Nonionic−Anionic Mixed Surfactants Cubic Mesophases. Part I

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Nonionic-Anionic Mixed Surfactants Cubic Mesophases. Part I: Structural Chaotropic and Kosmotropic Effect Rivka Efrat, Zoya Abramov, Abraham Aserin, and Nissim Garti* Casali Institute of Applied Chemistry, The Institute of Chemistry, The Hebrew UniVersity of Jerusalem, Edmond J. Safra Campus, Jerusalem 91904, Israel ReceiVed: April 27, 2010; ReVised Manuscript ReceiVed: June 8, 2010

We prepared and investigated cubic bicontinuous (V) phase from mixtures of nonionic monoolein (GMO) and anionic oleyl lactate (OL) surfactants in the presence of ethanol and water. The isotropic region and the composition of the V phase in the pseudoternary phase diagram vary with the nature of the hydrophilic headgroups and their charge. We examined three anionic species, acidic (HOL), partially neutralized (OL), and totally ionized (NaOL) forms. The largest swollen V region within the phase diagram was formed from the partially neutralized form. The V formation is dependent on the GMO/OL ratio. The largest isotropic region in the phase diagrams was found with GMO/OL at a 70:30 wt % ratio and in the presence of 5.0 and 38.5 wt % ethanol and water, respectively. The structural effect of OL was determined by small-angle X-ray spectroscopy, differential scanning calorimetry, and Fourier transform infrared. The results revealed that the structure is curvature-dependent. Mesophases made from 90:10 wt % GMO/OL showed phase transition from gyroid (G) to diamond (D) symmetry. Preparations made from 30:70 wt % GMO/OL exhibited coexistence of two mesophases, one (of low order) cubic and the other lamellar. Because of the overall gauche deformation growth, the hydrocarbon order decreased with the OL content increase. The GMO, sn2 and sn3 headgroups, and water structure vibration bands indicate a chaotropic effect as a result of the interdigitation of OL anions and Na+ and H+ counterions. Introduction The main families of amphiphiles defined as GRAS (Generally Recognized As Safe) are glycerol monofatty acids esters.1,2 Some self-assemble to form lyotropic liquid crystalline (LLC) structures in polar solvents. The mesophases appear in several arrangements such as lamellar (LR), hexagonal (HI and HII), and cubic (Q) mesophases. Cubic mesophases exhibit the most complex spatial organization of LLC classic arrangement and are classified as bicontinuous cubic (the V phase) and micellar cubic (the I phase).3-7 The structure of the cubic phase consists of a curved bicontinuous lipid bilayer extending periodically in three dimensions; hence they are called “Infinite Periodic Minimal Surfaces” (IPMS).8-10 The three common IPMS, relevant to lipid-water systems, are the Schwartz primitive surface (P) or Im3m space group, the Schwartz diamond surface (D) or Pn3m space group, and Shoen’s gyroid surface (G) or Ia3d space group.8,11 The different cubic mesophases can transform from one to another. The transitions are dependent on the temperature, components ratio, and coingredients.9,12-15 Incorporation of a third component, such as water channel diameter, rheology properties, or stability, influences the mesophase characteristics. It also may increase the number of mesophases or two-phases and three-phases coexist and become more extensive.8,16-18 These modified LLC phases with large surface area, elevated water and alcohol content, and high solubilization capacities can be used to entrap hydrophilic, amphiphilic, as well as lipophilic guest bioactive molecules.19-21 In recent years, the liquid crystalline phases have shown the ability to accommodate biologically active molecules such as * To whom correspondence should be addressed. Tel: +972-2-658-6574/ 5. Fax: +972-2-652-0262. E-mail: [email protected].

vitamins and enzymes.17,19,22-26 The hydrophilic/hydrophobic nature of the guest molecule determines whether the molecule will be preferentially located in the polar aqueous region, in the interface, or in the apolar hydrocarbon domains.22 Several studies were carried out on mixed surfactants with the intention to find synergism derived from mixed nonionic and anionic surfactants.27-29 The studies have shown that the interfacial activity of mixed surfactants exceeded that of single surfactant systems. Attractions between surfactants are dominated by any electrostatic interaction of the hydrophilic headgroups and their packing density. Aota-Nakano et al. investigated the effect of electrostatic interaction of charged amphiphiles on the cubic Pn3m phase composed of glycerol monooleate (GMO).27 To change the surface charge density in the GMO membrane, they added small amounts of oleic acid (OA) to the mixture. It was found that OA concentration induced phase transition from Pn3m to Im3m phase at pH 7. They concluded that the Im3m phase is more stable than the Pn3m due to the electrostatic repulsion between the headgroups of these lipids. They also found that lowering the pH induced phase transition from Im3m to Pn3m, and a phase transition from Pn3m to HII phase. Anionic surfactants combined with GMO are of interest to us since they offer some interesting applications in food and also provide some interesting structural characteristics along with the stability and transformations of cubic phases. In addition, these mixed surfactant systems can offer some solubilization and structural advantages that can be translated into enhancing delivery patterns. Finally, of great interest is the effect of the counterions of the anionic surfactant on the order/disorder of the cubic mesophases. In this study, we attempted to use a food-grade cosurfactant (oleyl lactate, OL) as a GMO replacement and to study the effect

10.1021/jp103799a  2010 American Chemical Society Published on Web 07/29/2010

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Figure 1. (A) Cubic mesophase regions in the pseudoternary phase diagrams of GMO-HOL/EtOH/water, GMO-NaOL/EtOH/water, and GMO-OL/ EtOH/water systems at 25 °C. AT, the total area in percentage of the isotropic cubic phase from phase diagram; ∆Wmax, the maximum water solubilized (in percentage); and ∆EtOHmax, the maximum percentage of ethanol. (B) Schematic presentation of gyroid structure consisting of nonionic and anionic surfactants, showing the difference between Na+ as a kosmotropic agent and H+ as a chaotropic agent.

of the headgroup and its charge on the formation of the bicontinuous cubic mesophases, their curvature characteristics, and possible transitions between mesophases. The more specific objectives of this study were (1) broadening the cubic isotropic region to enable more formulation flexibility (more water solubilization and more alcohol entrapment), by utilizing nonexpensive food-grade amphiphilic compounds along with GMO; and (2) examining the type of interaction and structure affected by the GMO/OL ratio in self-assembled aggregates of GMO mixed with OL in water and ethanol, using small-angle X-ray spectroscopy (SAXS), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) techniques. Experimental Section Materials. Monoolein, GMO, distilled glycerol monooleate that consists of 97.1 wt % monoglyceride and 2.5 wt % diglyceride and 0.4 wt % free glycerol (acid value 1.2, iodine value 68.0, melting point 37.5 °C) was obtained from Riken (Tokyo, Japan). Ethanol (EtOH) was purchased from Sigma Chemical Co. (St. Louis, MO). D2O (D, 99.9%) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). Sodium hydroxide (98%) and hydrochloric acid (6.0 N standardized aqueous solution) were purchased from Alfa Aesar, Ward Hill, MA). Water was triple distilled. The commercial sodium oleyl lactate (Olacta, OL was a gift of ADM, Archer Daniels Midland, Decatur, IL) is only partially neutralized. We prepared two additional oleyl lactate forms. One fully neutralized by sodium hydroxide as a counterion, that we termed NaOL, and another that was completely acidified with hydrochloric acid and termed HOL. Sample Preparation and Construction of Phase Diagram. In Figure 1 are shown the pseudoternary phase diagrams of the studied four-component systems. The phase diagrams were constructed in the following way: mixtures of surfactants, GMO + OL (at a constant weight ratio of GMO/OL for each diagram) ethanol, and water were prepared and sealed in culture tubes closed tightly with Viton-lined screw caps at different weight ratios of surfactants, EtOH, and water, depending on the phase diagram. Each pseudoternary phase diagram can be divided into water dilution lines that represent a constant weight ratio between the surfactants and EtOH, that is, water dilution line 6:4 indicates 60 wt % surfactant and 40 wt % ethanol. All samples were heated to a maximum of 80 °C for 2 min, stirred, and then kept in a 25 ( 0.5 °C water bath. The samples were allowed to equilibrate for at least 24 h before they were examined.

The different mesophases were determined by visual inspection and by SAXS. Cubic liquid crystalline samples were prepared by weighing appropriate quantities of the components (GMO-OL/ethanol/ water in a ratio of 57/38/5 wt %, respectively). D2O was used for ATR-FTIR measurements instead of ethanol and water. Methods Small-Angle X-ray Scattering (SAXS). SAXS measurements were used to identify the structure and to calculate the lattice parameters. The scattering experiments were performed using Cu KR radiation (λ ) 0.154 nm) from a Rigaku RAMicroMax 007 HF X-ray generator operated at a power rating up to 1.2 kW generating a 70 × 70 mm2 spot size and focus. ThedirectbeamthengoesthroughavacuumOsmicCMF12-100CU8 focus unit with a beam size at the sample position of 0.7 × 0.7 mm2. The scattered beam goes through a flight path filled with He and reaches a Mar345 Image Plate detector. The sample was inserted into 1.5 mm quartz capillaries that were then flamesealed. The temperature was maintained at 25 ( 1 °C with exposure time of ca. 1 h. The sample to detector distance was calibrated using silver behenate to 1840.5 mm. Differential Scanning Calorimetry (DSC). A Mettler Toledo DSC822 (Greifensee, Switzerland) measuring model system was used. The DSC measurements were carried out as follows: 5-15 mg of cubic liquid crystalline samples were weighed, using a Mettler M3 microbalance, in standard 40 µL aluminum pans and immediately sealed by a mechanical press. The samples were rapidly cooled in liquid nitrogen from +25 to -50 °C at a rate of 10 °C min-1. The samples remained at this temperature for 30 min and then were heated at a rate of 2 °C min-1 to 50 °C. An empty pan was used as reference. The instrument determined the fusion temperatures of the components and the total heat transferred in any of the observed thermal processes. The enthalpy change associated with each thermal transition was obtained by integrating the area of the relevant DSC peak. DSC temperatures reported here were reproducible to (0.2 °C. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR). An Alpha P model spectrometer, equipped with a single reflection diamond ATR sampling module, manufactured by Bruker (Ettlingen, Germany), was used to record the FTIR spectra. The spectra were recorded with 50 scans with spectral resolution of 2 cm-1 at room temperature. The absorbance intensities reported here were reproducible to (0.005.

Nonionic-Anionic Mixed Surfactants Cubic Mesophases

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TABLE 1: Summary of the Three Major Parameters of 90:10 Weight Ratios of the Three Anionic Surfactantsa GMO GMO:NaOL GMO:OL GMO:HOL

AT (%)

∆Wmax (%)

∆EtOHmax (%)

2.1 1.5 4.1 2.3

16 18 28 20

4 6 10 7

a AT, the total area in percentage of the isotropic cubic phase from phase diagram; ∆Wmax, the maximum water solubilized (in percentage) at a selected GMO/anionic surfactant that enables such solubilization; ∆EtOHmax, the maximum required percentage of ethanol to achieve minimum viscosity at any given GMO/anionic surfactant system.

Figure 3. The cubic mesophase regions in the pseudoternary phase diagrams of the GMO-OL/EtOH/water systems at 25 °C at different GMO/OL weight ratios.

TABLE 2: Summary of the Three Major Parameters That Were Measured for Each GMO/OL Ratioa Figure 2. The asymmetric carboxylate vibration bands of the three anionic surfactants; green, NaOL; red, OL; and black, HOL.

ATR-FTIR Data Analysis. Multi-Gaussian fitting was utilized to resolve individual bands in the spectra. The peaks were analyzed in terms of peak frequencies, width at half-height, and area.

GMO/OL

AT (%)

∆Wmax (%)

∆EtOHmax (%)

100:0 90:10 70:30 50:50 30:70

2.1 4.1 4.8 3.3

16 28 35 24 negligible

4 10 12 8

Results and Discussion

a AT, the total area in percentage of the isotropic cubic phase from phase diagram; ∆Wmax, the maximum water solubilized (in percentage) at a selected GMO/OL ratio that enables such solubilization; EtOHmax, the maximum required percentage of ethanol to achieve minimum viscosity at any given GMO/OL ratio.

Ionization Effect on the Solubilization Capacity. Cubic bicontinuous phase was prepared from mixed surfactants of nonionic surfactant, monoolein (GMO), and three types of anionic surfactants, oleyl lactylic acid (HOL), sodium oleyl lactate (NaOL), and partially neutralized oleyl lactate acid (OL) in the presence of water and EtOH. The phase diagrams of these systems were constructed (Figure 1). To better characterize the limits of the cubic mesophase area, we defined three parameters, AT, the area (in percent) of the isotropic cubic phase from the total phase diagram, and ∆Wmax (parallel to the x-axis, Figure 1) and ∆EtOHmax (perpendicular to the x-axis), the maximum water and ethanol content, respectively (Figure 1A). On the basis of the three defined parameters (AT, ∆Wmax, and ∆EtOHmax), the OL forms cubic bicontinuous phase in the largest compositional region (largest values, Table 1), while the NaOL forms the most restricted isotropic region (the smallest values). Yet, the major advantage of the HOL and NaOL were manifested in their effect on the solubilization capacity of the water and/or ethanol molecules (20 and 18 wt % for water and 7 and 6 wt % for ethanol, respectively). The dimensions of the cubic phase region in the phase diagram do not depend only on the degree of ionization (deprotonation) of the lipid. The vibration band (FTIR) intensity of the free asymmetric carboxylate (COOas-) at 1598 cm-1 reflects the degree of COOas- deprotonation.30-32 The highest intensity band is of a neutralized NaOL, while HOL is lacking the 1598 cm-1 vibration; the OL band appeared between these intensities (Figure 2). It appears reasonable to consider, therefore, that the difference between NaOL and HOL originates from the difference in their

choatropic/kosmotropic properties (based on Hofmeister series).33 Na+ is kosmotropic while H+ is a chaotropic agent (Figure 1B). But once the H+ and Na+ are present together, their character changes according to the ion concentrations and vicinal hydrated electrolytes, since the Hofmeister ions’ behavior is not absolute.34,35 Therefore, we can demonstrate that balanced selection of counterions on the cosurfactant (H+ and Na+) with their chaotropic/kosmotropic behavior within the same system yielded a synergistic effect and resulted in obtaining the greatest solubilization values. Phase Diagrams of the Pseudoternary Mixture at Selected GMO/OL wt % Ratios. Pseudoternary phase diagrams of the GMO/OL/EtOH/water system were constructed from 100:0 to 30:70 wt % ratios of GMO/OL in order to find the largest bicontinuous cubic area. It can be seen (Figure 3 and Table 2) that in the absence of OL the total isotropic region, AT, is relatively small, only 2.1% of the phase diagram, and the ∆Wmax along with ∆EtOHmax are of 16 and 4 wt %, respectively. Progressively replacing the GMO by OL yields a significant increase in all three parameters. The largest isotropic region is formed with 70:30 GMO/OL. The maximum solubilization values are 4.8, 35, and 12 wt % for AT, ∆Wmax, and ∆EtOHmax, respectively. Replacing an additional amount of GMO with OL caused a decrease in all three parameters. Once the GMO/OL ratio reaches 30:70, a single isotropic cubic mesophase is transformed into two phases (cubic and lamellar). This indicates that above certain levels of OL the “curvature is stressed” as a result of high frustration caused by the charged surfactant and deviation from their bilayer structure, the preferred energy

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Figure 4. SAXS diffractions of cubic mesophases composed of 6/4 GMO-OL/water and 5 wt % EtOH at 25 °C, where GMO/OL wt ratios were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, and 30:70 (see this SAXS data in Table 3).

conformational state.7,36 The cubic region decreases in size as a preliminary stage to formation of two coexisting mesophases. The transformation and the curvature frustration are probably derived from the electrostatic repulsive interactions and the water hydration effect, which increases with OL content (see FTIR results, items 2 and 3).

Efrat et al. Structural Effect of GMO/OL Ratios on Structure. The structure effect of progressive replacement of GMO by OL is shown in Figure 4 and Table 3 of samples from dilution line 60:40 GMO/OL weight ratio and 5 wt % ethanol (or surfactants/ water/ethanol at 57/38/5 wt %, respectively) at 25 °C. A system without OL (100:0) had shown SAXS diffraction with a spacing ratio of
3;
4;
6;
7;
8;
10;
11;
12;
13;
26 that corresponds to the Ia3d space group of cubic bicontinuous symmetry with lattice parameter of 123.39 ( 1.06 Å. Upon replacing part of the GMO by OL (80:20 weight ratio) the spacing ratios (reflections) changed to
2;
3;
4;
6;
8;
9;
10;
12, which corresponds to the diamond mesophase of space group Pn3m with lattice parameter of 98.38 ( 0.97 Å that increases with OL content to 102.45 ( 1.26 Å at 40:60 wt % GMO/OL ratio (Figure 4). Further addition of OL to 30:70 wt % ratio caused transformation of one phase into a mixture of the two phases, one of less ordered cubic (with spacing ratio of
3;
4;
9;
12) and the other a lamellar phase with lattice parameter of 53.86 ( 0.69 Å. A phase transition occurred probably as a result of a combined effect of electrostatic repulsion forces at the headgroups caused by the negative charged OL anion that stabilizes the phases with lower curvature and larger interface area. With low surface charge density, the electrostatic repulsion forces are not sufficiently strong to lead to significant change in the CPP; therefore, only minor change takes place, for example, transition between the cubic phases (Ia3d to Pn3m). Similar transition occurred in the case of the GMO/water system; the Ia3d phase transformed to Pn3m phase upon increasing the amount of water, that is, increasing hydration.37 Once a high negative surface charge density is obtained, the strong electrostatic repulsions enforce formation of lamellar phase with a flattened structure, to release the strain in the packing frustration. Similar transition of cubic to lamellar was reported by Gustafsson et al.38 as the GMO was replaced by sodium cholate in the binary GMO/water system. We also observed that samples with up to 30 wt % OL replacing the GMO, aged for a prolonged time (>2 months), showed a time-dependent stability; the structures remained cubic even after prolonged aging. On the other hand, samples richer in OL transformed faster into two phases, lamellar and less ordered cubic (data not shown). It was suggested that the high content of OL might be causing partial surfactant hydrolysis. We checked our samples for possible hydrolysis by measuring the free fatty acid that could occur and found it to be negligible (less than 0.1 wt %),39 showing that it is not the free fatty acids that are responsible for the transition effect. Therefore, we concluded that the effect is thermodynamically and kinetically controlled. The mesophases are not sufficiently ordered and tend with temperature and time to invert into other structures.

TABLE 3: Results from SAXS Experiments Performed on Cubic Bicontinuous with Increasing Concentrations of OL That Replacing in Part of the GMO GMO/OL

reflections

space group

lattice parameter (Å)

100:0 90:10 80:20 70:30 60:40 50:50 40:60 30:70


3:
4:
6
7:
8;
10
11:
12:
13:
26
3:
4:
7:
8:
10:
11:
12:
13
2:
3:
4:
6:
8:
9:
10:
12
2:
3:
4:
6:
8:
9:
10:
12
2:
3:
4:
5
6:
8:
9
2:
3:
4:
6:
8:
9
2:
3:
4:
6:
8
3:
4:
9:
12 1:2

Ia3d Ia3d Pn3m Pn3m Pn3m Pn3m Pn3m Pn3m lamellar

123.39 ( 1.06 120.12 ( 0.99 98.38 ( 0.97 98.70 ( 1.04 99.20 ( 1.12 101.24 ( 0.91 102.45 ( 1.26 104.23 ( 1.30 53.86 ( 0.69

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Figure 6. Infrared spectra of the wagging bands of cubic mesophases in GMO/OL ratios of 100:0, 90:10, 80:20, 70:30, and 60:40.

Figure 5. DSC thermograms of A) pure GMO, pure OL and their mixtures at the next weight ratios: 90:10, 70:30, 50:50, and 30:70; B) samples composed of 6:4 GMO:OL/water and 5 wt % EtOH, where GMO-OL wt ratios were: 100:0, 90:10, 80:20, 70:30, 50:50.

Thermal Behavior (DSC). To assess the interaction between GMO and OL, we monitored the main phase transitions of GMO/OL at different ratios (Figure 5A) and in the pseudoternary mixtures that contain GMO-OL/EtOH/water (Figure 5B) by conducting heating protocol in the DSC within -50 to +50 °C. GMO, as raw material, showed two endothermic melting peaks at 22.1 ( 0.2 and 31.7 ( 0.2 °C, while OL is liquid in this temperature range and does not exhibit any endothermic events (Figure 5A). Once the two components (GMO and OL) were mixed at 90:10 the phase is a gel at room temperature, and two endothermic transition events fuse into one broader peak that contains several phase transitions. This broadening of the peak indicates transitions (range of nearly 35 °C) that are characteristic of the heterogeneity of the sample. The first transition melting peak is the same as that of pure GMO and the three others are probably of the intermediate composition that can be interpreted as the formation of GMO/OL clusters.40-42 Further decrease in the GMO fraction (70:30 and 50:50 GMO/ OL wt % ratios) reveal a similar main phase transition with significant shift to lower temperatures (-11.8 and -16.1 ( 0.2 °C for 70:30 and 50:50 mixtures, respectively). Adding water and EtOH (38 and 5 wt %, respectively) to the surfactant mixtures yielded more ordered structures at any tested temperature; we did not identify intermediate compositions (Figure 5B and Table 4).

To assign the peak B (Figure 5B) we have replaced water for D2O and observed a shift of the peak B by +3.2 °C. This would confirm that the endothermic event was mainly due to the fusion of water.37 With the increase in the OL content the endothermic event B content was shifted to a lower temperature. These results indicate that OL influences headgroup interactions (see more information in the FTIR section). Peak C reflects the melting of tails of the crystalline lamellar mesophase. This melting also shifts to a lower temperature (from 6.4 °C to ca. -0.24 °C for 100:0 and 50:50 GMO/OL mixtures, respectively) with smaller ∆H values. This indicates a decrease in the hydrophobic interactions between the acyl chains that lead to additional disorder (see FTIR next section). An additional peak A appears around -25.8 and -27.34 ( 0.2 °C for the mixtures of 70:30 and 50:50 GMO/OL wt % that were used. It seems to represent a transition of one crystalline structure to another polymorph, which could account for interdigitated phases in the bilayer caused by the presence of sufficient amounts of OL. ATR-FTIR. ATR-FTIR reveals molecular interactions between the components of the LLC. As previously stated,43,44 the interactions within the cubic structure may be categorized into three distinct types, (1) those between the tails of the GMO and/or the OL; (2) those at the water-surfactant interface; and (3) those between at the headgroups of the surfactants and water. 1. Tail-Hydrocarbon Moieties of the Surfactants. Information on the hydrocarbon chain order conformations can be obtained from the 1400-1300 cm-1 wagging vibration bands of the nonplanar CH2 conformers. We identified three order sensitive bands: gauche-trans-gauche and the kink sequence conformer (gtg-gtg′) that appear together as a single small band (1367 cm-1), and the two adjacent gauche-gauche (gg) and end-gauche (eg) conformers (1354 and 1341 cm-1, respectively; Figure 6).19,45-49 An additional vibration (the fourth) was found at 1378 cm-1 of high intensity, reflecting the symmetric bending vibration of the end CH3 group (the umbrella deformation mode is of the CH3). The umbrella band is insensitive to the chain

TABLE 4: Melting Temperatures (°C) of Cubic Systems Composed of Various Ratios of GMO/OL GMO/OL (wt %)/Tm (°C)

100:0

90:10

80:20

70:30

50:50

Peak A Peak B Peak C

-2.9 6.4

-4.7 6.0

-5.4 3.6

-25.8 -5.9 0.21

-27.34 -6.5 -0.24

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TABLE 5: Intensities of the CH2 Wagging Bands (kink, gg, and eg) Normalized to the CH3 Bending Vibrations of Cubic Phase at Specified GMO/OL Ratios GMO/OL (wt ratio) I1367/I1378 (kink) I1354/I1378 (gg) I1341/I1378 (eg) 100:0 90:10 80:20 70:30 60:40

0.770 0.798 0.870 0.891

0.688 0.729 0.717 0.712 0.711

0.656

length and conformation, while the other bands were sensitive to both the configuration and packing changes. The umbrella band was used as an internal standard and normalized the intensity of the other sensitive conformation wagging bands, allowing quantitative calculation of the gauche “concentrations”.47,50 Such an approach was adopted in a study of chain conformations in micelle phospholipid bilayers.50-52 In our present study, the intensities were determined from curve-fitted spectra. The intensity of the umbrella mode was assigned a 1, and the intensities of the other three bands, sensitive to conformation, were normalized to this value (Table 5). The values indicate that the increase in the OL content increases the gtg-gtg′ conformation and very slightly decreases the gg deformation. In the presence of 40 wt % OL, we also identified the eg deformation. Since the magnitude of the gtg-gtg′ value increase is more than the decrease in the gg values the overall gauche deformation increases. We can claim that as the “concentration” of the gauche deformation per chain increases with OL content, the system becomes more disordered and the tail-tail interactions diminish. This is in line with DSC results showing that the temperature and enthalpy values decrease as the content of the OL tails increases. 2. Water-Surfactant Interface Groups. The carbonyl (CdO) stretching mode reflects changes at the polar/apolar interface. But the spectra of the investigated systems exhibit a water absorption band centered at about 1645 cm-l, which overlaps with the carbonyl band; thus to analyze the CdO band, the H2O was replaced with D2O. The carbonyl bands of samples with and without OL are composed of two superimposed bands at 1742 and 1722 cm-1. On the basis of previous investigations of the CdO stretching in liquid crystals formed from GMO/ H2O upon introducing cytochrome C and/or distearoylphosphatidyl-glycerol, the splitting of the carbonyl mode can be interpreted as a reflection of the coexistence of free and hydrogen-bonded populations.48,53 To extract quantitative information on the changes in these band profiles a Gaussian stimulation was performed. The curve-resolving procedure indicates that the wavenumber remains constant for any OL content. Calculated, normalized to the overall area, free and

Figure 8. Infrared spectra of carboxylate groups band vibrations. The intensity of this band grew with increase of OL content, 100:0, 90:10, 80:20, 70:30, 60:40, and 50:50.

bonded, of CdO (Afree/(Afree + Abonded) and Abonded/(Afree + Abonded)) exhibit a very slight decrease and increase for free and bonded carbonyl groups, respectively, with an increase of OL content (Figure 7A). As the width at half-height of the bound and free carbonyl modes increases, bonded carbonyl has a relatively significant width and the free carbonyl has only a slight width (7 and 2 cm-1 for bonded and free carbonyl peaks, respectively; Figure 7B). These changes in the line shape of the bonded and free carbonyl groups lead us to suppose that there are more bonded carbonyls and broad bands due to disruption of the intermolecular hydrogen bonding, which could indicate order reduction in the interface. Nilsson et al. suppose that broadening of the CdO stretching band may be due to a vibrational dephasing mechanism, where water would give a broader distribution of vibrational energy levels.44 The broadened band at the free carbonyl bond is negligible since this small value is within the device error. The carboxylate band at 1598 cm-1 in the system that contains D2O (replacing H2O; Figure 8) displays asymmetric stretching, while the carboxylate band of OL surfactant alone is 1602 cm-1. These values indicate that the carboxylate groups are being hydrated at the liquid crystalline structure. LLC vibration value does not change with OL content, only the intensity is affected. The intensity of this band grew with an increase of OL content, which indicates the degree of ionization or deprotonation. Meaning, the surface charge density, actually the electrostatic repulsive interaction, increases in the membrane interface with an increase in the OL content. The repulsion force probably causes modification of the local geometry (as we saw in SAXS results) and increases the headgroup area, which can lead to changes in the spontaneous curvature (H0) and packing parameter (V/al). The carboxylate intensity band of 70/30 GMO/OL ratio is higher than the corresponding band of a system that consists of 90:10 GMO/NaOL, although the cubic phase region in the phase

Figure 7. (A) Normalized to the overall area of free (O) and bonded (b) CdO (Afree/(Afree + Abonded) and Abonded/(Afree + Abonded)). (B) Width at half-height of the bound, free, and general (∆) carbonyl bands.

Nonionic-Anionic Mixed Surfactants Cubic Mesophases

Figure 9. Infrared spectra of the sn1 bands of cubic mesophases in GMO/OL ratios of (a) 100:0, (b) 90:10, (c) 80:20, (d) 70:30, and (e) 60:40.

diagram is larger. The reason could be that the electrostatic repulsion force of the OL anion was induced by the cations and their kosmotropic/chaotropic behavior. Kosmotropic/chaotropic behavior is influenced by the presence of its neighbors.54,55 Therefore, we can suppose that the large cubic area containing a high charge density at the headgroups becomes possible as a result of the synergistic effect produced from the integration of Na+ and H+. The secondary alcohol (β-C-OH or (sn2)) stretching peak frequencies remain unchanged, but the bandwidth and the intensity are slightly larger as the OL content increases. The remarkable change was recorded between systems with and without OL, whereas the differences between GMO/OL ratios is negligible. Thus, we suggest that the broadening of these bands reflects a lower order of the glycerol OH groups in the GMO-OL/EtOH/water systems. The spectral region of the primary alcohol appears at 1046 and also at 1088 cm-1. The last band corresponds to primary alcohol with lower polarity interaction than the previous band. This band becomes prominent with an increase in EtOH content but without change in the wavenumber (data not shown). Once OL replaces part of the GMO without any change in the other component content this band shifts toward higher wavenumbers (1088 to 1094 cm-1 for GMO/OL ratios 100:0 to 60:40, respectively) and becomes more prominent; even the OL molecule does not contain any primary alcohol (Figure 9). This can be interpreted as the presence of OL gradually reducing the hydrogen bonds of the EtOH, hence it is plausible that the EtOH location differs from a less polar environment that can lead to change at the interface configuration. Because the ethanol location varies according to the nature of the headgroups, orientation of the headgroup dipoles, and surfactant-water interaction, the EtOH molecule can interact with both the hydrophilic lipid groups at the interface and the methylene groups of the hydrophobic surfactant tail. Parallel to low polarity band change, the high polarity band (1046 cm-1) of the sn3 group exhibits only a slight change in width and a slight change in intensity, but without any change in the wavenumber. The intensity of sn2 and sn3 are almost unchanged even though their concentration decreases with a decrease in GMO content, which can indicate more hydration per GMO molecule. These results are associated with the phase transition to a larger interface that is reflected in the SAXS results. The hydroxyl hydration could be by the cations that are present in the system, meaning the hydronium ion, which can act as chaotropic due to its adsorption on the surfactant headgroups, but Na+ can also adsorb on the headgroup surface as we have shown in our previous investigation.21 3. The Headgroups. By replacing H2O with D2O we could study the O-D stretching vibrations that are affected by changes

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Figure 10. (a) O-D stretching vibrations of the cubic mesophases where GMO/OL wt ratios were 100:0, 90:10, 80:20, 70:30, 60:40, and 50:50.

in the water structures (H-bonding network). These vibrations are attributed to the tetrabonded, “icelike” water molecules and from a nonideal H-bonding arrangement (e.g., angular Hbonding disturbing).56,57 In our system, two major components appeared at 2485 and 2383 cm-1 for a system without OL surfactant. The gradual incorporation of OL increases the frequency of O-D stretching shift to 2502 and 2410 cm-1 as the GMO/OL ratio is decreased to 50:50 (Figure 10). This shift is characteristic of chaotropic or structure-breaking ions that disorder and/or weaken the hydrogen bonds between water molecules. They also can change the lipid surface potential and dipolar moment alterations.56,58 This chaotropic effect is in line with the SDC results, since the chaotropic effect leads to a decrease in the transition temperature from lamellar gel to lamellar liquid crystalline phase. It is also in line with SAXS results indicating that chaotropic ions have a tendency to increase the amount of interfacial water.58 Consequently, chaotropic ions stabilize the lipid phases having larger interfacial area per lipid molecule, thus they encourage the phase transformation of the gyroid to the body-centered cubic phase and this d-space increases upon added OL. The same effect has been reported from the study on the cubic phases of monoolein/oleic acid systems.59 Despite that Na+ is usually considered a kosmotropic ion (structure maker), the overall effect is choatropic since it is determined from the environment of both the anion and the cation of the total solubilized, ion concentrations, and the other molecules involved in the process. Conclusions It has been shown that the cubic bicontinuous mesophase composed of GMO/water and ethanol may also be formed if part of the GMO is replaced by an anionic surfactant. The cubic bicontinuous areas in the phase diagram are enlarged in all three parameters (AT, ∆Wmax, and ∆EtOHmax). The size of the cubic bicontinuous region depends on the counterion (cation). If NaOL and HOL had a relatively minor effects while the OL has a relatively large effect on the swelling of the cubic region as it could seen from the pseudoternary phase diagram. The presence of both Na+ and H+ ions yielded a synergistic effect on the cubic region as a result of their mixed chaotropic and kosmotropic character that they impart upon the system (Hofmeister series). The ratio of GMO/OL also modifies the size of the cubic phase region. The longer swollen isotropic region is obtained for the 70:30 wt % ratio. With the higher OL levels in the mixture a smaller cubic region is formed and the long-term stability is reduced.

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