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J. Phys. Chem. 1992,96, 10514-10520
0.; Wennerstrbm, 0.Synrh. Mer. 1989,31, 163. (c) Pei, Q.; Qian, R. Electrochim. Acra 1992,37, 1075. (6)Burgmayer, P.;Murray, R. W. J. Am. Chem. Soc. 1982,104,6139. (7) (a) Francois, B.; Mermilliod, N.; Zuppiroli, L. Synth. Mer. 1981,4, 131. (b) Baughman, R. H.; Shacklette, L. W. In Science and Applicarions of Conducring Polymers; Salaneck, W. R., Clark, D. T., Samuelsen, E. J., Eds.; Adam Hilger: Bristol, 1991;p 47 and references therein. (8) (a) Kanazawa, K. K.; Diaz, A. F.; Geiss, R. F.; Gill, W. D.; Kwak, J. F.; Logan, J. A.; Rabolt, J. F.; Street, G. B. J. Chem. Soc., Chem. Commun. 1979,854.(b) Bull, R. A,; Fan, F. F.; Bard, A. J. J. Electrochem. Soc. 1982, 129,1009. (c) Genies, E. M.; Pcrnaut, J. M. Synrh. Mer. 1984,10, 117. (d) Burgmayer, P.; Murray, R. W. J. Phys. Chem. 1984,88,2515. (e) Miller, L. L.; Zinger, B.; Zhou, X.-Q. J. Am. Chem. Soc. 1987,109,2267.(f) Pa&. C. D.; Pickup, P. G. J . Phys. Chem. 1988,92,7002. (9)(a) Tsai, E. W.; Jang, G. W.; Rajeshwar, K. J . Chem. Soc., Chem. Commun. 1987,1776. (b) Krishna, V.; Ho, Y. H.; Basak, S.;Rajeshwar, K. J. Am. Chem. Soc. 1991,113, 3325. (10)(a) Kaufman, J. H.; Kanazawa, K. K.; Street, G. B. Phys. Rev. L r r . 1984,53,2461.(b) Orata, D.; Buttry, D. A. J . Am. Chem. Soc. 1987,109, 3574. (c) Slama, M.; Tanguy, J. Synth. Mer. 1989,28, C171. (d) Naoi, K.; Lien, M. M.; Smyrl, W. H. J. E/ecfrwna/.Chem. 1989,272,273. (e) Baker, C. K.; Qiu, Y.-J.; Reynolds, J. R. J. Phys. Chem. 1991,95,4446.(f) Naoi, K.; Lien, M. M.; Smyrl, W. H. J . Electrochem. Soc. 1991,138,440. (g) Prezyna, L. A,; Qiu, Y.J.; Reynolds, J. R.; Wnek, G. E. Macromolecules 1991, 24, 5283. (h) Bilger, R.; Heinze, J. Synrh. Mer. 1991, 43, 2893. (11) Dcakin, M. R.; Buttry, D. A. AMI. Chem. 1989,61,1147A. (12) (a) Baughman, R. H.; Shacklette, L. W.; Murthy, N. S.;Miller, G. G. Mol. Crysr. Liq. Crysr. 1985,118,253. (b) Murthy, N. S.;Shacklette, L. W.; Baughman, R. H. J. Chem. Phys. 1987,87,2346.(c) Okabayashi, K.; Goto, F.; Abc, K.; Yoshida, T. Synrh. Mer. 1987,18,365. (d) Yoshino, K.; Nakao, K.; On&, M.; Sugomoto, R. Solid Stare Commun. 1989,70,609. (e) Ywhino, K.; Nakao, K.; Morita, S.;Onoda, M. Jpn. J . Appl. Phys. 1989, 28,L2027. (f) Winokur, M. J.; Walmsley, P.; Moulton, J.; Smith, P.; Heeger, A. J. Macromolecules 1991, 24, 3812. (13)Timoshenko, S.J . Opr. Soc. Am. 1925,11, 233. (14)Berry, B. S.;Pritchet, W. C. IBMJ. Res. Dev. 1984,28,662. (15) (a) Tong, H. M.; Saenger, K. L. J. Appl. Polym. Sci. 1989,38,937. (b) Tong, H. M.; Saenger, K. L. In New Characterization Techniques for
Thin Polymer Films; Tong, H. M., Nguyen, L. T., Eds.; Wiley: New York,
1990; p 29. (16)(a) Stoney. G.G. Proc. R. Soc. London, Ser. A 1909,A82,172.(b) Brenner, A.; Senderoff, S . J. Res. Narl. Bur. Stand. 1W9,42, 105. (c) Hoffman, R. W. In Physics of Thin Films; Hass, G., Thun, R. E., Eds.; Academic: New York, 1966;Vol. 1 1 1, p 211. (d) Sahu, S.N.; Scarminio, J.; Decker, F. J. Electrochem. Soc. 1990,137, 1150. (e) Fahnline, D. E.; Masters, C. B.; Salamon, N. J. J . Yac. Sci. Techno/. 1991,A9, 2483. (17) Jou. J.-H.; Chen, L.-J. Macromolecules 1992,25,179. (18) Yei, Q.;Inganiis, 0. Adu. Mater. 1992,4,277. (19)Jones, R.V. Bull. Insr. Phys. 1967,18,325. (20)(a) Heinze, J.; Stbrzbacb, R.; Mortensen, J. Ber. Bunsenges. Phys. Chem. 1987,91,960.(b) Zotti, G.; Schiavon, G. Synrh. Mer. 1989,31,347. (c) Baudoin, J. L.; Chao, F.; Costa, M. J. Chim. Phys. (Paris) 1989,86,181. (d) Odin, C.; Nechtschein, M. Phys. Rev. Lett. 1991,67,1 1 14. (e) Odin, C.; Nechtschein, M. Synrh. Mer. 1991,43,2943.(f) Odin, C.; Nechtschein, M. Synth. Mer. 1991, 44, 177. (21) (a) Fan, F. F.; Bard, A. J. J. Electrochem. Soc. 1986,133,301. (b) Iyoda, T.;Ohtani, A,; Shimidzu, T.; Honda, K. Chem. L r r . 1986,687.(c) Hirai, T.; Kuwabata, S.;Ikcda, 0. Chem. Leu. 1986,1243. Yoneyama, H.; (d) Shimidzu, T.; Ohtani, A.; Iyoda, T.; Honda, K. J. Electroanal. Chem. 1987,224,123. (e) Wernet, W.; Wegner, G. Makromol. Chem. 1987,188, 1465. (f) Bidan, G.; Ehui, B.; Lapkowski, M. J . Phys. D, Appl. Phys. 1988, 21, 1043. (8) De Paoli, M. A.; Panero, S.;Prospcri, P.; Scrosati, B. Elecrrochim. Acra 1990,35, 1145. (h) Li, F.; Albcry, W. J. J. Chem. Soc., Faraday Trans. 1991,87,2949. (i) Panero, S.;Prosperi, P.; Scrosati, B. Electrochim. Acra 1992,37,419. (22)Pei, Q.; Qian, R. C-MRS-International '90 Proceedings; Elsevier: Amsterdam, 1991;Vol. 3, p 195. (23) (a) Warren, L. F.; Anderson, D. A. J. Electrochem. Soc. 1987,134, 101. (b) Penner, R. M.; van Dyke, L.S.;Martin, C. R. J . Phys. Chem. 1988. 92,5274. (24)Carslaw, H. S.;Jaeger, J. C. Conduction of Hear in Solids, 2nd 4.; Clarendon: Oxford, 1959;Chapter 11. (25) Pei, Q. Ph.D. Thesis, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 1990. (26)Spiegel, M.R. Theory and Problems o f b p l a c e Tramform;Schaum Publishing: New York, 1965;p 223.
Phase Behavior of Mixed Systems of SOS and OS0 T. Koyano, I. Hachiya, Confectionery R&D Laboratories, Mevi Seika Kaisha Ltd., 5-3-1, Chiyoda, Sakado, 350-02, Japan
and K. %to* Faculty of Applied Biological Science, Hiroshima University, 724, Higashi- Hiroshima, Japan (Received: May 4, 1992)
The phase behavior of mixed systems of SOS (sn-1,3-distearoyl-sn-2-oleoylglycerol)and OS0 (sn- 1.3-dioleoyl-sn-2stearoylglycerol) was studied with X-ray diffractometry and thermal analysis by using pure samples (>98.0%). A molecular compound was formed at the mixture of equal weight ratio, giving rise to two monotectic phases of SOS/compound and compound/OSO in a juxtapositional way. The stable form of the compound, &, has an interlamellar distance of 44.72 A of double chain length structure, which is in contrast to the triple chain length structure of the stable forms of SOS and OS0 having the same interlamellar distance of 65 A. &was formed through specific molecular interactions between stearoyl and oleoyl chains connected to glycerol groups: stearoyl chains at the 1,3-positions of SOS and at the 2-pition of OS0 are packed together in one leaflet of the double chain length lamellae, whereas another leaflet contains oleoyl chains of the two molecules. In the monotoctic region of SOS/compound, two crystals are present below the melting point of Be, 36 O C . Above that temperature, the stable crystals of SOS equilibrated with mixed liquid are present. In the monotectic region of compound/OSO, 8, is present with mixed liquid above the melting point of the stable form of OSO, 25 OC, below which two crystal fractions of the compound and OS0 a r e present. 8, was expected to have a congruent melting point, according to its unique melting and crystallizing behavior. The compound formation having the double chain length structure was also observed in the mixtures of pOP/OSO and POS/OSO,in which P is a palmitoy1 chain. A molecular model of the compound crystal is proposed, based on the molecular structures of SOS,OSO, and principal monounsaturated fatty acids.
Iaboduction Molecular packings in fats and lipids have important implications for their physical Propertiesin crystalline, liquid crystalline, and membrane states.' Biologically important fats and lipids are usually composed not only of different chain lengths but of saturated as well as unsaturated chains. These chains are attached to the same backbones, such as glycerol groups, exhibiting close structural proximity. Aliphatic chain-chain interactions in these
fats and lipids are critical in terms of the structurtfunction relationship.2 In the case of acylglycerols, the packing conditions are dependent on the chemical natures of fatty acid moieties: chain length, parity (odd or even), unsaturation (cis or trans), etcq2 Particularly, the moiecularstruchves and t h e r " i c behavior are remarkably dependent on whether the acyl chains contain cis unsaturation or not. This is due to the fact that the aliphatic
0022-3654/92/2096- 10514$03.00/0 0 1992 American Chemical Society
Phase Behavior of Mixed Systems of
The Journal of Physical Chemistry, Vol. 96, No. 25, 1992 10515
SOS and OS0
sos a( 2 )
+
Y ( 3 )+pseud~-fl'(3 )+@ 3 )+/3,(
3)
os0 a(2)+/3'(2)--fl(3) Figwe 1. Polymorphic transformations of SOS9and OSO'o ( 2 ) double chain length structure; (3) triple chain length structure.
compounds containing the cis-unsaturated chains reveal quite diversified molecular structures. This fact has been demonstrated by structural variations of many &-monounsaturated fatty acids in their crystalline states.'" Oleoyl chains are the most abundant unsaturated aliphatic chains in nature, Specifically, triacylglycerols (TAGs) mainly with oleoyl chains are employed as fat phases in confectionery fats, pharmaceuticals, cosmetics, etc. The oleoylcontahhg TAGS are often employed to increase flexibility or softness, which is caused by o l e f ~ molecular c interactions. The stnrctural propertie of TAG molecules are reflected in polymorphism.2 In fact, the polymorphic behavior of the TAGs containing olefdc group are quite complicated compared with monosaturated acid TAGs, which normally reveal three forms (a,fl, and B)? For example, trioleoylglycerol (OOO) has five forms? SOS (1,3-distearoyl-2oleoylglycerol) also has five polymorph^,^ and OS0 (1,3dioleoyl-2-stearoylglycerol)has three polymorphs.1° In order to elucidate the properties of olefinic molecular interactions in TAGs, it is necessary to elucidate phase behavior of binary mixtures of oleoyl-containing TAGs. Precise study, however, on this approach at a molecular level has so far been lacking. In this view, a combination of SOS and OS0 is quite interesting, because the oleoyl and stearoyl chains are connected to a glycerol bone in an opposite manner in the two TAGs. Namely, stearoyl and oleoyl chains are placed at sn-1,3- and sn-2-positions, respectively, in SOS,whereas vice versa in OSO. Therefore, specific chain-chain packing is expected to occur in the SOS/OSO mixtures. This may be rationalized since the polymorphic behaviors of SOS9and OSO'O are quite different as illustrated in Figure 1. There are irreversible polymorphic transitions, occurring from the least stable form (aform for both TAGs) to the most stable form (8' for SOS and /3 for OSO), depending on incubation temperature and duration. During this transition, the lateral molecular packing mode as well as interlamellar stacking sequence, which is revealed in the chain-length structure, changes as a resuit of intra- and intermolecularforces between aliphatic chains, olefinic group, glycerol groups, and methyl end groups. Rossel reviewed diversified literature on mixed systems of TAG molecules." Among many types of phase behavior, he claimed the occurrence of compound formation in particular sets of TAG mixtures such as SOS/OSS or POP/OPO (P = palmitoyl). In his original article,I2 Moran indicated two binary systems at a juxtaposition of POP/compound and compound/OPO, in which the compound is estimated to form at an equal concentration ratio. More recently, Engstrom reported the mixed system of SOS and S O (1,2-disttatoyl-3-oleoylglycerol), claiming the formation of a compound around 1:l concentration ratio.I3 He also referred to compound formation in the SOS and OS0 mixture, but no details was presented about its total phase behavior. In the present study, we report the phase behavior of SOS and OS0 using pure materials. Attention has been paid to a total phase behavior involving compound formation and polymorphic transformation, being based on the newest studies of the polymorphic structures of S0S9and OSO.lo The compound formation of SOS/OSO is suggestivefor materials design of fat blends having enhanced fluidity and plasticity to be employed in many applications such as foods, cosmetics, and so on.
Materials and Methods The eamples of SOS and OS0 were provided by Fuji Oil Co., Ltd. POP,POS,and SOS were obtained by fractionation and purification by preparative HPLC. OS0 was produced by intermterification of SSS (tristearoylglycerol) and OOO (triol-
-1 0
0
50
100
OS0 Concentration (%I Figure 2. Melting points of the least stable polymorphs of SOS/OSO mixed systems.
eoylglycerol) and purified with HPLC. The purities of POP and SOS were 99% and POS and OS0 were 98.0%. The crystal structure was characterized by wide-angle powder X-ray diffraction (XRD, Rigaku, Cu Ka,monochronized by a carbon monochrometer) and small-angle scattering goniometery (Rigaku, RINTl5OO system). The detection of a scattered X-ray beam was done with a conventional photon counting system, taking data collection periods of about 30 min for each 213 scan. The thermal properties were examined with a differential scanning calorimeter (DSC, Seiko Denshi, SSC5200 Robot system). A sample of about 1 mg was put in an aluminum pan, and standard calibration was made by using gallium. DSC experimental procedures were as follows: a mixed sample was prepared by measuring the concentration of SOS and OS0 in weight percent. Since the difference in molecular weights of two TAGS is quite small (SOS= 888 and OS0 = 886), the weight percent concentration almost equals the molar percent. The mixed sample was put in a DSC pan and melted at 60 "C in a DSC head for 20 min to make a fully mixed liquid. After melting, the DSC head was cooled to -30 "C at a rate of 20 OC/min and held at this temperature for 5 min to crystallize the polymorph having the lowest melting point, e.g., the least stable form. The melting point of the least stable form was then measured by rapidly heating to 60 OC at a rate of 20 OC/min, so that the transformation to more stable forms during the DSC scan is minimized. To obtain the phase behavior of thermodynamically stable forms, the polymorphic transformation of the mixed solid was induced by incubating the samples at various elevated temperatures after quenching the mixed liquid at -30 "C. DSC and XRD measurements were done at certain periods of incubation. The DSC heating rate for the stable forms was 2 "C/min. ReSlllt!3 Figure 2 shows the melting points of the least stable polymorphs of the SOS/OSO mixed system. The melting point decreased linearly with increasing OS0 concentrations in the range 0-4046. In contrast, the melting point did not change around -7 "C in a range of OS0 concentrations of 40-100%. The melting point of OS0 d ois -7 "C. From this, it follows that the melting behavior of the least stable polymorph of the SOS/OSO mixed system showed two different profila, a solid-solution type at lower concentrations of OS0 and a monotectic type at higher OS0 concentrations. Figure 3 shows the DSC heating thermograms of various mixed systems of the most stable polymorphs that were formed by thermal incubation at 20 OC for 1 month. Figure 4 shows the melting points and heat of fusion. The latter is a total enthalpy in case of multiple heating profiles. Two melting peaks were observed except for OS0 = 0,50, and 10096. Single melting peaks of 41 and 25 "C were obtained at OS0 = 0 and 1001, respectively. At the equal ratio of OS0 and SOS (to be referred to as 1:1, hereafter), single melting peak at 36 "C appeared. Hence, the melting behavior of the two regions below and above 50% of the OS0 concentration are quite different. In a range of OS0 < 50%, the values of the higher melting points decreased with
Koyano et al.
10516 The Journal of Physical Chemistry, Vol. 96, No. 25, 1992
'I
1
10
1
20
28 0
20
40
60
30
(deg)
Figure 5. X-ray diffraction spectra of SOS/OSO mixed systems (incubated at 20 O C for 1 month): (a) SOS = 100%; (b) SOS/OSO = 90/10; (c) SOS/OSO = 70/30; (d) SOS/OSO = 50/50; (e) SOS/OSO = 30/70; (f) SOS/OSO = 10/90; (g) os0 = 100%.
Temperature ( "C )
Figure 3. DSC heating thermograms of the most stable polymorphs of
SOS/OSOmixtures. The ratios of SOS/OSOare (a) 100/0, (b) 90/10, (c) 70/30,(d) 50/50, (e) 30/70, (f) 10/90, and (8) 0/100.
2.00
0.08
-
28
10
0
50
6.00
(deg)
Figure 6. Small-angle X-ray scattering spectra of SOS/OSO = 1/ 1. 70 I
;201 c
4.00
I
0
100
f
OS0 Concentration (%) Figure 4. Me!ting points (0,for SOS and OSO: 0, for compound) and
50
heat of fusion (AH, 0 ) of stable polymorphs of SOS/OSO mixed systems.
increasing OS0 concentration. By contrast, no change was seen for the lower melting points lying around 35 "C. In a range of OS0 > SO%, the higher melting points decreased with increasing OS0 concentration from 36 to 32 OC,and the lower melting points did not change around 25 "C. For comparison, the heat of fusion (AH) was m e a s d for single melting peaks: AH = 155.2kl/mol for SOS, AIi = 135.3 kJ/mol at 1:l ratio, and AH = 137.8 kl/mol for OSO. The AHvalues except for these three positions are the sum of two melting peaks. The X-ray spectra at several concentration ratios are shown in Figure 5 . For long spacing, the pure SOS and OS0 samples showed the spectra corresponding to 65 A, which equals to the long spacing values of f12 of SOS9and 6 of OSOIo having triple chain length structure. By mixing the two samples, new spectra corresponding to about 45 A appeared. The strengths of these peaks increased on approaching the 1:l ratio from both sides of SOS and OS0 pure states, at the expense of the strengths of the peaks of 65 A. At the 1:l ratio, single peak of 45 A appeared
30
0
50
100
OS0 Concentration (%) Figure 7. Long spacing values of SOS/OSO mixed systems at different concentrations.
in Figure 5d. This peak was precisely measured by small-angle X-ray scattering, showing a d value of 44.72 A shown in Figure 6.
For short spacing, the pure SOS showed the typical pattern of &, which is the second stable form.9 It is reasonable to form this polymorph under thermal treatment described above, since the most stable form of SOS PI is obtained after incubation at 40 "C for a week? The short spacing pattern of OS0 componds to @.lo In the all mixture states, a strong peak at 28 = 19.3" (4.6 A) was always observed. This spectrum is characteristic of a triclinic subcell structure.14 The spectra around 28 = 20-25" became rather complicated or scrambled upon mixing. At the
The Journal of Physical Chemistry, Vol. 96, No. 25, 1992 10517
Phase Behavior of Mixed Systems of SOS and OS0
21
I.
‘EI.
I
C
I
?.ee
4.60
3.70
0
a
(C
X
1
1
10
20
30
28 (deg) Figure 10. Changes of X-ray diffraction spectra of SOS/OSO = 30/70 at (a) 15 O C and (b) 27 O C .
20
10
1
30
2 8 (deg) Rgun 8. Changes of X-ray diffraction spectra of SOS/OSO = 90/10 during incubation at 23 O C : (a) after quenching; (b) 3 days; (c) 8 days; (d) 11 days.
0
10
20
30
Temperature ( O C
40
50
1
Figwe 9. Changcs of DSC heating thermograms of SOS/OSO = 90/10 after incubation at 23 O C : (a) just after chilling; (b) 3 days; (c) 8 days; (d) 11 days.
1:l ratio, however, a unique short spacing pattern was obtained, detinitely differing from those of B2 of SOS and b of OS0 as well. Figure 7 shows long spacing values of the mixture at different OS0 concentrations. It is clear that the value around 45 A always appeared upon mixing and that the value of 65 A was always available except for the 1:l ratio. To more detail the phaw properties, precise analyses of the polymorphic transformations were done for two mixtures with S0S:OSO ratios of 9010 and 3070,which were chosen because the phase behavior is different below and above the concentration of OS0 = 50%. For the 9O:lO mixture, the results of XRD and DSC studies are shown in Figure 8 and 9, respectively. The sample was thermally treated under the following conditions; the mixture was melted at 60 OC for 60 min and quenched to -30 OC. Then, the sample was incubated at 23 OC for 11 days to aocomplish the phase transformations. a was formed immediately after quenching, as shown in the XRD spectra having a strong peak at 28 = 21.2O (d = 4.2 A) (Figure 8a). The long spacing value is 53 A, which is close to that of a of SOS (48 A). Some additional XRD peaks in the long and short spacing areas are due to the Occurrence of more stable forms during sample handling and XRD measure-
ment. This occurrence would be avoided by using a high X-ray detection system. The crystallization of a was confirmed by the DSC heating scan just after quenching. In Figure 9a, the melting peak at 20 OC is of a form. The melting peak of a was followed by exothermic peaks around 25 OC and, subsequently, by a large endothermic peak at 37 OC. This is caused by solidification (exotherm) and melting (endotherm) of more stable forms, which occurred in the DSC pan during the heating treatment. The XRD and DSC patterns revealed remarkable changes during the incubation at 23 OC, indicating independent transformations of the SOS and compound fractions of the mixed system. In Figure 8a, the XRD short spacing pattern was found to be a superpositionof y of SOS and B of the compound (referred bo hereafter). y of SOS was characterid by strong short spacing at 4.73 and 3.88 A. The short spacing peak of 4.60 A is of 8,. Correspondingly,two different sets of long spacing spectra are seen in Figure 8b, being indicated by a solid arrow for the compound and by a broken arrow for SOS. The solid arrow corresponds to 45 A of 8, (see Figure 5). The broken arrow is due to a long spacing of 70 A, which equals y of SOS. In Figure 9b, a sharp endothermic peak corresponds to the melting of 8, and y of SOS,which Occurred almost at the same temperature. After incubation for 8 days, as shown in Figure 8c and 9c, the SOS fraction transformed from y to pseudo-@’and b2. The XRD short spacing pattern is quite complicated, yet it actually contains three forms: y and pseudo-@’of SOS and 0., The three forms are independently specified in the followin SOS y by 4.73 and 3.88 A, SOS pseudo-@’by 4.18 and 3.70%,9 and & by 4.60A. It is clear that the intensity of the peaks of pseudo-@’of SOS increased at the expense of y of SOS in accordance with the transformation. Long spacing peaks did not change, since the long spacing values of y and pseudo-@’of SOS are 70 A.9 The DSC thermograms also showed the melting peaks of y and pseudo-@’ (36 “C) of SOS and 8,. The melting of b2 of SOS at 41 OC is also shown, although the existence of b2 was not shown clearly in the XRD pattern. This means successive transformations of y pseudo-@’- p2 of the SOS fraction induced by DSC heating. The incubation for 1 1 days gave rise to more stable forms of the fractions of SOS and the compound as shown in Fi ures 8d and 9d. The long spacing (a broken arrow) equals 65 which is the same value as that of p2 of SOS? The XRD short spacing spectra correspond to b2of SOS and 8,. The DSC melting peaks were clearly separated for the two forms. It was confirmed, although not given here, that the XRD pattern of 8, disappeared, but b2 of SOS remained, when the sample was raised above 35 OC. Consequently, thermal and X-ray analyses of the mixture S0S:OSO = 9O:lO proved that two crystal fractions of SOS and the compound are present below the melting point of &, but the crystal of SOS equilibrated with mixed liquid is present above that temperature. For mixture S0S:OSO = 30:70,Figure 10 shows the XRD patterns of (a) the sample incubated at 15 OC after quenching the mixed liquid at -30 OC and (b) the sample taken at 27 OC after the incubation at 15 OC. The differences between the two data are t h a t the XRD short spacing pattern in (b) is more simplified than in (a) and that long spacing spectra of 65 A
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1,
10518 The Journal of Physical Chemistry, Vol. 96, No.25, 1992
sos
Koyano et al.
OS0
A
10
50
30 Temperature ( O C
1
Figure 12. DSC heating thermogram of SOS/OSO = 40/60 of /3 of OS0 and 8,.
< 50%, the crystals of f12 of SOS and 8, coexist below 36 OC. Upon heating above 36 O C , 8, melts and thereby & crystals of 0 50 100 SOS coexist with liquid containing SOS and OSO. It must be noted here that p2 of SOS is not the most stable form of SOS, OS0 Concentration (%) which is fll having melting point of 43 O C . So the phase behavior F¶gw 11. Phase diagram of SOS/OSO mixed system. L, mixed liquid; in the range OS0 < 50% displayed in Figure 3 does not exactly SOS, stable crystal of SOS; C, stable crystal of compound; OSO, stable correspond to thermal equilibrium conditions. This is because crystal of OSO. the thermal incubation carried out in the present study was not long enough to achieve the complete & b1transformation of denoted by broken arrows in (a) disappeared in (b), presenting SOS. Hence, it is expected that a full thermal incubation may the long and short spacing spectra identical with that of &. It raise the equilibrium solid-liquid line of A-B, particularly with is clear that fl of OS0 corresponds to the XRD spectra that decreasing the OS0 concentration, as shown in Figure 1 1. In the disappeared at 27 O C . The melting of OS0 0 is also confirmed region OS0 > 50% the crystals of and @ of OS0 coexist below by the immase in the baclrground of the XRD short spacing region 25 O C , which is the melting point of j3 of OSO. Upon heating due to the presence of a liquid fraction. Therefore, it is concluded above 25 OC, 8, is in equilibrium with the OS0 liquid, giving a that, at the ratio S0S:OSO = 30:70, two crystalline phases 8, liquidus line of B-C. There is a single-crystal phase of at the and fl of OS0 are present below 25 OC,which is the melting point position of B. According to the unique figures in the melting of /3 of OSO. Above 25 O C , 8, equilibrated with mixed liquid behavior and X-ray diffraction pattern, it is highly possible to is present. assume that 6,is fairly stable at position B in Figure 11, where Dircdon the solid and liquid phases having the same composition coexist. Hence, the 1 :1 compound of SOS and OS0 is expected to have Pbue Behavior. In general, three typical phases are possible a congruent melting point at 36 O C . in binary solid mixtures, when the two components are miscible To confirm this, we measured the exact concentrationsof the in all proportions in a liquid state: solid solution phase, eutectic compound and of either single component at diffmnt initial mixing phase, and compound formation. The third case is further divided concentrations. Theoretically, at a given initial concentration of into two, congruent or incongruent, depending on whether or not SOS/OSO = m/n, the ratio of SOS/compound equals to (m the compound and liquid at equilibrium keep the same concenn)/2n in the range OS0 < 5076, whereas the ratio of comtration ratio at the melting point of the compound. pound/OSO equals to 2m/(n - m) in the range OS0 > 50%. This Various examples in the solid mixtures of fats and lipids can relation was clearly reflected in the DSC melting profiles. For be categorized in these three phases, reflecting spacific molecular example, at SOS/OSO = 90/10, the ratio of SOS and 8, is interactions. The solid solution phase is formed in paraffins whose estimated as 79/21 from the calculated AH value of each form chain lengths differ by 2.15 Even-numbered saturated fatty acids (Figures 4 and 9). This value almost equals the predicted ratio differing by two carbon atoms appear to indicate the compound of SOS/compound = 80/20. Another example is shown in Figure formation with an incongruent melting point.I6 X-ray study 12, in which the DSC heating endothermic peaks of & and B of indicated a molecular model of a compound with a head-to-head OS0 are shown for the SOS/OSO = 40/60mixture. The melting arrangement in double chain length structure.17 In the mixtures of saturated and unsaturated molecules such as allianes-alkene~~~ peaks give rise to the ratio compound/OSO = 78/22, which is almost the same as expected value of 80/20. and stearic acid4eic acid,I6 no interaction occuf8 in the crystalline In the SOS/SSO mixture,13 Engstrom reported two eutectic state, giving risc to monotdctic or eutectic phases. For acylglycerol phases, which are placed in the juxtapositional manner with the mixtures, phase behavior is rather complicated for two main formation of the compound at a ratio of SOS/SSO= 1 :1. Four m n s : polymorphism and acyl chain compositions attached to liquidus lines are produced: SOS-liquid and compound-liquid the glycerol group. For example, in the mixture of monosaturated in the eutectic phase of SOS-compound and compound-liquid acid TAGS, a eutectic phase with a limited region of solid solution and SSO-liquid in the eutectic phase of compound/SSO. This is formed for the stable form, when the chain length difference makes a good contrast to the prostnt study, mainly caused by the is not larger than 2 carbon atoms. However, metastable forms low melting point of 6,. Another difference between SOS/SSO of a and fl exhibit solid solution phases.I1 Similar results were and soS/OSO is seen in the least stable form. In the SOS/SSO reported for the mixtures of optically active diacylglycer~ls.'~As mixture, a completely miscible isomorphous phase was observed to the acyl chain composition, no solubility of the monosaturated for a. However, such a property for a was seen in SOS/OSO acid TAG was obtained in a crystal of &monounsaturated acid in a limited concentration region at OS0 5 30%. These differTAG." An iutereaw study, however,reported recently that the ences are not fully understood presently but may be attributed of a double bond is to modify effect of the tram conf".ion to some differences between molecular packing accommodations the phase behavior through partial solubilization in the mixtures EEE-OOO and EEE-SSS (E = elaid~yl).'~ in SOS/SSO and SOS/OSO. Finally, the X-ray structural data of SOS/OSOreported by Engstrom are identical with the present The phase behavior of the stable forms of the SOS/OSO mixture is schematically drawn in Figure 11. The most important StmebnlModelafthecOrapold Foramaidcriugthepasible finding is that the 1:l ratio gave rise to formation of a new structure of the SOS/OSO compound, XRD long and short compound having a melting point of 36 OC and enthalpy of Lw spacing and entropy of fusion are available. The long spacing = 135.3 kJ/mol. This compound exhibited two mixture phases value of 44.72 A means the double chain length structure,in which with SOS or OSO, giving two liquidus lines of below (A-B) and one repeat lamellae consists of two acyl chains. From this, we the OS0 concentration of 5096. In the region OS0 above (M) 20
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The Journal of Physical Chemistry, Vol. 96, No. 25, 1992 10519
Phase Behavior of Mixed Systems of SOS and OS0
(a)
SOS
(b)
OS0
\\\\ ( c ) Compound Figure 13. Structure model of stable polymorphs of (a) SOS,(b) OSO, and (c) compound.
can simply model the double chain length structure of 8, as shown in Figure 13, together with & of SOS and 8 of OS0 both having the triple cham length structure. In the latter two forms, stearoyl and oleoyl chains are segregated to different leaflets, being formed by chain sorting during polymorphic transformation. In SOS and OSO, metastable forms of a having the double chain length structure fmt occur after chilling the liquid and converted to the morc stable forms having the triple chain length structure during thermal incubation as illustrated in Figure 1. It is notable to mention that chain segregation was more manifest in SOS than OSO, since the double chain length structure is revealed only in a of SOS,but it is realized in a and 8’ of OSO. By mixing SOS with OSO, geometrical matching of three portions in a molecule, the stearoyl and oleoyl chains and the glycerol group, would play a decisive role in determining the most stable structure. The cis conformationof the oleoyl chain makes a bent geometry, which produces serious repulsive interaction with almost straight stearoyl chains, when the aliphatic chain packing approaches the most stable conformation. Since the total number of stearoyl and oleoyl chains becomes equal in the compound structure like Figure 13c, the steric hindrance must be “izad. Hagemann and Rothfus recently discussed the possibility of reducing energy barriers necasary for a change from double to triple chain length structure in SOS.20 The long spacing value of &, 44.72 A, indicates that both oleoyl and stearoyl chains are inclined with respect to the lamellar plane as shown in Figure 13c. This is rationalized by comparison with long spacing values of a forms of SOS (48.3 A)9 and OS0 (52 A),’O which are both of the double chain length structure having the aliphatic chains arranged normal to the lamellar plane. We assume that the chain inclination of 3/, may be similar to 8 forms of SOS and OSO. To confirm this, we examined the compound systems in POP/OSO and POS/OSO.In each system, double chain length structure for the most stable polymorph was available at a 1:l ratio, giving long spacing values of 42.8 (POP/OSO) and 44.1 A (POS/OSO).It is simply expected from the models of Figure 13a,c, that the increase in long spacing value due to the increase in the length of the saturated acyl chain must be twice in the triple chain length structure that in the double chain length structure, in the case where chain inclination with respect to the lamellar plane is similar. This was clearly seen in 8, of POP OS0 and SOS/OSO, the difference in long spacing being 1.9 ,which is just half of that between 8’ forms of POP and SOS,4 A.9 For the subcell structure, the triclinic subcell is highly like1 to occur, since a very strong XRD short spacing peak of 4.6 was obsmed in 8,. This is the reason that the stable form of the compound was called &. The same XRD data were also obtained in & of POP/OSO and poS/OSO. However, no convincing data are available as to whether the subcells in the stearoyl and oleoyl leaflets of the double-chain lamellae are identical or not. We assume that the stearoyl leaflet may be of typical TI subcell. For the oleoyl leaflet, Engstrom assumed 0)in the oleoyl leaflet in Wll was first observed in the lowthe SOS/SSO comp~und.’~ temperature form2’(named yZ2)of oleic acid and also in y forms
x
K
of erucic acid (cis-13-docosenoic acid)?’ palmitoleic acid (cis9-hexadectnoic and asclepic acid (cis- 11-octadecenoic acid).25 In all of these four unsaturated fatty acids, y revealed the same XRD short spacing spectra, among which a very strong XRD short spacing peak of 4.7 A is mast characteristic. However, & did not reflect this peak (set Figure 5). Therefore, no conclusion as to the existence of the VI,subcell could be drawn now for the oleoyl leaflet in 8,. More precise analysis using spectroscopic methods is needed. It is notable to mention that well-defined single crystals of 8, were grown from the melt phasesz6 So, structure analysis using single crystals may be possible. In connection with the possible subcell arrangement of the oleoyl leaflet, we note here the fact that a lateral packing mode, expreaped in the subcell, and a total molecular configuration of cis-monounsaturated chain largely varied due to the influence of the double bond positions, as observed in cis-monounsaturated fatty acids. For example, 0’,1 was confirmed in y of oleic acid,” as referred above, Very recently, Kaneko et al. have codirmed the presence of other sub cell^'^ in three polymorphs of two unsaturated fatty acids, using single-crystal X-ray analy~es:~’Mi and 0, in the HM form and O1 in the LM form of petroselinic acid (cis-6octadecenoic acid)28and TI,in yl of erucic acid.29 In the case of the HM form of petroselinic acid, the subcell M, is revealed in the chain segment between the double bond and a methyl end group (methyl-sided chain), whereas the carboxyl-sided chain was of the 0, subcell. In acoordance with the Werence in the subcell structure, the codiguration of the olefin group is different in the above three polymorphs, giving rise to the difference in a total molecular geometry and chain inclination with respect to the lamellar interface. This feature means flexibility of the cismonounsaturated acyl chain in forming the molecular packing under intra- and intermolecular influences. From this, it is reasonable to infer that the actual molecular packing of the oleoyl leaflet in the 8, form would be more modified than that modeled in F w r e 13c. This is because there are strong influences from adjacent oleoyl chains of SOS and OSO, which are differently connected to the glycerol groups. This factor is more complicated than the difference in the double bond position in the unsaturated fatty acids exemplified above. Therefore, the model depicted in Figure 13c may be too simplified, since we assumed that the structures of the oleoyl chains are identical with that of oleic acid?’ Plausible modifications of F i p 13c are found in (a) the subctlls of the stearoyl and oleoyl chains as well as the configurations of the double bond groups of SOS and OS0 molecules, (b) the positions of the double bonds, which would not be in the same plane, (c) the stacking modes of the methyl end groups at the lamellar interfaces, and so on. All of thest problems are open to future studies. The ordering state in the crystal phase can be discwed on the basis of the entropy of fusion, AS, which is 0.44 (kJ/mol)/deg for 8,. This value is smaller than those of OS0 (8: AS = 0.46 (kJ/mol)/deg) and of SOS (&: AS = 0.49 (kJ/mol)/deg). Entropy of fusion indicates the difference of ordering between crystal and melt, so 8, is more liquidlike than OS0 @) and SOS (&). We assume that this would be attributed to difficulty in arranging compact packing of oleoyl chains in the same leaflet, because of its bending geometrical structure. Finally, the compound formation of SOS/OSO, &, is useful in forming fat blends that are more plastic and flexible than SOS and SSS. This is due to the fact that the oleoyl chain may provide slipping interfaces in the fat blends. Furthermore, the fact that the melting point of 8, at 36 O C is close to body temperature may also be useful. Some developments on this approach are in progress. Ackmledgmenr. We are indebted to Fuji Oil Co. for providing us pure samples. References and Notes (1) Small, D. M.J . Lipid Res. 1984, 25, 1490-1500. (2) Small, D. M. The Physical Chemistry of Upfds;Plenum: N e w York, 1986; pp 345-394. (3) Kobayashi, M.; Kaneko, F.;Sato, K.;Suzuki, M. J. Phys. Chem. 1986, 90, 6371-6378.
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(4) Sato, K.; Yoshimoto, N.; Arishima, T. J. Disp. Sci. Technol. 1989, 10, 363-392. (6) _ _ Yoshimoto. N.; Suzuki, M. (7) Hagemann, J. W. In Crystallization and Polymorphism of Fars and ; Dekker: New York, 1988; pp Farry Acfds;Garti, N., Sato, K., Us.Marcel 9-95. (8) Hagemann, J. W.; Tallent, W. H.; Kolb, K. E. J . Am. Oil Chem. Soc. 1972,49, 118-123. (9) Sato, K.; Arishima, T.; Wang, Z. H.; Ojima, K.; Sagi, N.; Mori, H. J . Am. Oil Soc. 1989,66,664-674. (10) Kodali, D. R.; Atkinson, D.; Redgrave, T. G.;Small, D. M. J . Lipid Res. 1987, 28, 403-413. (1 1) Rossel, J. B. Adu. Lipid Res. 1%7, 5, 353-408. (12) Moran, D. P. J. J . A D D ~Chem. . 1963. 13. 91-100. (13) Engstrom, L. J . Far zci. Technol. 1992. 94. 173-181. (14) Hernovist. L. In ref 7. DD 97-137. ( l 5 j Small: D.'M. In ref 2; 183-232. (16) Lutton, E. S. In Forty Acids; Markley, E. S., Ed.;Wiley: Now York, 1967; Part 4, pp 2583-2641; (17) Degerman, G.; von Sydow, E. Acra Chem. Scand. 1958, 12, 1176-1182.
ii
(18) Iwahashi, M.; Ashizawa, K.; Ashizawa, M.; Kaneko, Y.; Muramatsu, M. Bull. Chem. Sot. Jpn. 1984, 57, 956-959. (19) Desmedt, A.; Culot, C.; Deroanne, C.; Durant, F.; Gibon, V. J . Am. Oil Chem. Sot. 1990,67, 653-660. (20) Hagemann, J. W.; Rothfus, J. A. J . Am. Oil Chem. Soc. 1992, 69, 429-437. (21) Abrahammson, S.; Ryderstadt-Nahringbauer, I. Acra Crysrallogr. 1962, 15, 1261-1268. (22) Suzuki,M.; Ogaki, T.; Sato, K. J . Am. Oil Chem. Soc. 1985, 62, 1600-1604. (23) Suzuki, M.; Sato, K.; Yoshimoto, N.; Tanaka, S.;Kobayashi, M. J . Am. Oil Chem. Sot. 1988.65. 1942-1947. (24) Hiramatsu, N.; Sato, Y.;Inoue, T.; Suzuki,M.; Sato, K. Chem. Phys. Lipids 1990, 56, 59-63. (25) Yoshimoto, N.; Suzuki, M.; Sato, K. Chem. Phys. Lipids 1991, 5 , 67-73. (26) Koyano, T.; Hachiya, I.; Sato, K. Chem. Phys. Lipids, submitted for publication. (27) Kaneko, F.; Kobayashi, M.; Kitagawa, Y.; Matsuura, Y.; Sato, K.; Suzuki, M. Acra Crystallogr. 1992, C48, 1054-1060. (28) Sato, K.; Yoshimoto, N.; Suzuki, M.; Kobayashi, M.; Kaneko, F. J . Phys. G e m . 1990, 94, 3180-3185. (29) Kaneko, F.; Kobayashi, M.; Kitagawa, Y.; Matsuura, Y.; Sato, K.; Suzuki, M. Acra Crystallogr. 1992, C48, 1060-1063.
A Study of the Interaction of Bilirubin with Sodium Deoxycholate in Aqueous Soiutlons M. D'Alagni,+,M. J Delfini? L. Galantini? and E. Giglio*,t Dipartimento di Chimica, Universitb di Roma "La Sapienza". P.le A. Mor0 5. 00185 Roma, Italy, and Centro di Studio per la Chimica dei Recettori e delle Molecole Biologicamente Attive del C. N . R., c/o Istituto di Chimica, Universitb Cattolica, Largo F. Vito I , 00168 Roma, Italy (Received: May 14, 1992; In Final Form: July 31, 1992)
The solubilization of bilirubin-IXa (BR) in submicellar and micellar aqueous solutions of sodium deoxycholate (NaDC) was investigated by circular dichroism (CD) and nuclear magnetic resonance (NMR) measurements. BR exhibits a bisignate CD Cotton effect in micellar solutions of NaDC at pH about 8, thus indicating that NaDC interacts preferentially with the BR molecule having left-handed chirality. However, the CD spectrum changes as a function of pH and BR concentration. Moreover, the spectrum becomes inverted at NaDC concentration below the critical micellar concentration. Potential energy calculations accomplished for the system formed by one deoxycholate anion (DC-) and the left- or right-handed BR molecule (LBR or RBR, respectively) show that the DC--RBR complex is more stable than the DC-LBR one and are in agreement with the CD results. Interaction models are proposed and checked by NMR measurements. The influence of pH and BR concentration on the CD spectra, NaDC being in submicellar and micellar state, is discussed.
Introduction Previously, the helical structures of the sodium and rubidium deoxycholate (NaDC and RbDC, respectively) micellar aggregates were proved by wide and small-angle X-ray ~cattering,l-~ extended X-ray absorption fine structure,4$ electron spin resonance? nuclear magnetic resonance (NMR)2v6q7and circular dichroism (CD)8,9 measurements. The 2-fold helix of RbDC (Figure 1) can be considered to be derived from the 6-fold helix of NaDC by means of slight movements.'*2 The helices are very similar and have the same left-handed screw sense. Their radii are nearly equal, namely, about 10 A. The repeat of six deox cholate anions (DC-, Figure 2) along the helical axis is 11.59 (RbDC) and 11.75 A (NaDC), whereas the area of the helical basis in the unit cell of the crystals is 342 A2 for RbDC and 345 A2 for NaDC. The helix is characterized by an interior part filled with cations surrounded by oxygen atoms and is strongly stabilized by ion-ion interactions between cations and carboxylate ions, by ion-dipole interactions between cations and water molecules or hydroxyl groups, and by a close net of hydrogen bonds (Figure 1). Astonishingly, the exterior lateral surface of the helix is covered by nonpolar groups, such as the methyl groups CIS and C,9. Its solubility can be accounted for since the water molecules of the
1
UniversitH di Roma 'La Sapienza".
* UniversitH Cattolica.
solvent can approach the interior polar groups of the helix through both the helical bases and the lateral surface, owing to a sufficient separation between two adjacent DC- along the helix. The bichromophoric bilirubin-IXa (BR, Figure 3), cytotoxic yellow pigment of jaundice, has drawn much attention because of its biomedical importance in the pathophysiology of abnormalities in bile pigment metabolism.'O The structure and conformation of BR were experimentally investigated by X-ray,"-I3 NMR,Iel9 and resonance Raman spectroscopic study20as well as by energy calculations.2'v22Moreover, complexes of BR with a wide variety of optically active molecules, such as, for example, amines and amino alcohols,23alkaloids,24alb~min:~-*~cyclode~trins,~." and bile ~alts,8Sz~~ were extensively studied by means of the CD technique. Two interconverting enantiomeric "ridge tile" conformations, stabilized by six intramolecular hydrogen bonds (Figure 3). were observed in two BR crystal structures"J2 and were invoked in order to explain many NMR and CD results obtained in experimental conditions that do not support the breaking of the intramolecular hydrogen bonds. These conformations are predicted to correspond to the lowest energy minima calculated for the isolated molecule.22 Unfortunately, the factors that govern the enantioselective complexation of BR to other molecules are not completely well understood, although a reasonable interpretation of the BR chiral recognition has been p r ~ p o s e d ? ~Previously, ,~~ the complexes of BR with NaDC,8.32933
0022-3654/92/2096-10520$03.00/00 1992 American Chemical Society
