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Department of Chemical Engineering, Indian Institute of Technology Bombay, ... and loose packing of molecules which would facilitate water transport a...
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Langmuir 2004, 20, 4816-4822

Articles Micro-Raman Investigations of Myelins in Aerosol-OT/ Water System M. A. Arunagirinathan,† Mainak Roy,‡ A. K. Dua,‡ C. Manohar,† and Jayesh R. Bellare*,† Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai-400 076, India and Novel Materials & Structural Chemistry Division, Bhabha Atomic Research Centre, Mumbai-400 085, India Received October 10, 2003. In Final Form: March 4, 2004 Surfactant outgrowth during dissolution as myelin figures, which happens on contact with water, is of prime importance in emulsification and detergency. Micro-Raman investigation of different lyotropic phases formed during dissolution of aerosol-OT (bis 2-ethylhexyl sulfosuccinate) in water during myelin formation reveals the flexible arrangement of the surfactant bilayers in myelin. The conformation around CC-CS bond and the hydrocarbon chains of aerosol-OT in the different liquid crystalline phases were identified from the fingerprints of CC-CS stretching, C-C stretching, C-H bending, and stretching frequencies. Existence of mixture of trans and gauche conformations around CC-CS bond and that of the hydrocarbon chains in myelin supports the fluid nature of bilayers by which it is made. Similar conformations of hydrocarbon chains in lamellar phase and in myelin support the concept of myelins being rolled up lamella. The observations are in line with the disorderness of the hydrocarbon chains in the bilayers of phospholipids that has been reported earlier. From the C-C stretching frequencies at the root of myelins, the kinked structure of the hydrocarbon chain is identified, and loose packing of molecules which would facilitate water transport across membranes is evident.

1. Introduction Self-assembly of surfactant molecules to form various equilibrium microstructures is a well known phenomenon and understood well; however, the nonequilibrium microstructure formation during the dissolution of surfactants is not yet explored in detail. The formation of “myelin figures” from dissolution of sparingly soluble surfactants such as phospholipids, aerosol-OT (AOT), and polyethoxyalkyl ethers in water, ethylene glycol, and glycerol have been studied by many researchers.1-8 The mechanism for appearance of myelin figures is probably due to the formation of insoluble lamellar phase and subsequent protrusion into the bulk aqueous phase in the form of snakelike structures9 instead of complete dissolution. The myelin figures are fingerlike extrusions having concentrically arranged alternating layers of bilayer of surfactant molecules and an aqueous layer with a central * Corresponding author. E-mail: [email protected]. † Indian Institute of Technology Bombay. ‡ Bhabha Atomic Research Centre. (1) Virchow, R. Virchow’s Archive 1854, 6, 652. (2) Sakurai, I.; Kawamura, Y. Biochim. Biophys. Acta 1984, 777, 347-351. (3) Mishima, K.; Yoshiyama, K. Biochim. Biophys. Acta 1987, 904, 149-153. (4) Mori, F.; Lim, J. C.; Raney, O. G.; Elsik, C. M.; Miller, C. A. Colloids Surf. 1989, 40, 323-345. (5) Lim, J. C.; Miller, C. A. Langmuir 1991, 7, 2021-2027. (6) Buchanan, M.; Arrault, J.; Cates, M. E. Langmuir 1998, 14, 73717377. (7) Haran, M.; Chowdhury, A.; Manohar, C.; Bellare, J. Colloids Surf., A 2002, 205, 21-30. (8) Dave, H.; Surve, M.; Manohar, C.; Bellare, J. J. Colloid Interface Sci. 2003, 264, 76-81. (9) Buchanan, M.; Egelhaaf, S. U.; Cates, M. E. Langmuir 2000, 16, 3718-3726.

core of water. The arrangement of surfactant molecules in the existing phase and its further evolution into the new phase from which the myelin figures form depends on the (i) conformations of the hydrocarbon chains present in the amphiphile, (ii) interactions between the polar headgroup and the counterions, and (iii) hydration of the hydrophilic part of the surfactant. Raman spectroscopy can be used as an analytical tool to identify the conformation of the hydrocarbon chains.10 The conformations of the hydrocarbon chains in lipids such as lecithin,10,11 cholesterol,12 and surfactants such as AOT13-15 have been studied by Raman spectroscopic investigations. Myelin formation in oleic acid/hydrazine system studied by Raman spectroscopy claims the presence of hydrogen bonds at the interfacial zone causes the stability of lipid sheets and in turn decreases the permeability for the aqueous medium.16 To our knowledge, so far Raman spectroscopic investigations on AOT have not been reported to understand the conformations of the hydrocarbon chains and the interactions of the polar groups in the myelin microstructure. Interestingly, AOT is the first amphiphile studied by Raman spectroscopy to show spectroscopic band variations in one region of the spectrum with changes only in phase (10) Larsson, K. Chem. Phys. Lipids 1973, 10, 165-176. (11) Lippert, J. L.; Peticolas, W. L. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 1572-1576. (12) Faiman, R. Chem. Phys. Lipids 1977, 18, 84-104. (13) Faiman, R.; Lundstrom, I.; Fontell, K. Chem. Phys. Lipids 1977, 18, 73-83. (14) Maitra, A.; Jain, T. K. Colloids Surf. 1987, 28, 19-27. (15) Nagasoe, Y.; Ichiyanagi, N.; Okabayashi, H.; Nave, S.; Eastoe, J.; O’Connor, C. J. Colloid Polym. Sci. 1999, 277, 947-956. (16) Gruger, A.; Vogel-Weill, C. Mol. Cryst. Liq. Cryst. 1994, 238, 227-239.

10.1021/la035898v CCC: $27.50 © 2004 American Chemical Society Published on Web 05/05/2004

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Figure 1. Phase succession observed by optical microscope in AOT/water system. H, reverse hexagonal phase; I, isotropic viscous phase; L+, positive birefringent lamellar phase; L0, myelin root (zero birefringent lamellar phase); M, myelin microstructure. The scale bar represents 100 µ.

structure and is also the first aqueous system that shows spectroscopic band variations within one phase region;13 AOT in the anhydrous form is a good Raman spectroscopic model for the study of cholesterols in the crystalline state.12 The variation in intensity of the 1460 cm-1 band occurred in the regions where changes have been observed in the optical birefringence, conductivity, and X-ray studies. The Raman investigations on straight-chain analogues of AOT by Nagasoe and co-workers reveal that in monohydrates the hydrocarbon tails become disordered at the CH2-CH2 single bond close to the terminal methyl groups and that in dihydrates the hydrocarbon chains exist in extended form. The interactions between the headgroup and the counterions have been studied in detail.15,17 Raman investigations of water-in-oil microemulsions of AOT have also been studied.14,18,19 Thus, the systematic investigation of the myelin microstructures formed from the solid AOT on contact with water was undertaken and reported here to understand the dynamic transformation of the conformations of the hydrocarbon chains with different mesophase formation. The conformations in the various phases formed during dissolution are studied by comparing the appropriate stretching and bending modes from this micro-Raman study. 2. Experiments 2.1 Materials. Aerosol-OT of approximately 99% purity was obtained from Sigma Chemical Co. The chloroform used is of GR grade of assay 99.4% obtained from Merck India Ltd., and distilled water was used for the studies. 2.2 Instrumentation. Raman microscopic investigations were carried out on the phases formed during dissolution of AOT. Micro-Raman investigations were performed on the localized area of the sample using optical microscope Olympus BX-40, 10× objective, connected to Raman spectrometer Labram-1. Ar+ 488nm laser source of intensity =10 mW was used. The spectra were recorded as the average of 20 scans with time span of 2 s and resolution of 2 cm-1. The obtained spectra were subjected to fivepoint FFT smoothening by Microcal Origin, version 6.0. 2.3 Method. A surfactant lump of AOT was made on the glass slide from two drops of 5 wt % AOT in chloroform, and the solvent was allowed to evaporate. The spacers of 150 µ was placed on both sides of the surfactant, and a cover slip was placed on the surfactant lump and pressed gently to make a thin sample of the surfactant. A few drops of distilled water were kept at the edge of the cover slip and allowed to contact with surfactant lump by capillary action. The myelin figures and the liquid crystalline phases formed during the dissolution of the AOT as shown in Figure 1 were studied by Raman spectroscopy by selecting the particular region in different liquid crystalline phases using optical microscope attached to the Raman spectrometer. (17) Nagasoe, Y.; Ichiyanagi, N.; Okabayashi, H.; Nave, S.; Eastoe, J.; O’Connor, C. J. Phys. Chem. Chem. Phys. 1999, 1, 4395-4407. (18) Jain, T. K.; Maitra, A. Colloids Surf. 1989, 36, 87-95. (19) Moran, P. D.; Bowmaker, G. A.; Cooney, R. P.; Bartlett, J. R.; Woolfrey, J. L. Langmuir 1995, 11, 738-743.

Figure 2. Raman spectra (up to 1800 cm-1) of different phases of AOT. H, hexagonal phase; L+, positive lamellar phase; L0, myelin root; M, myelin. The major peaks are identified in Table 1.

3. Results and Discussion AOT forms various liquid crystalline phases with concentration and temperature. The optical micrograph shown in Figure 1 is of typical myelin forming experiment. The water introduced from the right in to the surfactant 100% at the extreme left generated surfactant concentration gradient, in turn forming different lyotropic liquid crystalline phases starting from left to right as reverse hexagonal phase (H), isotropic viscous phase (I), lamellar phase, and reverse micellar phase. When viewed through cross polars, regions of positive birefringent lamellar phase (L+), zero birefringent myelin root (L0), and myelins (M) having negative birefringence were observed within the lamellar phase which conform with Rogers et al.20 Myelin is generated from a lamellar phase. The existence of lamellar phase appears to be a must for myelin formation.6,21 The lower frequency and higher frequency region of the Raman spectra obtained are shown in Figures 2 and 3, respectively. The tentative assignment of the Raman shifts obtained by micro-Raman investigations of the dissolution of AOT are given in Tables 1 and 2. The bands around 740-790 cm-1, 1050-1085 cm-1, 1450-1460 cm-1, and 2850-2950 cm-1 showed distinct features with phase change as discussed in the next section. As myelins are made up of concentrically arranged surfactant bilayers with alternating layers of water and central aqueous core, the arrangement of surfactant molecules in the bilayers may undergo transformation during rolling up of lamellar sheets into myelin tubes. The earlier studies on myelins by Buchanan21 showed that during myelin formation water enters through the root and not through the tip of the myelin. The arrangement of surfactant molecules at the root may differ from myelin because of the passage of water through it. The necessity of lamellar phase for the myelin formation also imparts definite arrangement of surfactant molecules to form lamellar phase and in turn myelin formation can be (20) Rogers, J.; Winsor, P. A. Nature 1967, 216, 477-479. (21) Buchanan, M. Dynamics of interfaces in surfactant lamellar phases. Thesis, University of Edinburgh, 1999.

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Arunagirinathan et al. Table 2. Tentative Assignment of Peaks in cm-1 for AOT in the High-Frequency Region with Relative Intensitya H

2730 s

2865 sh 2875 s 2898 s 2908 w 2938 vs 2960 sh

L+ 2689 m 2715 m 2734 ms 2756 m 2863 sh 2875 s 2898 s 2912 sh 2937 vs 2962 ms

Lo 2667 m 2686 m 2713 m 2738 ms 2756 m 2775 m 2805 m 2862 sh 2874 s 2901 s 2916 w 2937 vs 2963 ms

M

assignment

2731 ms 2752 m 2777 m

Fermi resonance, overtone

2865 sh 2874 s 2899 s 2915 ms 2936 vs 2960 ms

CH2 sym. stretch CH2 sym. stretch CH2 sym. stretch CH2 asym. stretch CH2 asym. stretch CH3 asym. stretch

a m, medium; v, very; w, weak; s, strong; sh, shoulder. H, hexagonal phase; L+, positive birefringent lamellar phase; Lo, zero birefringent lamellar phase (myelin root); M, myelin microstructure.

Figure 3. Raman spectra (2500-3100 cm-1) of different phases of AOT. H, hexagonal phase; L+, positive lamellar phase; L0, myelin root; M, myelin. The major peaks are identified in Table 2. Table 1. Tentative Assignment of Peaks in cm-1 for AOT in the Low-Frequency Region with Relative Intensitya H 222 w 545 w 562 w 734 vw 776 wb 791 w 829 w 901 sh 939 ms 966 ms 987 ms 1058 s 1094 sh 1140 sh 1271 w 1310 w 1449 s 1457 s

L+ 222 w 285 vw 545 w 560 mb 774 vw 793 w 827 w 907 ms 933 sh 946 w 988 w 1049 vs

Lo 221 w 319 w 535 m 555 m 786 w 806 w 827 w 900 w 920 sh 942 w 973 w 1045 s

1064 s 1083 ms 1087 vs 1146 m 1126 s 1207 m 1273 m 1274 vw 1302 m 1310 vw 1369 m 1365 vw 1453 s 1453 s 1582 m

1722 w 1729 vw 1734 w 1742 vw

M

assignment COO dimer, Ar+

540 wb 575wb 738 vw 794 w 827 w 905 sh

C-S stretch, CH2 rocking C-S stretch C-S stretch SdO sym. stretch C-C stretch

944 sh 989 s 1040 vs

C-C sym. stretch, SO3 sym. stretch 1074 ms C-C sym. stretch 1086 ms C-C sym. stretch 1132 sh C-C asym. stretch SO3 asym. stretch 1268 w, 1275 w SO3 asym. stretch 1297 w 1314 w 1372 w 1450 s CH2 deformn. CH2 deformn. 1467 sh CH2 deformn. 1592 w 1709 vw

1719 wsh 1732 m 1734 vw 1747 vw

CdO sym. stretch CdO sym. stretch

a b, broad; m, medium; v, very; w, weak; s, strong; sh, shoulder. H, hexagonal phase; L+, positive birefringent lamellar phase; Lo, zero birefringent lamellar phase (myelin root); M, myelin microstructure.

visualized by Raman microscopy. The arrangement of surfactant molecule is reflected by its conformation either as the tilted hydrocarbon chains having sufficient fluidity with kinked conformation or as elongated chains with

Figure 4. Raman spectra (expanded) of different phases of AOT in the C-S stretching region. H, hexagonal phase; L+, positive lamellar phase; L0, myelin root; M, myelin. The asterisk mark shows the peaks for trans conformer and the arrow corresponds to the gauche conformer. Both trans and gauche conformers are present in hexagonal phase and myelin figures, whereas in L+, L0 phase around CC-CS bond the trans conformer is present.

rigid trans conformation and can be identified from the Raman shift because of C-C backbone stretching, C-H bending, and stretching of surfactant hydrocarbon chains. The packing of surfactant molecules in the bilayers depend on the conformation of the hydrocarbon chains by which it is made. The polar moiety present in the molecule may also have definite arrangement that influences the fluid nature of the bilayers which can be identified by studying the conformations around CC-CS bond. 3.1 Conformation around CC-CS Bond. The local conformation around the CC-CS bond has been reported for sulfonates and disulfonates by Ohno et al.22-24 The C-S stretching bands around 800 and 740 cm-1 have been (22) Ohno, K.; Fukuda, M.; Yoshida, H.; Tamaoki, H.; Matsuura, H. J. Mol. Struct. 2000, 553, 49-59.

Micro-Raman Investigations of Myelins

Figure 5. Raman spectra (1000-1150 cm-1) of different phases of AOT in the C-C stretching region. H, hexagonal phase; L+, positive lamellar phase; L0, myelin root; M, myelin. The asterisk mark in the hexagonal phase denotes the trans conformer peak and the arrow represents the peaks corresponding to gauche conformer. Both trans and gauche conformers are in hexagonal phase, myelin root, and myelin figures, whereas only gauche conformer is in L+ phase.

assigned to the trans and gauche conformers in disodium R,ω-alkanedisulfonates and the peaks around 790-805 cm-1 and 728-766 cm-1 to the trans and gauche conformations around the CC-CS bond, respectively, in sodium 1-propanesulfonate and sodium 1-butane sulfonate. From the obtained spectra (Figure 4), peaks around 785-805 cm-1 and 735-745 cm-1 can be assigned to trans and gauche conformers around CC-CS bond in the different liquid crystalline phases of AOT. In the hexagonal phase of AOT, the presence of both trans and gauche conformers is evident from the bands at 791 and 734 cm-1. In other lyotropic phases such as positive lamellar phase and at myelin roots, the conformation around CC-CS bond is trans on the basis of the strong peak observed at 793 and 806 cm-1, respectively. The conformation at CC-CS seems to be a mixture of trans and gauche conformers in myelin figures which is evident from the peaks at 794 and 738 cm-1, respectively. 3.2 Conformation of Hydrocarbon Chains. The regions that are susceptible to the changes in the conformations of the hydrocarbon chains12,25,26 are discussed in detail in the following sections. 1. C-C skeletal stretching region (1000-1200 cm-1). 2. C-H bending region (1400-1500 cm-1). 3. C-H stretching region (2800-3000 cm-1). (23) Ohno, K.; Naganobu, T.; Matsuura, H.; Tanaka, H. J. Phys. Chem. 1995, 99, 8477-8484. (24) Ohno, K.; Mandai, Y.; Matsuura, H. J. Mol. Struct. 1992, 268, 41-50. (25) Rosenholm, J. B.; Stenius, P.; Danielsson, I. J. Colloid Interface Sci. 1976, 57, 551-563. (26) Abbate, S.; Zerbi, G.; Wunder, S. L. J. Phys. Chem. 1982, 86, 3140-3149.

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3.2.1 Confirmation of Conformer by C-C Skeleton Stretching. The region corresponding to skeletal vibrations of the carbon backbone 1000-1200 cm-1 has been used as the fingerprint to identify the conformations of the hydrocarbon chains in the system. The conformations are confirmed by the strong peaks in the above region because of symmetric stretching or in-phase C-C stretching and asymmetric or out-of-phase C-C stretching. The all-trans conformation has been confirmed by the presence of a peak at 1060 cm-1 along with 1070 and 1170 cm-1 peaks belonging to symmetric and asymmetric stretching in the AOT by Maitra and Jain.14 The peaks around 1040 cm-1 and 1080 cm-1 have been identified as the peaks belonging to gauche conformers and the peak around 1120 cm-1 to trans conformer by Raman studies on short-chain carboxylates.25 From the spectra in Figures 2 and 5, it can be seen that the peak pertaining to all-trans conformer is present at 1058 cm-1 as a very strong peak in the hexagonal phase of AOT. The peaks corresponding to symmetric and asymmetric stretching of the C-C skeleton appear as weak shoulders at 1094 and 1140 cm-1, respectively, and indicate the presence of gauche conformers along with the trans conformers. For the positive birefringent lamellar phase, the strong peak at 1049 cm-1 and the medium intensity peak at 1083 cm-1 along with the shoulder at 1146 cm-1 imply the presence of gauche conformers. At the myelin roots, the strong peak is observed at 1087 cm-1 along with two peaks of medium intensity at 1045 and 1064 cm-1 and a shoulder at 1126 cm-1. From the earlier peak assignments, it can be said that both trans and gauche conformers are present at the root of the myelin. For myelin figures, a very intense peak is observed at 1040 cm-1 along with a strong peak at 1086 cm-1 and a shoulder peak at 1132 cm-1. The shift in the peak position of the all-trans peak and the appearance of a peak at 1086 cm-1 with a decrease in intensity (Figure 6) on moving from hexagonal phase to the myelin figures confirm the existence of both trans and gauche conformers in the myelin figures with the larger proportion of trans conformer. 3.2.2 Confirmation of Conformer by C-H Bending. In addition to the C-C stretching, C-H bending is also used to identify the conformation of the hydrocarbon chains. The peak at 1460 cm-1 has been used as a tool to demarcate the trans conformer from the random configured conformer and on the basis of its intensity the amount of the particular conformer present in the system can be quantified.26 The intensity of the 1460 cm-1 peak has also been used to study the separation of chains in AOT/water system.13 From the Raman spectra (Figure 7), two peaks at 1449 and 1457 cm-1 with equal intensity are obtained unlike the single peak at 1450 cm-1 that has been observed earlier14,19 for the hexagonal phase of AOT in solid state at room temperature. The doubletlike appearance of the peaks might be due to the equal contribution of trans as well as gauche conformers. The peaks around 1450 and 1460 cm-1 have been attributed to the gauche and trans, respectively, as mentioned earlier. It seems that the AOT must have been hydrated and would have led to the splitting of the peaks as the experiments were carried out in the ambient atmosphere on the glass slide unlike the earlier reported experiments that have been carried out in the sealed capillaries. The spectra corresponding to positive lamellar phase of AOT, myelin root, and myelin indicate a single intense peak at 1450-1453 cm-1. The blown up spectra of the positive lamellar phase and myelin root show a weak

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Figure 6. Raman spectra of different phases of AOT in the C-C stretching region. H, hexagonal phase; L+, positive lamellar phase; L0, myelin root; M, myelin. The decrease in the intensity of the peak around 1083-1094 cm-1 indicates the increase in trans conformer content (L0 < L+ < H < M).

shoulder at 1443 cm-1, and for myelin figures at 1442 cm-1 in addition to a weak shoulder on the higher frequency side of the 1400 cm-1 at 1466 cm-1 for all phases of AOT. The presence of a weak shoulder at 1466 cm-1 implies the presence of trans conformer in the lyotropic phases of AOT, starting from the hexagonal phase to the myelin figures, and the peak near 1450 cm-1 indicates the presence of randomly oriented gauche conformers in myelin figures. The existence of both conformers signifies that the fluidity as well as rigidity of the surfactant bilayers in the myelin figures remain intact during transformation of solid AOT into the dynamic microstructure “myelin”. 3.2.3 Confirmation of Conformer by C-H Stretching. The C-H stretching region gives information on the contribution from the methyl symmetric and methylene antisymmetric vibrations in the molecule as a whole. In liquid crystalline phases with “melted” hydrocarbon chains, the average environment of the CH2 and CH3 groups can hardly vary much from one phase to another, and the observed differences in the Raman spectra should therefore be due to conformational differences.10 The contribution from the methyl groups appears to be linked with the degree of chain branching and hence chain disorder or gauche linkages in the side chains, although not directly to the number of methyl groups in the molecule.12 The C-H stretching region of the Raman spectra of the solid AOT and its lyotropic liquid crystalline phases is presented in Figure 3. From the spectra, it is clear that 2936-2938 cm-1 is predominant with very strong intensity in all phases and a weak shoulder is also observed at 2865 cm-1. From earlier studies on AOT system, the peaks at 2843-2863 cm-1, 2862-2882 cm-1, 2916-2936 cm-1, and 2952-2972 cm-1 have been assigned to CH2 symmetric stretching, CH3 symmetric stretching, CH2 asymmetric stretching, and CH3 asymmetric stretching vibrations,

Arunagirinathan et al.

Figure 7. Raman spectra of different phases of AOT in the C-H bending region. H, hexagonal phase; L+, positive lamellar phase; L0, myelin root; M, myelin. The doublet peak (1449, 1457 cm-1) in H indicates the presence of an equal amount of trans and gauche conformers, whereas the weak shoulder in the L+, L0, and M marked with arrows indicates the lesser amount of trans conformer. The strong peak around 1450 cm-1 in all phases indicates the presence of gauche conformer.

respectively.10 The peaks at 2904 cm-1 and 2936-2940 cm-1 have been assigned to CH2 symmetric stretching,13 whereas the 2870 and 2910 cm-1 peaks to symmetric and asymmetric CH2 stretching, respectively,14 and the 2875 cm-1 peak to CH2 symmetric stretching.19 On the basis of the earlier peak assignments, the peaks observed in this study at 2862-2865 cm-1, 2874-2875 cm-1, and 28982901 cm-1 are assigned for CH2 symmetric stretching while 2908-2916 cm-1 and 2936-2938 cm-1 to asymmetric CH2 stretching and 2962-2963 cm-1 for asymmetric CH3 stretching mode. For an ideally all-gauche polymethylene chain, the main peaks should lie near 2876, 2925, and 2940 cm-1 and when the concentration of the trans structure increases, the spectral feature of the infinite trans chain replaces the spectral pattern of all-gauche structure by downward shift of the peaks at 2876 and 2925 cm-1.26 From the spectra (Figure 8), it can be seen that the shoulder at 2865 cm-1 remains constant without any change intensity; alternately, the shoulder peak on the higher frequency side of the 2950 cm-1 starts growing on formation of myelin structures from solid AOT. The peak at 2962-2963 cm-1 increases in intensity and evolves as a well-resolved peak. The increase in intensity of this asymmetric CH3 stretching peak implies the formation of gauche conformers in the myelin figures. Similarly, the intensity of the 28982901 cm-1 peak increases with a decrease in intensity of the 2874-2875 cm-1 peak on going from solid AOT to its lyotropic phases. Also, the shoulder at 2912 cm-1 in solid AOT grows into a peak in the myelin. The increase in intensity of the symmetric stretching mode (2898-2901 cm-1) indicates an increase in the ordering of the hydrocarbon chains in the lamellar phases of AOT.

Micro-Raman Investigations of Myelins

Figure 8. Raman spectra (2850-2975 cm-1) of different phases of AOT in C-H stretching region. H, hexagonal phase; L+, positive lamellar phase; L0, myelin root; M, myelin. The asterisk mark shows the CH2 symmetric stretching peak, the singleheaded arrow denotes the CH2 asymmetric stretching peak, and the double-headed arrow represents the CH3 asymmetric stretching peak. The increase in intensity of the symmetric stretching peak (marked as asterisk) (H < L+ < L0 < M) indicates the increase in the ordered arrangement of hydrocarbon chains in different phases, whereas the increase in intensity of the asymmetric stretching peak (marked as single- and doubleheaded arrows) indicates the increase in fluidity of the hydrocarbon chains.

3.3 Discussion. The change in conformation around the CC-CS bond from mixture of trans and gauche in reverse hexagonal phase to trans on forming lamellar phase is due to steric hindrance because of the crowding of water molecules around the polar sulfonate group of AOT. In hexagonal phase (solid AOT), the surfactant would have undergone little or no hydration at all and the number of water molecules surrounding the polar group would have been less than six per AOT molecule that is required for complete solvation of the headgroup of AOT.19 The lesser hydration around the polar group would exhibit little restriction toward the freedom of CC-CS bond and hence the presence of both trans and gauche conformations. Also, the rodlike arrangement of surfactant molecules in the hexagonal phase supports the existence of both conformers which would be necessary for the compact arrangement of surfactant molecules in the rod form. On transforming to the lamellar phase which happens because of swelling of the hexagonal phase by water layers and the transformation of interwoven tubular arrangement of surfactant molecules and water layers of the isotropic viscous phase, the headgroup must have hydrated completely and the bulkier hydration sphere around the polar moiety would restrict the CC-CS bond to be in the gauche conformer which is a kinked form and allows the trans form to accommodate the bulkier aqueous sphere around the sulfonate group. The transport of water into the myelin takes place through the roots; hence, the hydration at the root will be comparatively larger than at the lamellar phase from

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where myelin forms. In addition to the continuous flow of water through the root, there will also be replenishment of water molecules around the polar group by continuously replacing old hydration sphere with incoming new water molecules. The hydration of polar group and the transport of the water molecules of the old hydration sphere imparts furthermore restriction on the freedom of the CC-CS bond at the myelin root by blocking the possibility of gauche conformation and allowing only the trans conformer. As surfactant bilayers rolled to form concentric lamellar sheets with alternating layer of water in the form of myelin tubes, the conformational freedom around the CC-CS bond increases by forming both trans and gauche conformers. The concentric arrangement of surfactant bilayers in myelin is similar to the rodlike arrangement of surfactant molecules in the reverse hexagonal phase except that the hydrocarbon tails are facing toward the periphery of the rod in reverse hexagonal phase and both toward and away from the periphery in bilayer sheet of myelin. Hydration around the polar groups of the surfactant molecules will be different if the hydrocarbon chains face toward or away from the periphery of the water layer. When hydrocarbon chains are away from the periphery of the water layer, then the polar group will have enough space to get hydrated and water molecules get packed themselves because of their bulkiness in the neighborhood of polar group and leave less space to have kinked conformation around CC-CS bond. For hydrocarbon chains facing toward the periphery of the water layer, compaction of water molecules near the headgroup will be very less which will create enough space by lesser steric interaction with the CC-CS leading to kinked conformation around CC-CS bond in addition to the existing trans conformation. By comparing the peaks at C-C stretching region in different phases, it appears that significant change occurs at 1040-1058 cm-1 and 1083-1094 cm-1 (Figure 5). The presence of kinked hydrocarbon chains in all phases is evident from the peaks on the lower side of the 1050 cm-1 and either a shoulder or peak around 1085 cm-1. The peak around 1085 cm-1 dominates in myelin root more than in other phases (see Figure 6). Brooker et al.27 have attributed the decrease in intensity of the peak around 1081 cm-1 to the presence of ordered conformers on attaining micellar solution from the sodium octyl sulfate solution. The shoulder or the peak around 1085 cm-1 with lesser intensity originates from the trans conformation of the C-C chain which indicates stretched hydrocarbon chains resulting from well-packed chains within the surface. At the roots, this contribution from trans is reduced drastically and gauche is increased indicating that chains are not well packed. Buchanan et al.9 on observing the diffusion of probe ploystyrene latex particles during myelin formation have come to the conclusion that water enters the myelin through the roots and not through the tips. The reason appears to be the loose packing at the roots. Most probably, roots are formed at the defects (holes) in the lamellae where packing is weak and water penetrates through these to swell the lamellae. The diffusion of the surfactant molecules along the myelin tube28 is expected starting from the root which necessitates the fluid nature of the hydrocarbon chains of the surfactant molecule; their assembly into concentric bilayered lamellar sheets proves the rigid nature of the hydrocarbon chains which is evident from the presence of (27) Brooker, M. H.; Jobe, D. J.; Reinsborough, V. C. J. Chem. Soc. Faraday Trans. 1984, 80, 73-86. (28) Sakurai, I.; Kawamura, Y.; Sakurai, T.; Ikegami, A.; Seto, T. Mol. Cryst. Liq. Cryst. 1985, 130, 203-222.

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a peak at 1126 cm-1 corresponding to trans in addition to kinked gauche conformations at the myelin root. The presence of the least intense peak around 1085 cm-1 in myelin proves that the larger proportion of hydrocarbon chains in concentric bilayer sheets of the myelin are of trans conformation. This is in line with compact packing of lamellar sheets in the myelin tubes as identified by cryoscanning electron microscopy.29 In the liquid crystalline phases or in the conformations that are liquidlike with the occurrence of relative chain disorder with predominant gauche conformers, a intense band with a broadened feature will be observed at 1448 cm-1.13 The intense peak near 1450 cm-1 in the lyotropic phases of AOT confirms the presence of gauche conformers which is indication of randomly oriented conformation with bending and kinking in the hydrocarbon chain, in turn this imparts fluidity to the constituent bilayers in lamellar phase and in myelin. Raman spectroscopic studies on various straight-chain hydrocarbons in triglycerides, soaps, and phospholipids showed that whenever relative chain order occurs in the crystalline state, such as in the triclinic chain packing where all-trans conformations of the chains predominate, the intense band in the methylene deformation/rocking region occurs between 1434 and 1444 cm-1 with a fairly intense shoulder band or doublet at approximately 1456 cm-1.13 The overlapping feature of the intense bands at 1449 and 1457 cm-1 in the hexagonal phase of AOT which is similar with the above peak assignment reveals the triclinic packing with all-trans conformation. The two hydrocarbon tails of AOT seem to split apart at lower concentrations13 which might be the cause for negative birefringence of myelin figures and is supported by the presence of both gauche conformers and trans conformers. The splaying of the hydrocarbon chains would increase the area spanned by them, which in turn increases the freedom for free movement and the probability to have either trans or gauche conformation, resulting in increased fluidity of the bilayers in myelin figures. The peak pattern in the C-H stretching region shows that the asymmetric stretching increases on formation of myelin figures from solid AOT which is an indication of increase in fluidity of the hydrocarbon chains and larger freedom for the methyl groups attached to the carbon skeleton. The anticipated increase in intensity of the 2898-2901 cm-1 band confirms the increase in the ordered arrangement of the surfactant molecules, whereas the increase in 2908-2916 cm-1 peak suggests the randomly oriented conformers in the myelin figures. The fluidity of the bilayers with sufficient rigidity in myelin figures and (29) Sakurai, I.; Suzuki, T.; Sakurai, S. Biochim. Biophys. Acta 1989, 985, 101-105.

Arunagirinathan et al. Table 3. Conformations of Hydrocarbon Chains and Conformations around CC-CS Bonda conformation around CC-CS conformation by CC stretching conformation by CH bending conformation by CH stretching

H

L+

Lo

M

G+T G+T G+T T

T G G+T T

T G+T G+T G+T

G+T G+T G+T G+T

a H, hexagonal phase; L , positive birefringent lamellar phase; + Lo, zero birefringent lamellar phase (myelin root); M, myelin microstructure. G, gauche conformer; T, trans conformer.

in other phases is supported by the C-H stretching frequencies. The coexistence of ordered as well as randomly oriented conformations of the hydrocarbon chains at the root and in myelin supports the passage of surfactant and water entering into the myelin through the root of the myelin and not through the tip, lateral, or longitudinal diffusion of surfactant molecules along or around the myelin tubes. 4. Conclusions Raman microscopy is used to investigate the myelin formation in AOT/water system. Comparison of the spectra in reverse hexagonal, lamellar, myelin roots, and myelins leads to the following conclusions on the conformations of the hydrocarbon chains and around the polar group (see Table 3). 1. Hydration accompanied by steric interactions determines the conformation around CC-CS bond in different lyotropic phases of AOT. 2. Spectrum arising from C-C skeletal stretching region shows drastic reduction in trans conformation of chains in myelin roots compared to other phases with a similar increase in gauche conformation. 3. Spectral comparison of lamellar and myelin phases shows similarities supporting the concept that myelin could be regarded as a rolled up cylinder of lamella. 4. Coexistence of ordered and randomly oriented conformations of hydrocarbon chains in the myelin figures supported by C-H bending and stretching frequencies signify the fluid nature of hydrocarbon chains in myelin. 5. Loose packing of molecules in the myelin roots in contrast to lamella indicate this to be a likely site for water penetration. Acknowledgment. We would like to thank the referee for constructive suggestions. The discussions with Dr. Ramola D’Cunnha and Dr. P. A. Hassan are gratefully acknowledged. We would also like to thank Dr. Dhanuka and Dr. Peter Garrett of Unilever for supporting this work. LA035898V