Evidence for Multilamellar Vesicles in the Lamellar Phaseof an Electrostatic Lyotropic Ternary System. A SolidState 2H-NMR and Freeze Fracture Electron Microscopy

StudyFrederic Auguste, Jean-Paul Douliez, Anne-Marie Bellocq,* and

Erick J. Dufourc*

Centre de Recherche Paul PascalsCNRS, av. A. Schweitzer, 33600 Pessac, France

Tadek Gulik-Krzywicki

Centre de Genetique MoleculairesCNRS, 91190 Gif-sur-Yvette, France

Received July 11, 1996. In Final Form: November 12, 1996X

Deuterium solid state NMR and freeze fracture electron microscopy experiments have been carried outin the lamellar LR phase of the water-sodium dodecyl sulfate-octanol system. Within the lamellar phasetwo types of bilayer organizations have been found. At high surfactant and alcohol concentrations, thelamellar phase ismade of a stack of flat parallel bilayers while in the dilute part, it consists ofmultilayeredvesicles of large radius (ca. 10 000 Å). These latter structures, commonly called spherulites, which appearspontaneously at low octanol contents and/or for high water dilution, can be described as textural defectsof the lamellar phase. The location of this onion-like structure region is discussed within the frameworkof the membrane elasticity theory. As a side result, the presence of 33% glycerol in some of the freezefracture experiments is shown to barely affect the bending modulus of the film but rather disorder themolecular packing of the bilayers.

Introduction

Amphiphilic molecules in solution form awide range ofself-assembling structures which have motivated manytheoretical and experimental developments.1,2 Uponspecific conditions of composition and temperature, thesemolecules aggregate into bilayers sometimes designatedas membranes. These two-dimensional aggregates canorganize in space either as a long range ordered structure,the lamellar phase LR,3,4 or as isotropic disordered phaseslike the vesicle L4

5-9 or the sponge L310,11 phase. In the

vesicle phase, the bilayers form unilamellar or multila-mellar spherical objects, while they are randomly con-nected in the sponge phase. Theoretical studies9,12-14

based on the framework of the membrane elasticitytheory15 have shown that the ultimate structure of theaggregates existing in solution depends strongly on twoimportant intrinsicparametersof thebilayers, thebendingelastic constants κ and κj which control respectivelythe mean curvature and the Gaussian curvature. Themean bendingmodulus κ characterizes the rigidity of thebilayer, it is generally of the order of kBT in alcohol-surfactant-water systems.16 The Gauss-Bonnet theo-rem17 states that the integral of the Gaussian curvature

* Author to whom correspondence should be addressed. Fax: 5684 56 00.

X Abstract published in Advance ACS Abstracts, January 15,1997.

(1) See: Physics of amphiphilic layers; Meunier, J., Langevin, D.,Boccara, N., Eds.; Springer-Verlag: New York, 1987.

(2) See: Micelles, membranes, microemulsions and membranes;Gelbart, W. M., Ben-Shaul, A., Roux, D., Eds.; Springer-Verlag: NewYork, 1994.

(3) Bellocq, A. M. In Microemulsions: fundamentals and appliedaspects; Kumar, P., Mittal, K. L., Eds.: Marcel Dekker: New York, inpress.

(4) Ekwall, P. In Advances in liquid crystals; Brown, G. M., Ed.;Academic Press: New York, 1975.

(5) (a) Brady, J. E.; Evans, D. F.; Kachar, B.; Ninham, B. W. J. Am.Chem. Soc. 1984, 106, 4279. (b) Dubois, M.; Zemb, T. Langmuir 1991,7, 1352.

(6) Hauser, H.; Gains, N.; Lasic, D. In Physics of amphiphiles:micelles, vesicles and microemulsions; Degiorgio, V., Corti, M., Eds.;North Holland: Amsterdam, 1985.

(7) Kaler, E. W.; Murthy, K. A.; Rodriguez, B. E.; Zasadzinski, J. A.N. Science 1989, 245, 1371.

(8) (a)Munkert,U.;Hoffmann,H.; Thunig,C.;Meyer,H.W.;Richter,W. Prog. Colloid Polym. Sci. 1993, 93, 137. (b) Hoffmann, H.; Munkert,U.; Thunig, C.; Valiente, M. J. Colloid Interface Sci. 1994, 163, 217.

(9) Herve, P.; Roux, D.; Bellocq, A. M., Nallet, F.; Gulik-Krzywicki,T. J. Phys. II 1993, 3, 1255.

(10) Porte, G.; Marignan, J.; Bassereau, P.; May, R. J. Phys. (Paris)1988, 49, 511.

(11) Gazeau, D.; Bellocq, A. M.; Roux, D.; Zemb, T. Europhys. Lett.1989, 5, 447.

(12) Huse, D. A.; Leibler, S. J. Phys. (Paris) 1988, 49, 605.(13) Cates, M. E.; Roux, D.; Andelman, D.; Milner, S. T.; Safran, S.

A. Europhys. Lett. 1988, 5, 733.

(14) Simons, B. D.; Cates, M. E. J. Phys. II 1992, 2, 1439.(15) Helfrich, W. Z. Naturforsch. 1973, 28c, 693.(16) For a review see: Roux, D.; Safinya, C.; Nallet, F. In ref 2, p 303.(17) See for instance: David,F. InStatisticalmechanics ofmembranes

and surfaces; Nelson,D., Piran, T.,Weinberg, S., Eds.;WorldScientific:Singapore, 1989.

Figure 1. Partial phase diagram of the water-SDS-octanolsystem at T ) 25 °C: L1, micellar phase; L4, vesicle phase; HR,hexagonal phase; LR, lamellar phase; region I, stack of parallelflat bilayers; region II, multilayered vesicles. The dashed lineseparates region I from region II and the dotted-dashed linerepresents a typical dilution line in our experiments (see text).

666 Langmuir 1997, 13, 666-672

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for agivensurface isa topological invariantwhichdependsonly onnc andnh, thenumber of disconnected componentsof the surface and the number passages or handles,respectively. As a consequence, the Gaussian curvatureis onlya functionof the topologyand theGaussianbendingconstant κj plays an important role in phase transitionswhich involve topological transformations. When κj issufficientlynegative, the surface formsmanydisconnectedaggregates suchas vesicles; in contrastwhen κj is positive,highly connected surfaceswithmanyhandles are favoredand the sponge phase is stabilized. These considerationsindicate that while increasing κj we expect the followingsequence of phases: vesicle phase (L4)-lamellar smectic(LR)-sponge phase (L3).Experimentally the L4-LR-L3 sequence of phases has

been found in the sodiumdodecyl sulfate (SDS)-octanol-brine system.9 At low SDS content (<10% inweight), theincrease of the cosurfactant concentration in the mem-brane induces changes from vesicles to planar lamellaeand to multiconnected membranes. In addition to thesethreephasesofbilayers, ithasbeen foundthat the lamellarphase, LR, of the above system can be divided into twodistinguishable regionswhere the bilayers have differenttopologies.18 Observations by optical polarizing micros-copy have revealed two types of focal conic domains: atlowalcohol content,Maltese crosses corresponding to largemultilamellar vesicleswithapositiveGaussian curvaturereferred to as spherulites are found, while oily streaksassociated with usual focal domains with negative Gauss-ian curvature are seen at high alcohol content. Thesetwo kinds of textures have also been observed in othersystemsmadeof brine-alcohol-surfactant.19-23 Fromallthe above results, i.e., the sequence of phases L4-LR-L3on one hand, and the appearance of spherulites on the

other hand, one can conclude that in salted systems theGaussianmodulus κj is governed by the composition of themembranes and changes its sign fromnegative to positiveas the alcohol-to-surfactant ratio, R, increases.18,19,21-23

Up to now, the experimental studies reported on thepure water-SDS-alcohol systems have not providedevidence for the above important polymorphism asobserved in salted systems. For thewater-SDS-octanolsystem one finds a lamellar phase which, upon dilution,undergoes a phase transition toward a dilute phase ofvesicles (L4). Observations by optical microscopy did notreveal the presence of spherulites in the lamellar phaseLR. As we will point out in this paper, deuterium solidstate NMR (2H-NMR) and freeze fracture electron mi-croscopy (FFEM) show that the lamellar phase alsoseparates into two regions in pure water-octanol-SDSsystems. Wewill see in the following thatNMRdata giveevidence for the presence of a marked change in theevolution of both the spectral shape and the quadrupolarsplitting upon dilution, and that FFEM experimentsenable attribution of this change to the appearance ofspherulites in the lamellar phase. In addition, 2H-NMRleads to the calculation of C-D bond order parameters,24of 2H-labeled molecules (SDS, octanol) embedded inlamellar phases. This order parameter may be linked tothe fluctuations of the membranes25 or to local ordering

(18) Herve, P.; Bellocq, A. M.; Roux, D.; Nallet, F.; Gulik-Krzywicki,T. To be submitted for publication.

(19) Auguste,F.;Bellocq,A.M.;Nallet,F.;Roux,D.;Gulik-Krzywicki,T., To be submitted for publication.

(20) Benton, W. J.; Miller, C. A. J. Phys. Chem. 1983, 87, 4981.(21) Gomati, R.; Appell, J.; Bassereau, P.; Marignan, J.; Porte, G. J.

Phys. Chem. 1987, 91, 6203.(22) Porte,G.; Appell, J.; Bassereau, P.;Marignan, J.J.Phys. (Paris)

1989, 50, 1335.(23) Boltenhagen, Ph.; Lavrentovich, O. D.; Kleman, D. Phys. Rev.

A 1992, 46, 1743.(24) Davis, J. H. Biochim. Biophys. Acta 1983, 737, 117.(25) Auguste,F.;Barois,P.;Fredon,L.;Clin,B.;Dufourc,E.J.;Bellocq,

A. M.; J. Phys. II 1994, 4, 2197.

Figure 2. Variation of the quadrupolar splitting vs the smectic repeat distance for the water-SDS (R-deuterated)-octanol systemalong the four dilution lines defined as (a) A/S ) 1.8, (b) A/S ) 2.4, (c) A/S) 2.6, and (d) A/S ) 2.9. Arrows show the position ofthe discontinuity in the variation (see text). In (b) is also shown the variation obtained for the water+ glycerol (33%)-SDS-octanolsystem.

Multilamellar Vesicles Langmuir, Vol. 13, No. 4, 1997 667

properties,26 thus affording the calculation of aliphaticchain length and conformations of chain segments. In arecent paper25we indeeddemonstrated, on sampleswhichcan be easily swollen, that one can determine the meanbendingmodulus, κ, from fitting thequadrupolar splittingvariation versus the lamellar repeat distance, d.

Experimental SectionSamples. We have investigated the lamellar phase of the

ternary systemcomposed ofwater, sodiumdodecyl sulfate (SDS),and octanol. For NMR experiments, we have used an R-deu-terated surfactant, synthesizedaccording to standardmethods,27or a perdeuterated surfactant, purchased from Commissariat al’Energie Atomique (France). Octanol was purchased fromAldrich (France); water was purified via an ion exchangepurification train (Milli-Q system, Millipore); protonated SDSwas obtained fromTouzart etMatignon (France). Sampleswereprepared with 50 mg of perdeuterated SDS, or 200 mg ofR-deuterated SDS, mixed with the appropriate quantity ofprotonated SDS, in order to give a total weight of about 1-2 gof lamellar phase. Alcohol and water are first added to make“stock solutions”, composed of 40-45%water. After a sufficientdelay (1weekto reachtheequilibrium), thesesolutionsaredilutedwith water in order to prepare samples on dilution lines defined

by a constant alcohol-to-surfactant (A/S) ratio. The samples aresealed in order to prevent solvent evaporation and centrifuged.In order to work with samples of known orientation, we have

used a cell made of a stack of about 20 glass plates of 0.2 mmthickness (microscope coverslips). Samples of LR phase weresandwichedbetween these sheets. Puremechanical interactionsbetween the glass lamellae and the lamellar phase are assumedto be sufficient to enable annealing of the lamellae. In order toimprove the orientation, samples were sealed in the cell andsubmitted to a thermal variation in the magnetic field (4.7 T)from 90 to 25 °C. Because SDS may hydrolyze at hightemperature, it was reneutralized. In addition NMR spectragave no evidence (vide infra) for such a thermal degradation.Such a chemical transformation indeed should lead to anadditional set of quadrupolar splittings (for the longchainalcohol)which is clearly not detected.SolidStateNMR. Experimentswere carried out onaBruker

MSL 200 spectrometer, operating at 30.7 MHz for deuterium.The temperaturewas regulated to 298( 1K. NMRsignalswererecorded with either a quadrupole echo technique24 (π/2 pulsewidth of 6 µs, pulse spacing of 25 µs) or a composite quadrupoleecho pulse sequence.28 Data treatment was accomplished on aVAX/VMS 8600 computer (DEC, USA).FreezeFractureTransmissionElectronMicroscopy. In

order to achieve the best preservation of the sample structureupon cryofixation,we replacedwaterwithawater-glycerol (33%in volume) solution and checked that this replacement does notmodify dramatically the phase diagram. A thin layer of thesample (20-30 µmthickness) was placed on a thin copper holderand then rapidly quenched in liquid propane. The frozen samplewas then fractured at -125 °C, in a vacuum lower than 10-6

Torr, with a liquid-nitrogen-cooled knife in a Balzers 301 freeze-etching unit. The replication was done using unidirectionalshadowing, at an angle of 35°, with platinum-carbon, 1-1.5 nmof mean metal deposit. The replicas were washed with organicsolvents and distilled water and were observed in a Philips 301electron microscope.

Results

Phase Diagram. Figure 1 shows a partial phasediagram of the ternary system water/SDS/octanol. Wehave focused on the part containing the LR phase, i.e., thealcohol poor side of the diagram. In this part four phaseshave been identified: At low octanol content, we observethe hexagonal phase HR in the high surfactant contentpart of the diagram and the micellar phase, L1, in thediluted part. For higher alcohol content, we find thelamellar phase, LR. The extension of this phase is verylarge, both in membrane composition and in dilution,which enables experiments to be carried out on dilutionlines with samples containing from 40 to 90% water (wt%) and to change membrane composition from 1 to 3octanol molecules per SDS (A/S ratios). For very highdilution (<0.3 wt % SDS), a vesicle phase, L4, appears.The lamellar phase was characterized by 2H-NMR, X-raydiffraction, and optical microscopy.25 Deuterium NMRexperiments (vide infra) carried out on dilution lines inthe LR phase have led us to separate this phase in twosubregions, as represented in the diagram by the dashedline. As we will see later on, the lamellar phase consistsin region I of a stack of planar bilayers, while in regionII it contains the so-called spherulites dispersed in alamellar matrix. One must note, that for this systemoptical polarizingmicroscopy does not enable detection ofthe presence of the multilamellar vesicles, the texturesappearing the same in both regions I and II (data notshown).

2H-NMR Experiments on Samples ContainingLabeledSDS. Measurements of quadrupolar splittings,∆νQ, of theR-deuterated SDShave been carried out in thelamellar phase along dilution lines and plotted vs therepeat distance, d, of the stack of lamellae, which was

(26) (a) Douliez, J. P.; Bellocq, A. M.; Dufourc, E. J. J. Chim. Phys.1994, 91, 874. (b) Douliez, J. P.; Leonard, A.; Dufourc, E. J. Biophys.J. 1995, 69, 1727.

(27) Dreger, E. E.; Keim,G. I.;Miles, G. D.; Shedlovsky; Ross, J. Ind.Eng. Chem. 1944, 36, 610. (28) Levitt, M. H. J. Magn. Res. 1982, 48, 234.

Figure 3. Solid state 2H-NMR spectra of water/SDS (perdeu-terated)/octanol for A/S ) 2.4: (a) region I, 45% water; uppertrace, powder (randomly oriented) spectrum; lower trace,oriented sample spectrum (normal to the microscope plateoriented at 0° with respect to B0); (b) region II, 66% water;upper trace, powder spectrum; lower trace, oriented samplespectrum as in (a). Typical experimental parameters were asfollows: spectral width of 250 kHz; recycle delay of 2 s; 2000acquisitions; temperature 25 °C.

668 Langmuir, Vol. 13, No. 4, 1997 Auguste et al.

independentlymeasured by X-ray diffraction.25 Figure 2shows such a plot for four dilution lines located in thelamellar phase of the water-SDS-octanol system formolar A/S ratios of 1.8, 2.4, 2.6, and 2.9. One sees that∆νQ decreases as d increases, i.e., as the water volumefraction increases. It has been already shown25 that thisdecrease can be linked to the undulations of the mem-branes, i.e., to themean bendingmodulus κ. For the fourdilution lines, we have pointed out the presence of amarked changeof slope in theevolutionof thequadrupolarsplitting vs d. The position of this break, shown by anarrow in the Figure 2, is water-composition dependent.Its position corresponds to 48, 62, 68, and 72% water, forA/S ratios respectively equal to 1.8, 2.4, 2.6, and 2.9. Theevolution of this singularity with the membrane composi-tion is reported by a dashed line in the phase diagramsketched on the Figure 1 and appears to delimit twodistinct regions. Here, it is important to mention thattheX-ray scattering study of these samples shows regularbehavior of the lamellar spacing with the water fractionincreasing all along the dilution lines investigated.25Wehave tried to prepare oriented samples, as described

in the Experimental Section, for the two regions labeledI and II in Figure 1. Figure 3 shows the solid state 2H-NMR spectra of water-SDS (perdeuterated)-octanol inthese regions, for A/S ) 2.4. In both cases spectra forrandomly oriented samples (upper traces) and for sampleswhere thenormal to themicroscopecoverslipswasorientedat 0° with respect to the magnetic field direction (lowertraces) were recorded. Powder spectra are easily recog-nizable due to the presence of both the major peaks andtheshoulders respectively corresponding tobilayernormaloriented at 90° and 0° with respect to the static magneticfield direction. One can note that for the concentratedsample (Figure 3a, region I, 45% water) the lower tracerepresents an almost totally oriented sample spectrum,whereas for the diluted sample (Figure 3b, region II, 66%water)most of the sample remains essentially as apowderasevidencedbya close similaritybetweenupperand lowertraces of Figure 3b. One must however mention that a

small but detectable part of the sample orients in betweentheplates as seenby thepeaks detected in the “shoulders”of the lower trace spectrum. Since samples in regions Iand II have undergone the same treatment, the aboveresult demonstrates that region II is made of structuresthat do not easily orient in between plates.Because solid state NMR powder pattern line shapes

are sensitive to vesicle size and to the presence of manyconcentric multilayers,26 spectral simulations have beencarried out on 2H-NMR powder spectra of R-deuteratedSDS in systems coming from regions I and II (Figure 4aand Figure 4b, respectively). Experimental spectra(dashed lines) were acquiredwith the same experimentalparameters in both regions, and a marked increase inspectral intensity near the Larmor frequency (set to zeroin the x-scale of Figure 4) may be noted for the spectrumrepresentative of region II. Because details for spectralsimulationshave beendiscussed elsewhere,26wewill onlymention that Figure 4a spectrum has been calculatedassuming an extended set of lamellae randomly distrib-uted with respect to the magnetic field whereas that ofFigure 4b has been simulated assuming an externalspherulite diameter of 10 000 Å, made of 70 concentricvesicleswithamembrane thickness of 24Åanda lamellarrepeat distance of 70 Å. The C-D bond order parameterfor both cases was taken to be 0.14, as deduced frommeasurement of the peak-to-peak separation. Note thatfor these simulations, the increase in intensity near theLarmor frequency as detected in region II spectra comesfrom the presence of vesicles of smaller radius (those ofthe inner core). These vesicles, if they were observed asisolated species, would be characterized by a broadisotropic line whose width depends on the vesicle size, innormal conditions of temperature and viscosity.The above results led us to examine by transmission

electronmicroscopy samples in the two identified regionsof the lamellar phase.Freeze Fracture Transmission Electron Micros-

copy. Figure 5 represents the electron micrographsobtained for three samples located along the line A/S )

Figure 4. Experimental (dashed lines) and calculated (solid lines) 2H-NMR spectra of R-deuterated SDS in the water-SDS-octanol system forA/S) 2.4: (a) region I, 45%water; (b) region II, 66%water. Acquisition parameters are as in Figure 3. Simulationparameters are (a) C-D order parameter of 0.14, extended (diameter >100 000 Å) lamellae randomly distributed with respect tothe magnetic field direction and (b) C-D order parameter of 0.14, 70 concentric spherical vesicles of 10 000 Å external diameter,membrane thickness of 24.0 Å lamellar repeat distance of 70 Å. Temperature and lateral diffusion coefficient of molecules in thebilayer are respectively 25 °C and 100 × 10-8 cm2 s-1.

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2.4. Figure 5A shows a picture from a sample with awater + glycerol weight fraction, XWG, of 45%. It showsa typical texture for a lamellar phase presenting only fewdefects; throughout the replica we observe stacks ofparallel bilayers. As thewater+ glycerol weight fractionXWG is increased above 60%, the structure of the sampleis completely different. At XWG ) 75%, multilamellar

vesicles are clearly observed as dispersed in a lamellarphase matrix (Figure 5B). The largest spherulites are of4 µm in diameter. As the solvent content is furtherincreased (Figure5C), oneobserves thecoexistenceof largemultilayered spheruliteswith smaller onesandwith smallunilamellar vesicles. This lastmicrograph obtainedwitha sample at the boundary of the lamellar phase revealsthat the system is biphasic at this dilution. The smallunilamellar andmultilamellar vesicles come respectivelyfrom the L4 and LR phases. It is worth noting that thesameevolution in the organization of the bilayers is foundalong a path at constant SDS concentration. At lowoctanol-SDS ratio, the structure consists of a densepacking of spherulites (Figure 6A), while at higheroctanol-SDS ratio, a perfect stacking of flat bilayers isobserved (Figure 6B).Because such samples are prepared in the presence of

glycerol (33% inwater, v/v),wehavedetermined the effectof the latter bothon the smectic periodandon the observedquadrupolar splitting by performing X-ray and 2H-NMRexperiments on the (water + glycerol)-SDS-octanolsystem along the dilution line defined by the ratio A/S )2.4.The Effect of Glycerol. X-ray Measurements. The

first and second harmonics of the structure factor areobserved on the X-ray spectra for lamellar samples inregions I and II, in the presence and absence of glycerol.For glycerol-containing samples, the peaks appear athigher wave vector q than those in samples lackingglycerol. This indicates that the interlamellar periodicitybecomes smaller in the presence of glycerol. Figure 7displays the smectic period, obtained from the first-orderBragg peak (d ) 2π/q0), as a function of 1/Φm. Thereciprocal of the membrane volume fraction, Φm, isobtained from the sample composition assuming, to a firstapproximation, that there is no glycerol in the surfactant/octanol bilayer.29 For samples in regions I and II, in thepresence and absence of glycerol, note that the smecticperiod follows the classical swelling law (d ) δ/Φm) asexpected for a lamellar phase. This variation is based ona simple geometrical model of a periodic stack of flatlamellae of thickness δ and of volume fraction Φm.16 Asobtained from the slopes of Figure 7, δ decreases from23.4 Å in the absence of glycerol to 22.0 Å in its presence.Within our assumption that glycerol is exclusively inwater, the observed decrease in d would suggest a slightincrease in area per surfactant head group in the bilayer.Alternatively, the decrease in the smectic period atconstant alcohol plus surfactant concentration could alsoarise froma cosurfactant-like behavior of glycerol, i.e. thepenetration of glycerol in the bilayer.In order to check the latter hypothesis, we have

performed two sets of 2H-NMR experiments on water-glycerol-SDS-octanol systemsby followingeither labeledSDS or D2O.

2H-NMRExperiments. Figure8aandFigure8bdisplayspectra of the systemswater-SDS-octanol and (water+glycerol (33%))-SDS-octanol, respectively. Inboth casesH2O has been replaced by D2O. An anisotropic powderpattern with a 1.4 kHz peak-to-peak separation, ∆νQ,dominates the spectra of Figure 8a. A minor componentwith ∆νQ ) 13.9 kHz is also detected on increasing thevertical scale (right panel). The major component isunambiguously assigned to bound water whereas thesmallerPakedoublet comes fromtheO-Dbond (chemicalexchange with D2O) of octanol. As already docu-mented,30,31 the observed water quadrupolar splitting isthe result of a fast exchange, on theNMR time scale (10-3

s), betweenwater bound to the interface (anisotropic) and

(29) The following densities are used to calculate Φm: dSDS ) 1.16,doctanol ) 0.827, dwater+33%glycerol ) 1.063.

Figure 5. Freeze fracture electron micrographs of water +glycerol (33%, v/v)-SDS-octanol for samples in the LR domainalong the dilution line A/S ) 2.4 (dotted-dashed line of Figure1): (A) 45%water+ glycerol; (B) 75%water+ glycerol; (C) 90%water + glycerol. Bar size = 0.5 µm.

670 Langmuir, Vol. 13, No. 4, 1997 Auguste et al.

water undergoing fast isotropic tumbling in the inter-lamellar space. In the presence of glycerol (Figure 8b),four quadrupolar splittings (26.5, 13.0, 3.1, and 1.6 kHz)plus a small (2%) isotropic line are observed. The ∆νQ of1.6 and 13.0 kHz are assigned to D2O and octanol (OD)by analogy with the system lacking glycerol. The new

components may be attributed to the glycerol hydroxylgroups which have undergone isotopic exchange with theOD groups of heavy water and demonstrate unambigu-ously that there is some anisotropic glycerol, i.e., boundto the interface. They could represent two populations ofglycerol bound to the interface and in slow exchange inthe NMR time scale. Although this possibility cannot betotally ruled out, it appears unlikely that a situation ofslow exchange is reached for this small molecule. Alter-natively it is reasonable to assume fast exchange betweenbound and free molecules in the interlamellar space, thetwo quadrupolar splittings of 26.5 and 3.1 kHz then beingrespectivelyattributed to the central and terminal glycerolOD groups; the two terminal OD groups are equivalentand lead to the most intense signal. Because of thisexchange situation, it is not possible with this set ofexperiments to determine the amount of glycerol presentin the membrane. The small amount of isotropic line(Figure 8b) can be attributed to water in an isotropicenvironment.InFigure2b is reported theevolutionof thequadrupolar

splittingvsd forR-deuteratedSDS in the (water+glycerol(33%))-SDS-octanol system. One remarks that thepresence of glycerol leads to a marked decrease (7-10%)of∆νQ but does not seem to affect the position of the breakobserved in its absence. Interestingly, this decreaseparallels that of the octanol (OD) quadrupolar splittingas observed in the presence of glycerol (Figure 8). Thisindicates that the presence of glycerol in the bilayer doesnot greatly affect the bending modulus of the film butrather disorders, at the molecular level, the membranepacking.Stability of Vesicles. Thermal variations on diluted

samples of the A/S ) 2.4 line have been carried out. Intheseexperiments the samplespreviously sealed inaglasscapillarywereheated to a temperatureabove the isotropictransition temperature, i.e., around 120 °C for the mostdiluted samples. It is important to note that in theseexperiments, the temperature of samples is much higherthan in attempts of macroscopic orientation in betweenplates, in the magnetic field, as previously described formore concentrated samples. Then a slow cooling downfrom the liquid state enabled the system to build up in atextural state different from the initial one: the opticaltexture found after this thermal treatment is differentfrom that observed just after the preparation; it exhibitsoily streaks, specific of lamellar phases. This resultsuggests that the spherulites are not thermodynamicallystable and confirms that the difference between regionsI and II is a matter of texture rather than structure. The

(30) Ulmius, H.; Wennerstrom, G.; Lindbloom, G.; Arvidson, G.Biochemistry 1977, 16, 5742.

(31) Faure, C.; Tranchant, J. F.; Dufourc, E. J. Biophys. J. 1996, 70,1380.

Figure 6. Freeze fracture electron micrographs of water +glycerol (33%, v/v)-SDS-octanol for samples in the LR domainalong the line at 20% SDS: (A) 15% octanol; (B) 25% octanol.Bar size = 0.5 µm.

Figure 7. Variation of the smectic period, d, vs the reciprocalof the volume fraction, 1/Φm, along the dilution line A/S ) 2.4in the presence (b) and absence (O) of glycerol. d is obtainedfrom the first-order Bragg peak of X-ray spectra (d ) 2π/q0).

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formation of the multilamellar vesicles depends on theway the samples have been prepared and in particular onthe energy supplied either by heat or shear. RecentlyDiat et al. have found that in a certain range of shearrate the lamellar phase of various systems organizesinto spherical multilamellar vesicles of well-controlledsize.32

DiscussionAll the above results clearly give evidence for the

existence of spherulites in a very largepart of the lamellarphase of the water-SDS-octanol system. As alreadymentioned in the Introduction, the two topologies of thebilayerssclosed and flatsobserved in the water-SDS-octanol systemhave been already found inmany systemsmade of brine-surfactant-alcohol.9,18-23 Theywere alsoidentified in the lamellar phase of double chain surfactantsystems known to form vesicles. In the case of didode-cyldimethylammoniumbromide (DDAB), the spherulitesformed in the binary system DDAB-water change intostacks of flat bilayers by addition of methanol.33 In thesesystems, as in the system studied here, the separationbetween the lamellar phase and the spherulites ismainlycontrolled by the amount of alcohol in the membranes.The existence and the location of this region of texturaldefects can be linked to the conditions of stability ofsurfactant membranes in solution. As pointed out in theIntroduction, the topological transformation from spheri-cal to planar structures is triggered by an increase of theGaussiancurvaturemodulusκj. Porteandco-workershavefirst proposed that this Gaussian curvature modulus isdetermined by the membrane composition and increaseswith the A/S ratio.22 Our observations are partly inagreementwith this prediction, indeed at high surfactant

concentration, the spherulites exist at low A/S, but theycanalso be obtainedathighA/S ratio by varying thewaterdilution.In the last few years, the effect of electrostatic double

layers on the bending elasticity of fluid membranes hasbeen examined inmany theoretical papers.34-38 Both thebending rigidity κ, and theelasticmodulus of theGaussiancurvature κj as well as the spontaneous curvature C0 canbeaffectedby thepresence of anelectrostatic double layer.In particular, the dependence of the electrostatic contri-bution to the bending modulus κ of charged surfactantbilayers in lamellar phases on the interlamellar spacinghas been considered.36,37 To our knowledge, the corre-sponding behavior for κj has not been predicted. Ourresults suggest that κj decreases as the distance betweenthe charged layers increases.Moreover, our results indicate that glycerol behaves as

a cosurfactant and disorders themolecular packing of themembrane. This effect arises from a partitioning of theglycerol between the water layer and the alcohol-SDSbilayer. Interestingly, the presence of glycerol does notappreciably modify the mechanical properties of themolecular film, i.e., the bending modulus. This is of firstimportancewhenonewishes to study systemswith freeze-fracture electron microscopy techniques.

Acknowledgment. The authors wish to thank J. C.Dedieu for his excellent technical assistance in freezefracture electronmicroscopy andM.Maugey, G. Raffard,and C. Faure for help in performing X-ray and NMRexperiments.

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(32) Diat, O.; Roux, D. J. Phys. II 1993, 3, 9.(33) Dubois, M.; Gulik-Krzywicki, T.; Cabane, B. Langmuir 1993, 9,

673.

(34) Lekkerkerker, H. N. W. Physica A 1989, 159, 319.(35) Mitchell, D. J.; Ninham, B. W. Langmuir 1989, A140, 520.(36) Higgs, P. G.; Joanny, J. F. J. Phys. (Paris) 1990, 51, 2307.(37) Pincus, P.; Joanny, J. F. Andelman,D.Europhys. Lett. 1990, 11,

763.(38) Winterhalter, M.; Helfrich, W. J. Phys. Chem. 1992, 96, 327.

Figure 8. 2H-NMRpowder spectra of the systemsD2O-SDS-octanol (a) and (D2O+ glycerol (33%))-SDS-octanol (b). Right handside spectra are vertical expansions (×8) of left panel spectra. Experimental parameters are as in Figure 3.

672 Langmuir, Vol. 13, No. 4, 1997 Auguste et al.