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CHEMISTRY AND

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Chemistry and Physics of Lipids 82 (1996) 63 72

PHYSICS OF LIPIDS

Pressure effect on phase behavior of binary mixturesof cis-unsaturated fatty acids

Tohru Inoue a,*, Isamu Motoda ~, Nobuyasu Hiramatsu b, Masao Suzuki ~,
Kiyotaka Sato d
'~Department ~?! Chemistry, FaculO' t~f Science, Fukuoka ~)~iversity, Nanakuma, Jonan-ku, Fukuoka 814-811, Japan ~Department ~?/ Applied Physics, FaculO, ~?fScience, Fukuoka UniversiO', Nanakuma, Jonan-ku, Fukoda 814-80, Japan ~Oleochemical Research Laboratoo', Nippon Oil a;td Fats Company, Ohama, Amagasaki 660, Japan dFaculty of Applied Biological Science, Hiroshima Unit~ersity, Hi,,.,ashi-Hiroshima 724, Japan

Received 6 March 1996: accepted 22 April 1996

Abstract

The solid-to-liquid phase transition of binary mixtures of cis-monounsaturated fatty acids was investigated as a function of their composition under various pressures up to about 200 MPa by means of a high pressure differential thermal analysis. Two mixture systems were examined; one is the mixture composed of oleic acid (OA; c#-9octadecenoic acid) and palmitoleic acid (POA: cis-9-hexadecenoic acid), and the other is that composed of asclepic acid (APA; cis-I l-octadecenoic acid) and POA. The components in the former mixture differ in the length of cJ-chain (the aliphatic chain segment between the double bond and methyl end group) by two methylene units, whereas those in the latter mixture differ in the length of A-chain (the chain segment between double bond and carboxyl group) by two methylene units. In both mixtures, the temperature of solid-liquid phase boundary, i.e. solidus and liquidus lines, was drastically increased by the increase in pressure. On the other hand, the shape of the phase boundary remained almost unaltered regardless of the pressure. The characteristic features in solid phase of these fatty acid mixtures, i.e. the molecular compound formation for OA/POA mixture and the highly restricted mutual miscibility tbr APA/POA mixture, are retained even under high pressure. This indicates that the intermolecular interaction of cis-monounsaturated fatty acids responsible for determining the phase behavior of binary mixture systems is rather insensitive to pressure, at least in the range up to 200 MPa.
Kern'orals" Unsaturated fatty acids: Fatty acid mixture; Solid-liquid phase diagram; High pressure

* Corresponding author. Department of Chemistry, Faculty of Science. Fukuoka University, Nanakuma, Jonan-ku, Fukuoka 814-8t), Japan. 0009-308496/$15.00 <~ 1996 Elsevier Science Ireland Ltd. All rights reserved PII S(1009-3084196)02562- 5

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7". hToue et al. / CTtemistry and Physics c?f Lipids 82 (1996) 63 72

1. Introduction Phase science of unsaturated fatty acids has been attracting considerable attention in connection not only with the biophysics of membrane but also with the practical use of lipid materials in oil and fat technologies. Unsaturated fatty acids occupy about one half of fatty acid species involved in membrane-forming phospholipids, and the physico-chemical characteristics of the unsaturated fatty acids should be relevant to the physical properties and biological functions of biomembranes. In the practical applications of fats and fatty acids, the elucidation of phase behavior of unsaturated fatty acids in solid state is of great importance because of its significant influence on the physical properties of end products such as edible solid fats, confectionery fats, and cosmetics. Since ultrapure samples became available, the phase behavior of unsaturated fatty acids has been extensively investigated, in which are included oleic acid (OA; cis-9-octadecenoic acid) [1-3], asclepic acid (APA; cis-ll-octadecenoic acid) [4], palmitoleic acid (POA; cis-9-hexadecenoic acid) [5], petroselinic acid (cis-6-octadecenoic acid) [6-8], erucic acid (cis-13-docosenoic acid) [5,9,10], and so on. The most interesting phenomenon observed in these fatty acid species is the diverse polymorphism in solid phase. Two modifications of several polymorphic forms appearing in cis-monounsaturated fatty acids are named 7- and ~-forms [1]. In the low temperature form, ;,-form, both ~o- and A-chains have ordered all-trans conformation, while the high temperature form, ~-form, has a disordered to-chain retaining ordered A-chain [3], where ~o- and A-chains refer to the aliphatic chain segment between double bond and methyl end group and that between double bond and carboxyl group, respectively. It is a characteristic feature of the ~-modification that the ~o-chain has a 'liquid-like' nature even being in the 'solid' phase, In addition to the physical properties of pure fatty acid systems, attention has also been directed to phase behavior of binary mixture systems [11-13]. It is essential to clarify the phase behavior of fatty acid mixtures for fundamental

biophysical science as well as for practical applications of lipid materials, because, in these fields, we mostly treat the multicomponent lipid mixtures, and their phase behavior determined by the acyl-acyl interaction between different fatty acid species should participate in physical properties of supramolecular aggregates composed of fats and fatty acids. In previous works, we investigated the phase behavior of OA/POA mixture [12] and APA/POA mixture [13] in terms of a binary phase diagram constructed by a differential scanning calorimetry (DSC). The fatty acid components in the former mixture have the same A-chain but differ in the length of ~)-chain by two methylene units, while, in the latter mixture, the two components differ in zl-chain length by two methylene units having the same ~Jg-chain. The phase diagram obtained for the OA/POA mixture suggested that molecular compounds are formed in both ~- and 7-phases (the solid phases constituted from ~- and i'forms), the stoichiometry of which depends on the polymorphic form of the fatty acids [12]. The shape of the phase boundary also demonstrated that the molecular compounds are miscible with each pure component in the ~-phase, whereas they are perfectly immiscible in the ;:-phase. On the other hand, the APA/POA mixture provided a eutectic-like phase diagram, indicating that the two fatty acid species exhibit quite restricted miscibility even in ~-phase [13]. This difference in phase behavior in solid phase between the two mixture systems may be attributed to the difference in the intermolecular interaction among the fatty acid species between the two cases, which originate from the difference in the ~,~-chain length or A-chain length between the fatty acid components. The phase state of molecular assemblies is a function of pressure as well as temperature and, in the case of mixtures, the composition. The study of pressure effect on the phase behavior of lipid systems is of interest in relation to the physiological adaptation of barophilic organisms of ocean depth [14,15], the sensitivity of excitable cell membranes to pressure [16], the pressure antagonism of anesthetic action [17 19], and so on. In addition to these biophysical significance, the pressure

T. lnoue et al. / Chemistry and Physics q[ Lipids 82 (1996) 63 72

65

study on unsaturated fatty acid systems may provide some useful information to understand their rather complex phase behavior. From these points of view, we have carried out pressure studies on some unsaturated fatty acid species with a special attention focused on the pressure effect on the polymorphic transformation, using a highpressure differential thermal analysis ( D T A ) [ 2 0 22]. As an extension of this series of study, we intended to investigate the pressure effect on the phase behavior of unsaturated fatty acid mixtures. In the present communication, we report the binary phase diagrams obtained for OA/POA mixture and APA/POA mixture under various pressures up to about 200 MPa by means of a high-pressure DTA.

the sample was pressurized again to higher pressure, and the cooling-heating cycle was repeated. Pressure was raised up to about 200 MPa by repeating these procedures. The temperature difference between the sample and the reference material (silicon oil) during the temperature scan was recorded on an X-Y recorder as a function of the sample temperature by the use of Chromel/ Alumel thermocouple junctions.

3. Results and discussion

3.1, Construction of phase diagram ./)'om DTA curves

Typical DTA thermograms obtained with APA/POA mixtures of several compositions under various pressures are shown in Fig. 1. The small endothermic peaks appearing at lower ternperatures in pure POA and pure APA are ascribed to the ),-u transformation characteristic of these fatty acid species, while the large endotherms at higher temperatures are due to the melting of the ~-polymorph [21,22]. In mixture systems, the 7-~ transformation temperature was lowered drastically below the lower temperature limit of the present DTA apparatus (for detail of the ,.,-:~ transformation temperature as a function of the composition, see [21,22]). Thus, our attention was addressed only to the melting behavior of the ~-form as a function of pressure and the composition of the mixture. It can be seen in Fig. 1 that the endothermic peaks due to the melting of the u-form arc much broader for mixture systems than for pure components, indicating that the melting proceeds over a wide temperature range in the fatty acid mixtures. In addition, the temperature at which the melting occurs is shifted toward higher temperature by the increase in the pressure, while the shape of the endothermic peaks remains rather unaltered. The onset and completion temperatures of the melting were determined from these DTA thermograms as the temperatures corresponding to the intersections between the baseline and the tangential lines drawn through the descending and ascending arms of the melting DTA curves, and were plotted

2. Materials and methods Three fatty acid samples, OA, POA and APA, were supplied by Nippon Oil and Fats Company (Amagasaki, Japan). Their purities were confirmed to be better than 99.9% by capillary gas-liquid chromatography (Shimadzu GC-14A, column: SP-2560, Supelco Inc.). The fatty acid mixtures were prepared by weighing appropriate amounts of two fatty acid species into a glass vial to give a desired composition, followed by immersing the vial in hot water (50 60C) with gentle stirring in order to facilitate the homogeneous mixing of the two components. The mixture samples thus prepared were employed for DTA measurements. The solid-liquid phase diagrams of binary fatty acid mixtures under high pressure were constructed from DTA curves obtained at various pressures. A high-pressure DTA apparatus equipped with a DTA cell for liquid sample designed tbr high-pressure use has been described elsewhere in some detail [20,23]. The sample of" fatty acid mixture (approx. 0.1 ml) was introduced into the DTA cell and heated well above the melting temperature, and then was pressurized up to a desired pressure. After cooling far below the crystallization temperature, the sample was heated at the rate of lC/min to the temperature sufficiently above the melting temperature. Then,

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T. lnoue et al./ Chemistry and Physics qf Lipids' 82 (1996) 63 72

as a function of pressure; some examples of the plot are depicted in Fig. 2. It can be regarded that the solid and liquid are coexisting in the temperature range between the onset and completion temperatures of the melting, and hence, these temperatures define the boundaries among phase regions of solid, liquid, and their coexistence. Thus, the melting behavior of mixture systems obtained by DTA measurements as a function of the composition can be used to construct a solid-liquid phase diagram, For pure materials, the onset and completion temperatures of the melting should be, in principle, equal to each other, because in one-component system, the solid and liquid coexist only at a definite temperature under a given pressure ac-

~~
2 _ i __ _
0.1

X,pA:0 -:~ ,s6 XApA~0.23

. , ~ -

0 ~

XAPA:0.47 ,s~l6 50 ,0.

cording to the phase rule. However, experimental transition curves detected by DTA are usually accompanied by a finite temperature width, within which heat absorption continues due to the progress of melting, as is seen in Fig. 1 for pure POA and pure APA. The origin of this transition width observed even with pure materials is attributed to some undefined factors such as finite sample size, which causes a temperature gradient within the samples, and heat dissipation associated with a phase transition. This apparent temperature width resulting from the methodological origin may be kept approximately constant when the experimental condition and sample condition, such as the sample size, scanning rate, heat capacity of the sample, etc. are kept nearly constant. Thus, a convenient and frequently adopted method to construct a solid-liquid phase diagram of mixture systems is to correct the onset and completion temperatures of melting for mixtures by formally subtracting the weighted average of the temperature width observed for pure components [24]. The solid-liquid binary phase diagrams of the present fatty acid mixtures were constructed by this procedure. The onset and completion temperatures of the melting at given pressures were read from such curves as shown in Fig. 2, and after correction for the transition width of the pure components, these temperatures were plotted as a function of the composition of the mixture to obtain the phase diagrams.
3.2, APA/POA mixture

~'~

XApA = 0.81

---~--'~
vo'l

6~'~ ~ ' ~ '1 XAPA:|.0 1,8

,F .... v
-20

~L__~}~/~ ,o~ 0.,~ ~0~-~ ~ ~ 20 J ~ 40 i i 0 Temperature(OC)

60

Fig. 1. DTA thermograms obtained for some APA/POA mixture systems under various pressures. The composition of the mixture is indicated in the figure by the tool fraction of APA, xae n. The pressure corresponding to the completion of the endotherm due to melting is also indicated in the figure; the pressure is expressed in MPa.

Fig. 3 shows binary phase diagrams of APA/' POA mixture obtained by the above-mentioned procedure under various pressures, in which the composition is expressed in terms of the mol fraction of APA in the mixture, XApA. The points corresponding to lower and higher temperatures define solidus and liquidus lines, respectively. The shape of the phase boundary obtained by the present DTA experiments at atmospheric pressure is in good agreement with that previously obtained by a differential scanning calorimetry (DSC) [13]. T h i s p r o v e s t h e applicability o f t h e present DTA method as well as DSC t o c o n s t r u c t t h e liquid-solid phase diagram.

T. Inoue et al. / Chemistrl' and Physics (~/' Lipids 82 (1996) 63 72

67

60

60

50 ~"
40 ~

XAPA---0

50
40

XAPA:0.60

30
. 20 10
0

30
20 10
0

-10 -20 0
60
I I I I

-10 -20 250

60
I I I I

50

100

150

200

50

100

150

200

250

50
40

XAPA=0.23

50
4O

XAp~

~'~ 30 ~ ~ [" 20 10
0

30 20 10
0

-10 -20 60 50
40

-10 I 50 I 100 I 150 I 200 250

1 -20 / 0 60

I 50

I 100

I 150

1 200

250

~ X A P A = 0 " 4 7

50
40

30
"~ 20 E [" 10
0

30
20 10
0

/APA = 1 .0

-10 -20 0
I I I I

-I0
-20 I l I 1

50

100 150 200 Pressure (MPa)

250

50

100 150 200 Pressure (MPa)

250

Fig. 2. Plot of the onset (' i) and completion (O) temperatures of the melting against pressure for several APA/POA mixtures with different compositions. The composition is indicated in the figure by the tool fraction of APA.

It can be seen in Fig. 3 that the temperatures of the solidus and liquidus lines increase remarkably

with the increase in pressure, whereas the shape of the phase boundary remain almost unaltered. The

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T. lnoue et al. / Chemistry and Physics q[' Lipids 82 (1996) 63 72

50-

-I

"-"

30 -

0 t
)
/ ~

[]

:
~%-4%"

200

10 -

-10

~. 1 0.6 0.8 1.0

0.0

0.2

0.4

XAPA Fig. 3. Solid-liquid binary phase diagrams of A PA/POA mixture at various pressures. Pressures in M Pa are 0.1 (.~"), 50 (A), 100 ([]), 150 (O), and 200 (V). Solid lines are phase boundaries calculated from the thermodynamic relations derived by applying the Bragg-Williams approximation to evaluate the non-ideality of mixing (for detail see text).

pattern of a solid-liquid binary phase diagram reflects the mixing behavior of the two components in both phases. An eutectic-type shape of the phase boundary for APA/POA mixture demonstrates a low miscibility of the two fatty acid species in the solid phase. A rather insensitive feature of the shape of phase boundary to pressure indicates that the mixing behavior of POA and APA is not influenced appreciably by pressure, at least, in the pressure range up to 200 MPa. The mixing behavior of different chemical species is, roughly speaking, understood in terms of the difference in the pair-interaction energies between the like-pair and the mixed-pair formed in

the mixture. When both pair-interactions are identical, the different species would mix randomly; this corresponds to an ideal mixing. The non-ideal behavior of mixing appears when the two pair-interaction energies become to differ from each other. If the mixed-pair formation is energetically more favorable than the like-pair formation, the two components would arrange in some ordered manner. Contrary to this situation, if the interaction energy of mixed-pair is higher than that of like-pair, domains in which either of the two components is richer than the other would appear in the mixture, and as the limiting case, demixing of the components, or phase separation, would occur.

T. Inoue et al. / Chemistry and Physics oJ Lipids 82 (1996) 63 72

69

The phase diagram of the APA/POA mixture, which reveals highly non-ideal mixing behavior, was analyzed by evaluating the non-ideality of mixing within the framework of the BraggWilliams approximation [25]. According to this treatment, the temperature, T, at which the solid and liquid are coexisting in equilibrium is related to the composition of the two phases through non-ideality parameter, P0, by the following equations [24,26]: v (L' ~ALI) 2 - - p~0S)(1 -- X~AS)) 2 ___ ~ + p[(~(1 - ~ In .v},~/ RT AH A 1 1 ~- ( ~ - ~-~A)
-

I -

x~?

, , ' ~ ' ,~A ~ ' ~ ' ~ J -- p ~ , ~ ( x ~ ) ~ ~o

In I -

x~i~'~ + AH B 1

RT

1 ~ ( ~ - ~-~R)

values of non-ideality parameter of mixing: Tr, oA = 275.2 K, TApA = 287.0 K, zJHpo A = 32.1 k J m o l - J , A HApA = 39.8 kJmol t p l~l = _ 2.1 k J m o l - l , and pl s~ = + 5.0 kJmol ~. The resultant phase boundary is shown in Fig. 3 by solid line. The agreement between the experimental and calculated phase boundaries is satisfactory. Generally speaking, the heat of fusion and non-ideality parameter of mixing would both depend on pressure, although may be not so strong function of pressure as the melting temperature. We attempted to analyze the phase diagrams obtained at various pressures by assuming that A H and p. are invariant with pressure. The phase boundaries calculated based on this assumption are drawn by solid lines in Fig. 3 for phase diagrams under high pressure. The experimental phase boundaries are sufficiently well reproduced by the calculated ones at respective pressures. This suggests that the intermolecular interaction among these fatty acid species is rather insensitive to pressure in both liquid and solid phases, at least, within the pressure range up to 200 MPa.
3.3. O A / P O A mixture

In the above equations, XA represents the tool fraction of component A , A H A and A H B are the molar heats of fusion, TA and TB the melting temperatures, for respective components, R the gas constant, and superscripts (L) and (S) refer to the liquid and solid phases, respectively. The nonideality parameter is defined by
Po = Z(UAB U,4A + UI~I3)

2 where z is the first coordination number and UAA, UBR, and UA~ are the molar energies of A - A , B - B , and A - B pair interactions. When the heats of fusion and melting temperatures for each cornponent are known, and if the values of p(0L~ and p~0 s) are given, the above simultaneous equations can be solved to provide the relations between x~L~ and T (i.e. liquidus line) and between X(A s~ and T (i.e. solidus line). Usually, the heats of fusion and the melting temperatures are readily obtained from DSC experiments. The P0 values can be determined so as that the calculated phase boundary reproduces the experimental one most satisfactorily. As reported in a previous paper [13], the phase boundary of APA/POA mixture at atmospheric pressure was calculated from the above equations using the following thermodynamic data and the

The solid-liquid binary phase diagrams of OA/ POA mixture under various pressures are shown in Fig. 4. The characteristic feature of these phase diagrams is that the solidus and liquidus lines converge at XOA = 0.60 from both sides of the composition axis. This suggests the molecular compound is formed in solid phase between OA and POA, the stoichiometry of which is given by OA3POA2. The overall feature of the phase boundary can be regarded as a juxtaposition of an azeotropic-type phase diagram of the mixture composed of POA and the molecular compound OA3POA2 and a cigar-shaped phase diagram of the mixture of OA3POA2 and OA. The azeotropic-type phase boundary in the composition range 0 < XOA < 0.60 indicates that POA and OA~POA2 form a solid solution, although the mixing is highly non-ideal. In the region 0.60 < XOA --< 1, the solid solution is also formed between OA and OA3POA 2, the mixing of which is closer to ideal than the case of the mixture of POA and OA3POA2, as demonstrated by a cigarshaped phase boundary. The molecular corn-

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T. lnoue et al./ Chemistry and Physics of Lipids 82 (1996) 63-72

50 -

"~

_
j~I -

40

t_

~" 20 -

200

10 -

//~,

J 2

'

'

~q~

o
-10 0.0
,

.
i , I , I i I

~ s s~

L~O,1
1.0

0.2

0.4 xOA

0.6

0.8

Fig. 4. Solid-liquid binary phase diagrams of O A / P O A mixture at various pressures. Pressures in M Pa are 0.1 (), 50 (A), 100 ([~), 150 (O), and 200 (V),

pound formation and the solid solution formation ocurring in ~-phase of OA/POA mixture was also supported by an X-ray diffraction study [27]. The diffraction pattern corresponding to the short spacing was rather diffuse for OA/POA mixture of xoA = 0.33, reflecting the solid solution formarion. On the other hand, the sharp diffraction pattern was observed for the mixture of XOA = 0.60, indicating the formation of the molecular compound, OA3POA2. As is shown in Fig. 4, the temperature corresponding to the phase boundary is largely increased with the increase in pressure, whereas the shape of the phase boundary is almost independent of the pressure. Thus, it can be recognized again for OA/POA mixture that the pressure do not affect appreciably the intermolecular interaction among these fatty acid species, which is

responsible for the compound formation and the mixing behavior in solid phase. It is of interest to notice the pressure dependence of the melting temperature of the molecular compound. From the pressure dependence of the melting temperature, the volume change associated with the melting, A V, can be estimated with the aid of the knowledge of heat of fusion, A H, according to the Clausius-Clapeyron equation, dp 3H
dT TA V

The melting temperature of OA3POA2 is plotted in Fig. 5 as a function of pressure. From this plot, the value of d P / d T at atmospheric pressure was determined to be 5.18 MPaK-~ by a leastsquares curve fitting procedure, assuming that the melting temperature is a quadratic function with respect to the pressure. The molar heat of fusion

T. Inoue et al. / Chemistry and Physics o! Lipide" g2 (1996) 63 72

71

of OA3POA2 was obtained to be 165.2 kJmol ' from the DSC measurements at atmospheric pressure. Substituting these values and the melting temperature at atmospheric pressure into the Clausius-Clapeyron equation, the volume change for the melting of OA3POA 2 was estimated to be 114.0 cm3mol 1 It is suggestive to compare the A V value for OA3POA 2 with those obtained for respective fatty acid species, which have been reported previously [20,21]. The sum of the corresponding AV values for 3 tool OA and 2 mol POA is calculated to be 134.8 cm 3, while the A V value for OA3POA 2 is 114.0 cm3mol 1 as mentioned above. This means that the molecular compound OA3POA2 is packed in solid phase with excess void volume of 20.8 cm3mol 1 compared with the situation that OA and POA are packed separately in different phases. On the other hand, OA and POA differ in their acyl chain length by two methylene units, and the volume occupied by the two methylene units is estimated to

be 33 cm3mol 1 from the molecular weight and density data for OA and myristoleic acid (cis-9tetradecenoic acid) [28]. Thus, if the void space due to the chain length difference of the two fatty acids is kept in the molecular compound, the A V value for OA3POA2 would differ by 66 cm3mol -j from that expected for the case in which OA and POA are packed separately. The actual difference in A V, i.e. 20.8 cm)mol ', is considerably smaller than the above value. This suggests that OA and POA molecules are arranged in the compound in a manner so as to fill up the void space originating from the acyl chain difference between the two fatty acid species.

4. C o n c l u s i o n

40

3o
0 ~ .~ =

2o

In the present work, we investigated the effect of pressure on the solid-liquid phase diagrams of APA/POA mixture and OA/POA mixture. The results obtained for both mixture systems revealed that the increase in pressure induces large shift of the phase boundary toward higher temperature, while the shape of the phase boundary is not influenced appreciably by the pressure. On the viewpoint that the shape of the phase boundary, which reflects the mixing behavior of the two components, is governed by the difference in the pair-interaction energies between like-pair and mixed-pair formed in the mixture, the present obserwttion indicates that the intermolecuhtr interaction acting alnong cismonounsaturated fatty acid molecules is rather insensitive to the pressure.

Acknowledgements

so

too

~so

zoo

2so

Pressure (MPa)
Fig. 5. Plot of melting temperature of the molecular cornpound OA3POA 2 against pressure. Solid line is the best-fit curve obtained by a least-squares method fitted to a quadratic

This work was supported by Grant-in-Aid for Scientific Research (No. 06660168) from the Ministry of Education, Science and Culture, Japan, and by the Japan Private School Promotion Foundation. The support of the Central Research Institute of Fukuoka University for this study is also gratefully acknowledged.

function of pressure,

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T. lnoue et al. / Chemistry and Physics of Lipids 82 (1996) 63 72

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[14] A,R. Cossins and A.G. Macdonald (1989) J. Bioenerg. Biomembr. 21, 115 135. [15] J.R. Hazel and E.E. Williams (1990) Prog. Lipid Res. 29, 167-227. [16] K.T. Warm and A.G. Macdonald (1980) Comp. Biochem. Physiol. 66A, 1-12. [17] F.H. Johnson and E.A. Flagler (1950)Science 112, 9192. [18] J.R. Trudell, D.G. Payan, J.H. Chin and E.N. Cohen (1975) Proc. Natl. Acad. Sci. USA 72, 210-213. [19] H. Kamaya, I. Ueda, P.S. Moore and H. Eyring (1979) Biochim. Biophys. Acta 550, 131 137. [20] N. Hiramatsu, T. lnoue, M. Suzuki and K. Sato (1989) Chem. Phys. Lipids 51, 47 53. [21] N. Hiramatsu, T. Sato, T. lnoue, M. Suzuki and K. Sato (1990) Chem. Phys. Lipids 56, 59-63. [22] N. Hiramatsu, T. Inoue, T. Sato, M. Suzuki and K. Sato (1992) Chem. Phys. Lipids 61, 283-291. [23] N. Hiramatsu and T. Inoue (1988) Rev. Sci. Instr. 59, 671-672. [24] A.G. Lee (1977) Biochim. Biophys. Acta 472, 285 344. [25] W.J. Moore (1972) Physical Chemistry, Prentice-Hall, Englewood Cliffs, N J, pp. 229 278. [26] B.G. Tenchov (1985) Prog. Surface Sci. 20, 273--340. [27] N. Hiramatsu, K. Uchino, T. lnoue, S. Ueno, M. Suzuki and K. Sato (1994) Rep. Prog. Polym. Phys. Jpn. 37, 259 262. [28] D.M. Small (1986) The Physical Chemistry of Lipids, Plenum Press, New York, p. 589.