircular cylinders still would be able to form a hexagonal phase, provided that the cross section of the cylinders fluctuated in time so that on average they had a circular shape. As the axial ratio grows, a transformation to either a rectangular phase or a lamellar phase occurs. This description accounts for the observed phase succession, while at the same time it allows the aggregates to change shape in a more or less continuous way. With this picture at hand, we now move on to discuss the intermediate phases found in the present study. The Rectangular Phase. There exist several examples of models that relate NMR and SAXS observables to the (26) Henriksson, U.; Blackmore, E. S.; Tiddy, G. J. T.; Soderman, O. J. Phys. Chem. 1992, 96, 3894-3902. (27) Hagsla¨tt, H.; So¨derman, O.; Jo¨nsson, B. Liq. Cryst. 1994, 17 (2), 157-177.
Ora¨ dd et al.
size and shape of the noncylindrical cross sections of the cylinders in rectangular phases.13,14,17,25 In this paper, we have chosen to follow the so-called hexagon-rod model developed by Hagsla¨tt et al.25 Within this model, SAXS data can be used to calculate the axial ratio, F, of the hydrocarbon core of the noncircular rods in the rectangular phase. This has been done for samples 4-6, and the values have been included in Table 2. It can be seen that F increases as X decreases but that the change is rather small. The increase in F reflects the tendency to move toward smaller average curvature as the concentration of the lamella-forming EPC increases. The analysis gives a length of the smallest dimension of the aggregates, rs, of about 19 Å. As this is the length of the molecule excluding the polar headgroup, it should roughly correspond to a fully stretched hydrocarbon chain. Results published from a variety of systems have shown that rs is about 80% of the all-trans length of the chain and that F lies between 1.2 and 2.25 Our results thus fall in the same range as results obtained earlier (an all-trans 16 carbon long hydrocarbon chain is calculated to be approximately 21.8 Å).28 Using the hexagon-rod model, it is also possible to relate the axial ratios calculated from SAXS data to the normalized residual anisotropy, Ar/AN, and the asymmetry parameter, η, obtained from the NMR data. For 31P, AR and AN can be replaced by the residual csa and the local csa of the surface, respectively. Equations 19-26 in Hagsla¨tt et al.25 give the relations between F and AR/AN and η for the case of uniform distribution and nonuniform distribution of the molecules along the flat and curved surfaces. The expressions can be generalized to include a “uniformity parameter”, W, which gives the population weight on the flat surface compared to that on the curved surface. The uniform and nonuniform cases used by Hagsla¨tt then correspond to W ) 1 and 2, respectively. The corresponding general expressions are
ηSDR ) 3 - 9[3 + Wx3(F - 1)]-1 1 e F < 1 + x3/2W (3) ηLDR ) 9[3 + 4Wx3(F - 1)]-1 ASDR ) -AN/2
F > 1 + x3/2W
1 e F < 1 + x3/2W
(4) (5)
9 ALDR ) AN 1 - [3 + Wx3(F - 1)]-1 4 F > 1 + x3/2W (6)
(
SDR and LDR denote the small deformation regime and the large deformation regime, respectively. In Figure 6, these equations are plotted for F-values ranging from 1 to 3 for two values of W along with the experimental points of samples 4-6. For the experimental points, the local csa has been estimated to be equal to the csa of sample 1, which forms bilayers without defects where (ideally) no additional averaging should occur. Although this is clearly only a crude estimate of the local csa, the reasonable values obtained for AR/AN give some credibility to the chosen value. It can be seen that the points agree qualitatively with the curves for W ) 3, which means that the surface density of EPC is 3 times higher in the flat regions than in the curved parts. Because the headgroup area typically is twice as large in a hexagonal phase (curved) than in a lamellar phase (flat), one would expect W to be 2 if EPC (28) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: London, 1991.
Intermediate Phases in Egg Lecithin/CTAC/Brine
Figure 6. The normalized residual anisotropy, AR/AN (solid line), and the asymmetry parameter, η (dashed line), as a function of the axial ratio, F. The curves are calculated according to the hexagon-rod model. Normal and bold lines correspond to W ) 1 and 3, respectively. The experimental points correspond to samples 4-6. The values of AR/AN (circles) and η (triangles) are determined from 31P NMR, and F is calculated from SAXS data.
would distribute evenly in the two regions. The even larger value of W reflects the tendency of the EPC molecules to reside preferentially in the flat region of the cylinders and is thus an indication that molecular segregation takes place. The Lamellar Mesh Phase. The line shapes obtained from samples in the LRh region depart from the powder pattern which would result from an isotropic orientational distribution. In particular, the 90° peaks of the 31P and 2 H line shapes were often seen to be enhanced in the lamellar mesh phase. This has been observed earlier4,11,29 and is probably an effect of a decreased bending rigidity of the bilayers because of the defects. As the membranes get more flexible, they can rearrange more easily in the magnetic field to obtain the preferred perpendicular orientation of the acyl chains. (The line shapes from the biaxial phase also showed some deviations from the powder pattern, which is attributed to partial orientation. In this case, it is probably not due to magnetic susceptibility effects as these samples are rather stiff and would have difficulties in reorienting spontaneously. The deviation from the powder pattern is most probably an effect of sample treatment, e.g., centrifuging the sample up and down to obtain a good mixture could result in sample orientation along the tube walls. This is supported by the fact that rotation of the sample by 90° in the magnetic field changes the line shape intensities.) According to the model proposed by Bagdassarian and co-workers,30 the density of defects should increase as the fraction of hydrocarbon volume, φ, is decreased in the system. This effect, which is attributable to the increased interbilayer interactions, is reflected by changes in the apparent bilayer thickness, dapp, measured as dapp ) φd. For an ideal bilayer, dapp should be constant as φ is varied, but if defects appear in the bilayer dapp will decrease, and the relative decrease in this parameter is directly proportional to the volume fraction of solvent-filled defects in the bilayers.29 For all the amphiphilic compositions of Table 3, dapp is increasing with increasing φ, and in addition (29) Gustafsson, J.; Ora¨dd, G.; Nyden, M.; Hansson, P.; Almgren, M. Langmuir 1998, 14 (18), 4987-4996. (30) Bagdassarian, C. K.; Roux, D.; Ben-Shaul, A.; Gelbart, W. M. J. Chem. Phys. 1991, 94 (4), 3030-3041.
Langmuir, Vol. 17, No. 11, 2001 3233
dapp is also seen to decrease with increased X at constant solvent content. Both these trends for dapp are well in line with the model mentioned above which states that the density of defects should grow with increasing aggregate curvature, here X, and decrease with φ. Table 3 also shows the corresponding shifts in the position of the broad reflection D, that is, the characteristic distance that originates from scattering from the bilayer defects. A broad reflection of this type has been observed on several occasions in the scattering patterns of defective lamellar phases.31-35 The general interpretation given is that the scattering is of intrabilayer origin and that the low q-position of the broad reflection corresponds to the nearest neighbor distance of defects within the bilayer.33-35 The observed shift to smaller D with increasing X is in line with such a picture because it suggests that the distance between defects should decrease as the volume fraction of defects within the bilayer increases. Such a decrease in D with increasing aggregate curvature is in accordance with observations made with other defective lamellar systems.34,35 However, a pure intrabilayer origin of this broad reflection is not compatible with the other trends shown by D in Table 3, namely, (i) that D actually is decreasing when dapp is increasing with increased φ at fixed X and (ii) that samples 7 and 10 may show similar dapp but significant differences in D. These two observations show that D is likely to contain a dependence on d, the layer repeat distance, which in turn indicates that the broad scattering peak results from correlations between defects in adjacent layers. We note that even though this suggestion of an interbilayer correlation does not rhyme with the conclusions normally made with defective lamellar phases,33-35 there are also examples of scattering studies of lamellar phases where the diffuse scattering at low q vectors is shown to originate from structural defects that are correlated between adjacent layers.32 However, in these cases the defective lamellar phases were at high φ, and it is indeed suprising that the present system seems to show layer correlations also at large bilayer separations (>60 Å). In this connection, it should be mentioned that a rhombohedral phase recently was shown to display a layer-correlated structure at rather high solvent contents.36 Interestingly, the three-connected mesh structure which forms the bilayers of this particular rhombohedral phase corresponds to the ordered version of the disordered mesh structure that the present defective lamellar phase has displayed in its dispersed state at high dilution.4 It is thus possible that the broad scattering peak originates from both interbilayer and intrabilayer correlations of the defects in precursors of an intermediate phase, such as the rhombohedral or the tetragonal phase. If we model the structure as a centered tetragonal phase (I4mm) with a ) x2d and c ) 2d, we would get the same nearest distance (x2d) between the pores within one bilayer as between bilayers while keeping the bilayer distance at d. Comparing the values of d and D in Table 2 shows that the ratio is indeed close to x2. Similar estimations can also be made for a rhombohedral structure. It is also possible to imagine precursors for twodimensional structures, such as the H1 or R phases, by (31) Hendrikx, Y.; Charvolin, J. Liq. Cryst. 1988, 3 (2), 265-273. (32) Ke´kicheff, P.; Cabane, B. J. Phys. 1987, 48, 1571-1583. (33) Holmes, M. C.; Smith, A. M.; Leaver, M. S. J. Phys. II 1993, 3, 1357-1370. (34) Holmes, M. C.; Smith, A. M.; Leaver, M. S. Langmuir 1995, 11, 356-365. (35) Zipfel, J.; Berghausen, J.; Lindner, P.; Richtering, W. J. Phys. Chem. B 1999, 103, 2841-2849. (36) Leaver, M. S.; Fogden, A.; Holmes, M. C.; Fairhurst, C. Langmuir 2001, 17, 35-46.
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inter- and intrabilayer correlation between elongated pores (slits). Phase Transitions. The lamellar mesh phase is stable only in a narrow range of values R. Below R ) 1.8, bilayers presumably free from defects are stable and swell only moderately in brine. The LRh phase is stable to a ratio of about 2.2, where a phase transition occurs in which the defect bilayers transform into a rectangular, a hexagonal, or a micellar solution phase depending on the solvent content. It thus seems as though the defect bilayer has the ability to mediate the transformation from ordinary bilayers into three different phases in a smooth way. The transformation into a micellar solution phase was discussed in earlier work.4 There is no distinct two-phase region, and also cryo-TEM micrographs suggested a smooth transition. This seems reasonable because the peculiar structures observed by cryo-TEM at surfactantto-lipid ratios just above 2.2 may equally well be described as threadlike micelles interwoven into lacelike structures or as pieces of defect bilayers dispersed in the L1 phase. This would allow for a smooth transition, and if there is a two-phase area it is narrow without a distinct boundary to the micellar phase. From the fact that the defect lamellar phase transforms into hexagonal and rectangular phases at lower aqueous content, one may be inclined to conclude that the defects should be slit defects instead of pores. Correlation of slits into long parallel rows would give structures similar to the hexagonal and rectangular phases built up of long rods. The hexagonal phase close to the boundary of the rectangular phase has been proposed to consist of cylinders with transient noncircular cross sections.9,27,31,32 Some evidence for this is found also in this system as the SAXS diffraction lines from hexagonal samples close to the rectangular phase are broadened toward higher q-values, indicating noncircular cylinders.32 This might also be indicated in the fact that the radius of the hydrocarbon core cylinders in the hexagonal phase, calculated from SAXS data, is significantly larger (24.4 Å) than expected from the length of the hydrocarbon chains.
Ora¨ dd et al.
As φ increases, a transformation from slits to pores is anticipated on theoretical grounds,30 and when the intrabilayer interactions between pores in different bilayers becomes important one would expect the pores to correlate both within and between bilayers, thereby forming threedimensional space groups such as the rhombohedral or the tetragonal phases.12 Such a phase would give a uniaxial 2H or 31P NMR spectrum resembling that of the lamellar phase, provided that the molecules probe the whole surface on the NMR time scale. Sample 3 seems to adhere to this picture because it gives a uniaxial 31P spectrum and SAXS shows several reflections that cannot satisfactorily be indexed to the rectangular, lamellar, or hexagonal phases. It seems probable that at least part of this sample contains stacked bilayers with defects correlating between the bilayers, thereby forming a rhombohedral or a tetragonal phase, but no satisfactory indexation has so far been found. All data taken together, the system seems to conform to the general picture of defect formation in bilayers.27,30 Starting at high water content, the lamellar phase can be pictured as a two-dimensional network of interconnected rods which transforms into a micellar phase of elongated rods as the CTAC content increases. At lower water content, the lamellar phase includes defects that, upon an increase in X, can correlate in orientation to form precursors to either two-dimensional (H1 or R) or threedimensional (tetragonal or rhombohedral) structures, depending on the water content. Acknowledgment. We would like to thank Bertil Halle and Stefan Gustafsson for valuable discussions regarding the NMR line shapes and also for providing the Matlab code for simulating NMR line shapes in biaxial and uniaxial phases. Thanks also to Gordon Tiddy for letting us use some of his SAXS beamtime at Daresbury Labs. This work was supported by the Knut and Alice Wallenberg Foundation. LA001772B
