Synchrotron Radiation X-ray Diffraction Study of Liquid Crystal

Polymorphic crystallization of SOS (sn-1,3-distearoyl-2-oleoyl glycerol) has been studied with a time-resolved synchrotron radiation X-ray diffraction...
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J. Phys. Chem. B 1997, 101, 6847-6854

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Synchrotron Radiation X-ray Diffraction Study of Liquid Crystal Formation and Polymorphic Crystallization of SOS (sn-1,3-Distearoyl-2-oleoyl Glycerol) Satoru Ueno and Akiyoshi Minato Faculty of Applied Biological Science, Hiroshima UniVersity, Higashi-Hiroshima 739, Japan

Hideki Seto Faculty of Integrated Arts & Sciences, Hiroshima UniVersity, Higashi-Hiroshima 739, Japan

Yoshiyuki Amemiya Faculty of Engineering, UniVersity of Tokyo, Bunkyo-ku, Tokyo 115, Japan

Kiyotaka Sato* Faculty of Applied Biological Science, Hiroshima UniVersity, Higashi-Hiroshima 739, Japan ReceiVed: May 9, 1997; In Final Form: June 25, 1997X

Polymorphic crystallization of SOS (sn-1,3-distearoyl-2-oleoyl glycerol) has been studied with a time-resolved synchrotron radiation X-ray diffraction method (SR-XRD) using pure samples. SOS has five polymorphs: R, γ, β′, β2, and β1. Emphasis was placed on analyzing the events of melt-mediated transformation, in which the crystallization of more stable forms (γ, β′, and β2) was induced by rapid melting of the less stable forms. This mode of crystallization is closely related to a long running dispute concerning a “memory effect” possibly occurring in the polymorphic crystallization of triacylglycerols from liquid. Two types of thermal processes were applied: (a) a rapid heating soon after the least stable R form was crystallized from liquid and (b) a rapid heating after thermal annealing of the R form for 20 min before its melting. The SR-XRD measurements were monitored at a time interval of 10 s. The main results are as follows: (1) the formation of lamellar ordering of SOS occurred more rapidly than that of subcell packing, as exhibited in the earlier occurrence of SR-XRD long spacing spectra in comparison to the short spacing spectra, (2) the R-melt mediation without thermal annealing unveiled the formation of two types of liquid crystalline structures having long spacing values of 5.1 and 4.6 nm, (3) the γ form directly occurred in the R-melt mediation involving the thermal annealing, which generated γ-embryos in the solid phase of R to serve as seed materials, and (4) the formation of the second most stable β2 form of SOS was obtained after quite long incubation of the β′-melt mediation, yet the most stable β1 did not crystallize. The present study has given deeper insights of the polymorphism of SOS in comparison to previous studies with optical, calorimetric, and conventional XRD techniques: in particular the presence of the liquid crystalline form and the formation of γ-embryos were directly observed for the first time.

1. Introduction Polymorphism of triacyl glycerols (TAGs) is an important phenomenon which influences the physical chemical properties of fats employed in foods, pharmaceuticals, and cosmetics.1 Compared to monosaturated acid TAGs, in which three fatty acid moieties are all saturated fatty acids of the same type,2,3 saturated-unsaturated mixed acids (Sat-U-Sat) TAGs have been less understood. Special attention should be paid to Sat-U-Sat TAGs, since they are abundantly present in natural fats and oils. Especially sn-1,3-saturated acyl, 2-oleoyl glycerols (Sat-O-Sat) should be studied in single and mixed systems systematically, because they are major components of vegetable fats, which are attracting new interest in view of nutritional4 and industrial applications.1,5 Three fundamental subjects may be inherent to the polymorphism of Sat-U-Sat TAGs: (1) molecular-level structural analysis,6-8 (2) kinetics of polymorphic crystallization9-13 and (3) dynamics of polymorphic transformations in crystalline states.6 The second subject is related to the * Corresponding author: TEL +81-824-24-7935; FAX +81-824-227062; e-mail; [email protected]. X Abstract published in AdVance ACS Abstracts, August 1, 1997.

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melt crystallization techniques of multiple polymorphs of TAGs, which have widely been applied in confectionery and food industry.1,5 In this connection, molecular-level understanding of the polymorphism of various unsaturated fatty acids has been elucidated using advanced spectroscopic techniques in recent years.14-17 Koyano et al. reported kinetic properties of the melt crystallization of POP (sn-1,3-dipalmitoyl-2-oleoyl glycerol), SOS (sn1,3-distearoyl-2-oleoyl glycerol),10 and POS (sn-1-palmitoyl2-oleoyl-3-stearoyl glycerol),11 all of which are main component Sat-O-Sat TAGs in cocoa butter. Using polarizing optical microscopy, they examined two modes of melt crystallization: simple cooling and melt-mediated crystallization (melt mediation). In the former process, neat liquid was quickly cooled to crystallization temperatures (Tc). In the latter process, the crystallization of the more stable form was induced by rapidly melting the less stable forms. Striking differences were revealed between the two crystallization processes: (1) the occurrence of more stable polymorphs was enhanced in the melt mediation compared to the simple cooling, and (2) the rate of the meltmediated crystallization was much higher than that of the simple © 1997 American Chemical Society

6848 J. Phys. Chem. B, Vol. 101, No. 35, 1997 cooling when compared at the same Tc. The same behavior was carefully analyzed for POP, SOS, and POS by other researchers using a combined technique of optical microscopy and calorimetry.12,13 Two physical interpretations have been raised to account for the curious crystallization behavior: the presence of an ordering structure in liquid and embryo formation during the melt mediation. As to the first point, the presence of the ordering structure in the liquid state of TAGs has already been discussed for many years, since Larsson proposed the model representing a liquid crystalline state of smectic nature.18,19 Callaghan et al. supported the existence of the ordering structure in the liquid state of tristearin (SSS) by means of a 13C-NMR study.20 On the contrary, Cebula et al. have performed small angle neutron scattering experiments of neat liquid of trilauroylglycerol (LLL), claiming that no effect indicating the organized molecular aggregates of the smectic liquid crystals was observed.21,22 Instead, they postulated a molecular arrangement like a nematic phase of liquid crystals.22 In response to Cebula’s assertion, Larsson argued that the ordering structure in liquid of TAGs was like the L2 phase, because of the analogy between the XRD low-angle diffraction spectra exhibited by TAGs23 and L2 phases of polar lipids.24 As to the embryo formation, Sato25 speculated the formation of the crystal nuclei of the more stable forms in the crystals of the less stable form during the heating process. The embryos may serve as precursors of crystal nuclei which promote the melt-mediated crystallization. It has highly been anticipated to elucidate the polymorphic crystallization of TAGs with newer techniques, which may shed light on kinetic aspects of complicated crystallization from the melt phase, providing precise structural information. Recently, X-ray diffraction with the synchrotron radiation source (SR-XRD) has been applied to the dynamic processes of polymorphic transformations of TAGs. SR-XRD has the best facility to assess the rapid crystallization, providing highly accurate structure information. Kellens et al. studied tripalmitin (PPP), tristearin, and their mixture systems (PPP-SSS).26-28 The SR-XRD study of cocoa butter crystallization was examined by Gelder et al.29 The binary mixture systems of POP-PPP30 and POP-PPO (sn-1,2-dipalmitoyl-3-oleoyl glycerol)31 have recently been examined by the SR-XRD by the present authors. All of these studies have given fruitful information for the molecular-level understandings of the kinetics processes occurring in the polymorphic crystallizations and transformations of TAGs. In this paper, we report the dynamics of polymorphic crystallization of SOS examined by the time-resolved SR-XRD technique. SOS has five polymorphs (Table 1).6 The main aim of the present study was to clarify the mechanism of the meltmediated crystallization, since it is related to a so-called “memory effect”,32,33 which involves several meanings such as the presence of ordered structures in liquid (as mentioned above), seed crystals or embryos formed by the melting of preexisting solid phases, etc. The crystallization of the least stable R form from neat liquid by simple cooling and the occurrence of γ, β′, and β2 forms via R-melt mediation were examined on a time scale of 10 s by the SR-XRD technique. 2. Experimental Section The sample of SOS of more than 99% purity was supplied by Fuji Oil Co. and used without further purification. SR-XRD experiments were carried out at the beamline BL15A of the SR source which was operated at 2.5 GeV at the National Laboratory for High Energy Physics, Tsukuba, Japan.

Ueno et al. TABLE 1: Polymorphic Forms of SOS (001) long spacing (nm) ref 6a present

form

melting point (°C)

R

23.5

4.83

γ

35.4

7.05

5.3b 4.4 5.0c 7.3

β′

36.5

7.00

7.1

β2

41.0

6.50

6.6

β1

43.0

6.50

a

short spacing (nm)a ref 6 present 0.421(vs) 0.472(s) 0.450(m) 0.388(s) 0.430(m) 0.415(m) 0.402(s) 0.395(m) 0.383(m) 0.458(vs) 0.400(m) 0.390(m) 0.375(m) 0.367(m) 0.458(vs) 0.402(w) 0.397(w) 0.385(w) 0.380(w)

0.43(m)b 0.41(m) 0.42(m)c 0.47(s) 0.45(w) 0.39(s) 0.45(w) 0.44(w) 0.42(s) 0.38(s) 0.46(s) 0.40(m) 0.39(m) 0.38(m) 0.37(m)

vs, very strong; s, strong; m, medium; w, weak. b Figure 3. c Figure

6.

Figure 1. Instrument of X-ray diffraction with synchrotron radiation source.

The double focusing camera was operating at a wavelength of 0.15 nm. The SR-XRD instrument set up at BL-15A is shown in Figure 1. The X-ray scattering data were detected by position sensitive proportional counters (PSPC) for the small and wide angle positions at the same time. The distances between the sample and PSPC were 1.28 and 0.27 m for the small and wide angle positions, respectively. The sample was placed in the center of a stainless steel sample cell with kapton film windows, which was sandwiched in a brass jacket. The temperature of the sample was controlled by thermostated water circulating between the sample cell and the brass jacket. A quick temperature jump was performed by utilizing two thermostated water baths connected by a magnetic switch. Temperatures of the sample were measured by thermocouples dipped in the sample. The thickness of the sample was 2 mm. It should be noted here that the SR-XRD spectra were taken at 10 s intervals. However, in the present paper, all spectra were not reproduced in the figures, because the XRD patterns were too complicated to be read clearly. Two types of thermal processes were applied, as shown in Figure 2. The first process, Figure 2a, was performed as follows: quenching of the liquid from 50 °C to about 10 ( 1 °C, which is far below the melting point of the least stable form R (melting point, Rm: 23.5°C), and jumping to the target temperature (Tt > Rm), 5 min after the formation of the R form by the quenching. The second process, Figure 2b, involved

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Figure 3. SR-XRD spectra of the R-melt-mediated crystallization at Tt ) 30.0 °C without annealing.

Figure 2. Diagrams of the two types of thermal processes and melting temperatures of R (Rm), γ (γm), and β′ (β′m): (a) without annealing, (b) with thermal annealing at 22 °C.

TABLE 2: Rates of Temperature Jumping experiments

quenching

Figure 3

50 °C/min (50 f 10.5 °C) 48 °C/min (50 f 11 °C) 50 °C/min (50 f 11 °C) 49 °C/min (50 f 11 °C)

Figure 4 Figure 5 Figure 6

annealing

heating rapidly

14 °C/min (10 f 22 °C)

24 °C/min (10.5 f 30 °C) 24 °C/min (10.5 f 31.5 °C) 27 °C/min (11 f 33 °C) 12 °C/min (22 f 30 °C)

thermal annealing at 22 °C before jumping to Tt after the quenching at 10 ( 1 °C. The annealing time at 22.0 °C was 20 min. The rates of temperature changes for the quenching, annealing, and jumping are shown in Table 2. 3. Results Figures 3, 4, and 5 show the time-resolved SR-XRD data performed in the thermal processes denoted in Figure 2a with three different Tt values. In the three experiments, the common result was the formation of the R form by quenching around 10 °C. However, the behavior of the R-melt-mediated crystallization was dramatically influenced by changing the Tt values. Figure 3 shows the R-melt-mediated crystallization at Tt ) 30.0 °C. The R form was crystallized at 10.5 °C, showing the long spacing values of 5.3 and 4.4 nm and short spacing values of 0.41 and 0.43 nm. This feature of R was always observed in the SR-XRD data, soon after the liquid was quenched around 10 °C. However, the conventional XRD study revealed the long and short spacing values of 4.83 nm and 0.421 nm, respectively.6 Ten minutes after the temperature jump to Tt ) 30.0 °C, the β′ form started to crystallize, as revealed in two small angle diffraction peaks corresponding to the long spacing of 7.1 nm of the triple chain length of the β′ form.6 The wide angle diffraction peaks at 0.45, 0.44, 0.42, and 0.38 nm also correspond to the short spacing values of β′. It is interesting

Figure 4. SR-XRD spectra of the R-melt-mediated crystallization at Tt ) 31.5 °C without annealing.

to note that a lamellar structure with a long spacing value of 5.1 nm occurred, during the period after the R melting and before the β′ crystallization at 30 °C. In more detail, the small angle diffraction peaks of R immediately vanished by heating to 30 °C, and a new peak at 5.1 nm appeared at the expense of R. The value of the lamellar distance means the double chain length structure. In the wide angle region, the short spacing XRD spectra of R disappeared upon heating, and no peak appeared until the β′ form was observed, although the long spacing spectrum of 5.1 nm was clearly detectable. As the β′ form grew at 30.0 °C several minutes after the temperature jumping, the long spacing spectrum of 5.1 nm gradually decreased in intensity and eventually disappeared. This result was repeatedly confirmed by 10 experiments, in which Tt ranged from 24.5 to 30.5 °C. Therefore, we concluded that the presence of the liquid crystalline phase was unveiled during the R-melt mediation under the thermal treatments shown in Figure 2a. The liquid crystal thus defined is tentatively called LC1.

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Figure 5. SR-XRD spectra of the R-melt-mediated crystallization at Tt ) 33.0 °C without annealing.

TABLE 3: Occurrence of Lamellar Structure range of target temperature (°C)

lamellar structure (nm)

23.5 < Tt e 31 31 < Tt < 32.5

5.1

32.5 < Tt < 35.4

4.6

occurrence β′ form γ form β′ form γ form β′ form

Figure 4 shows the R-melt-mediated crystallization performed by the temperature jumping from 11 °C to Tt ) 31.5 °C. Although the R-melt mediation crystallized the β′ form, the results of Figure 4 are different from those of Figure 3 in three respects. In the first, the lamellar structure having the lamellar distance of 5.1 nm was not detectable. Secondly, γ appeared about 20 min after Tt was reached. This is evidenced by the long spacing spectrum of 7.3 nm and short spacing of 0.47 and 0.39 nm.6 Thirdly, the β′ form occurred several minutes after the occurrence of the γ form. The occurrence of γ and β′ was remarkably retarded, in comparison to the β′ crystallization shown in Figure 3. Figure 5 shows the third type of R-melt mediation with Tt ) 33.0 °C. A new type of liquid crystal was formed, and γ and β′ forms crystallized at the same time after the occurrence of this liquid crystal. In more detail, no diffraction peak was detectable during the initial 40 min after Tt was reached. After about 45 min, however, the new lamellar structure having the long spacing spectrum of 4.6 nm started to occur and increased its peak intensity with time, whereas no short spacing spectra were detectable until those of γ and β′ started to occur. The long spacing peak of 4.6 nm means the double chain length structure, corresponding neither to γ nor to β′, which are both of triple chain length. Similarly to the LC1 form defined in Figure 3, the liquid crystal structure with the long spacing value of 4.6 nm is called LC2. The long and short spacing spectra of γ and β′ started to appear 40 min after the LC2 form occurred. The results shown in Figures 3-5 are summarized in Table 3. It is clear that the behavior of the R-melt mediation was highly dependent on Tt and that two types of liquid crystalline structures were observed during the intermediate period after the R melting and before the occurrence of γ or β′. The absence of the liquid crystal formation by the R-melt mediation at Tt )

Figure 6. SR-XRD spectra of the R-melt-mediated crystallization of SOS at Tt ) 30.0 °C, with thermal annealing for 20 min at 22.0 °C.

31.5 °C (Figure 5) may mean that 31.5 °C is between the melting temperatures of LC1 and LC2, as will be discussed later. Figure 6 shows the result of the R-melt mediated crystallization, in which the annealing process for 20 min at 22 °C occurred the temperature jumping to Tt ) 30 °C. It is clearly shown that γ appeared during the annealing at 22 °C through the solid state transformation from R. The small angle diffraction peak at 7.3 nm means the triple chain length γ form, which increased in intensity at the expense of R (5.0 nm). Correspondingly, the wide angle short spacing diffraction peaks at 0.47 and 0.39 nm of γ increased with time at the expense of R (0.42 nm). After the jumping to 30 °C, R disappeared, and γ remained and continued to grow. Upon further heating to 50 °C, the γ form disappeared. The same results were obtained for the annealing temperatures of 20 and 20.5 °C, and Tt at 34 °C, although not shown. The result of Figure 6 suggests that the melt-mediated crystallization from R to γ through annealing is totally influenced by the γ-embryos. The γ-embryos were formed in the solid phase of R and served as seeding materials to crystallize the γ form after the melting of R. By contrast, no embryo was formed in crystals of R in the R-melt mediations without the annealing displayed in Figures 3-5. In the cases of Figures 3 and 5, the rapid melting of R formed the liquid crystalline phases, which might be precursors for the occurrence of the more stable γ and β′ forms. Concerning the structural changes in the R form during the thermal annealing (Figure 6), one may note that the two peaks of the long and short spacings gradually coincide to the single peaks of 5.0 and 0.42 nm, respectively, which are typical values of the R form observed by the conventional XRD.6 This means the relaxation process of the R form of SOS. As the final experimental finding, an interesting feature was observed in the order of appearance of long and short spacings during the melt-mediated crystallization. It was clarified that, irrespective of the polymorphic forms and the method of crystallization, the long spacing spectra first appeared before the appearance of the short spacing spectra, as shown for β′ in Figure 3, γ and β′ in Figures 4 and 5. In these cases, however, time lags between the long and short spacing spectra were minimized, because the total event of crystallization was quite

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Figure 7. SR-XRD spectra of β2 form occurring from the liquid formed by the β′ melting at 38.0 °C.

rapid. A more enhanced process was revealed in the crystallization of β2 from the liquid formed by the β′ melting (β′melt-mediated crystallization), as shown in Figure 7. After the melting of β′ (not shown), the (001) spectrum of 6.6 nm of β2 first appeared, soon followed by the (002) spectrum. This long spacing value means the triple chain length of β2.6 Over 20 min after the (001) spectrum was revealed, no short spacing spectra were detectable. The first appearing short spacing spectra was 0.46 nm, which was followed by four short spacing spectra. This result means that the arrangement of coherent net planes from the liquid formed by the melting of β′ is first completed in the stacking sequence of lamellar planes, and thereafter, the lateral packing within the lamellar leaflet is formed. This property was unveiled in the present study for the first time. 4. Discussion The present experiments have revealed newer aspects of the polymorphic crystallization and the formation of liquid crystals as well, which have never been unveiled by previous studies even involving the SR-XRD analysis of SSS and PPP.26-28 Four main points must be discussed here: (i) the formation and property of liquid crystals, (ii) the presence of embryos of the more stable forms prepared during the solid state transformation, (iii) the ordering sequence of lamellar stacking and subcell packing, and (iv) the structures of R. Liquid Crystal Formation. As to the liquid crystalline formation, LC1 and LC2 were characterized by their structural periodicity of 5.1 and 4.6 nm of the lamellar distances, respectively, with no definite periodicity in the lateral packing. This means that LC1 and LC2 correspond to the liquid crystals of a smectic type.34 The liquid crystals are defined as thermodynamically stable phases which have definite Gibbs energies comparable to those of crystalline phases and neat liquid as well.34 The occurrence behavior of small angle XRD spectra of the lamellar distances during different thermal treatments in Figures 3 and 5 indicated that SOS has two liquid crystalline forms as stable phases. As to the transformations from LC1 and LC2 phases to the neat liquid, only preliminary results are available. Figure 8a shows the “melting” of the LC1 form around 32 °C, as revealed in the

Figure 8. Melting behavior of LC1 at 32 °C: (a) SR-XRD spectra of the small angle region of the R-melt-mediated crystallization at Tt ) 33.0 °C without annealing; (b) a stepwise temperature variation of the SR-XRD measurement of part a.

small angle SR-XRD spectra taken during a stepwise temperature variation shown in Figure 8b. The first temperature rise from 11 to 25 °C (∼18 °C/min) caused the simultaneous occurrence of the LC1 and β′ forms at the expense of R. The LC1 and β′ forms maintained the spectral intensity when the temperature was kept at 25 °C. Upon the second temperature rise from 25 to 33 °C (∼14 °C/min), the spectrum of 5.1 nm of LC1 abruptly vanished at 32 °C, while the spectra of β′ maintained their intensity, although somewhat decreased, until it melts at 36.5 °C upon further heating. This indicates that the LC1 form transforms to liquid around 32 °C. Consequently, Gibbs energies (G) of LC1 and LC2 are tentatively illustrated in Figure 9, together with five polymorphic forms, R, γ, β′, β2, and β1. The five solid phases exhibit a monotropic nature;35 namely there are no crossing points of the G values among the polymorphic forms below their melting points. However, it seems that the LC1 and LC2 phases may reveal an enantiotropic nature with respect to the crystalline R form, because of their higher values of enthalpy and entropy. LC1 is more stable than the R form in a range of temperature of 25-31 °C. This is because the R-melt mediation without the thermal annealing caused the formation of LC1 at lower Tt values (23.5-31 °C). LC1 melts around 31-33 °C, as demonstrated in Figure 8. As to the LC2 form, it occurred at higher Tt values (33-35.4 °C) and transforms to the more stable crystals. No data of the transformation from LC2 to liquid were available, probably because the phase changes to γ and β′ are thermally activated, in comparison to those from LC1 to β′. The two liquid crystalline phases are less stable than γ and β′,

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Figure 11. Extended all-trans conformation of SOS: (a) regular conformation, (b) irregular conformation.

Figure 9. Phase stability of the five polymorphs and two liquid crystal forms of SOS.

Figure 10. SR-XRD spectra of the small angle region of the rapid cooling of isotropic liquid from 46 to 25 °C.

since the latter two forms were crystallized at the expense of LC1 and LC2. The activation free energies necessary for the transformations from the two liquid crystals to the polymorphic crystals must be so small, even compared to those from R to LC1 as well as from R to γ and β′, that it is quite difficult to maintain the forms of LC1 and LC2 in their single phases, in particular for the LC2 form. The phase stability depicted in Figure 9 must also be reflected in the crystallization behavior in the simple quenching of isotropic liquid, not alone in the R-melt-mediated crystallization. Such work was not fully performed in the present experimental series, mostly because of quite limited machine times available in the SR experimental station at Tsukuba. However, preliminary data were obtained by a rapid cooling of isotropic liquid from 46.0 to 25 °C, as shown in Figure 10. The rate of cooling was 19 °C/min. Upon cooling, the long spacing spectrum of 5.1 nm started to occur at 26.5 °C and increased its intensity with time at 25 °C, until the long spacings of γ and β′ appeared about 10 min later. No short spacing spectra were detectable

before the γ and β′ form crystallized, during the period when the long spacing of 5.1 nm was observed (not shown). This result was exactly the same as that displayed in Figure 3, where the R-melt mediation with no passage of the annealing of the R form let the LC1 phase occur. The two results of Figure 3 and Figure 10 indicate that the liquid crystalline phase having the lamellar distance of 5.1 nm is present in the supersaturated liquid of SOS at 25 °C as expressed in the tentative phase relationship of Figure 9. More precise experiments using the SR-XRD technique on the simple cooling of isotropic liquid as well as sophisticated melt mediation at different Tc values and different cooling rates are in progress, in an attempt to resolve the phase stability problems raised in the above discussion. As to the detailed molecular structures of LC1 and LC2, no convincing data except for the lamellar distance values are available. However, it may be claimed that the liquid crystalline phases may not be of the nematic type, as pointed out by Cebula et al.,22 because the long spacing spectra are quite strong and sharp in shape. According to the XRD study of the L2 phase of monoglyceride-water mixed systems, the long spacing peak was broadened and slightly shifted during the conversion from LR to L2.19,24 Similar variation was observed in Figure 3: for example, the long spacing spectra of the LC1 were decreased in intensity compared to the R form during the R-melt-mediated transformation to the LC1 phase. Further studies using polarizing microscopic observation, high-sensitive DSC, NMR, neutron diffraction using selected deuterized sample and microprobe polarized Fourier transformed infrared (FT-IR) spectroscopy, etc., should be needed to resolve the molecular structures of LC1 and LC2. The dimensions of the lamellar distance, 5.1 nm for LC1 and 4.6 nm for LC2, mean the double chain length structure, in which stearoyl and oleoyl chains are packed in the same leaflet. Figure 11 illustrates the molecular structures possibly present in the liquid crystals of the double chain length. Figure 11a assumes an extended all-trans conformation of the hydrocarbon chains, with the two stearoyl chains placed at the same side and the oleoyl chain at the opposite side. In this structure, the lamellar distance value may be around 4.8 nm. However, this assumption cannot be applied to LC1 and LC2, because the hydrocarbon chains must be conformationally disordered, not in an all-trans conformation, and thereby the chain length should be shortened. In this respect, it seems that LC2 has a reasonable value of long spacing, 4.6 nm, whereas the value of 5.1 nm of LC1 means some anomaly in the molecular conformation involving the glycerol groups, such as all three extended glycerol carbons as illustrated in Figure 11b. However, this structure gives rise to instability in the lamellar stacking, glycerol structures, and olefinic group. Further clarification is necessary to assess the structures of LC1 and LC2.

Polymorphic Crystallization of SOS Concerning the relative thermodynamic stability of the crystalline R form and liquid crystalline LC1 form (Figure 9), an important question may arise: why is liquid crystal LC1 more stable than the R crystal? The experimental results have proved this stability relation, since LC1 was formed after the rapid melting of R (Figure 3), and by the quenching of isotropic liquid at 25 °C which is above the melting point of R (Figure 10). No reasonable answer is given to this question yet. The R crystal of SOS contains conformationally disordered hydrocarbon chains involving the olefinic group, as revealed in FTIR8 and 13C-NMR36 studies. It is expected that this disordering may also occur in the LC1 form, and some other factors, such as higher entropy terms, may stabilize the LC1 phase at elevated temperatures. As to the generalization of the occurrence of the liquid crystal in the TAGs, two possibilities can be considered: (1) all TAGs have liquid crystals,: (2) the liquid crystals occur only in the mixed acid TAGs containing oleoyl acyl chains. No definite solution can be drawn yet, since the present work is the first to show the liquid crystal in SOS. We need more work, in particular on the other monosaturated acids TAGs such as PPP and SSS, to compare with the present result. γ-Embryo Formation. The R-melt mediation of SOS through the annealing process before the R melting has shown that the solid embryos of γ, formed via the R-γ solid state transformation, played the role of crystal seeding for the γ crystallization, as shown in Figure 6. This is the reason that only the γ form is crystallized after the annealing, whereas competitive nucleation of β′ and γ was induced without annealing. It was observed that the transformation rates of SOS in crystal are highly temperature dependent.6 The completion of the R-γ transformation occurs over 12 h at 15 °C. This rate is raised with increasing temperature, and vice versa. Therefore, the annealing at 22 °C, just below Rm, must activate the formation of the γ crystals, and in turn no γ-embryo is formed after the quenching at 10 °C, as observed in Figures 5 and 3, respectively. This process must also occur in the R-meltmediated crystallization of POS, which was reported quite recently.13 Ordering Sequence. As for the ordering sequences of the lamellar stacking and the lateral packing, it was shown that the stacking of lamellar structures occurred faster than the lateral packing formation (Figures 3, 4, 5, and 7), as revealed in time lags of the appearance of the XRD spectra between the long and short spacings. In more detail, the time lags are 10-30 s for γ and β′ and 20 min for β2. As to the R form, it seems that the two processes occurred simultaneously, since the time lag, if any, was less than 10 s, which is the maximum time resolution. This property may be interpreted in the following. van der Waals interactions may operate more effectively along the normal to the long chain axes than along the long chain axes, because of the rodlike shape of SOS. Therefore, the lamellar structure is formed in the first, and the stable subcell packing arrangements are stabilized next. r Form Structures. A final discussion is given to the strange diffraction peaks of the R form shown in Figures 3-6 and 8. Conventional XRD showed the sharp diffraction peaks of 4.8 nm (long spacing) and 0.421 nm (short spacing) (Table 1).6 Regardless of the differences in the absolute values, one may note that both the long and short spacings split in two peaks for the R form formed soon after the quenching at 10 °C: a sharp 5.3 nm and a broad 4.4 nm peak for the long spacing, and 0.43 and 0.41 nm peaks for the short spacing. However, the thermal annealing of R at 22 °C (Figure 6) and heating (Figure 8) to 25 °C caused the coincidence of the split peaks to

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Figure 12. Two types of R form: (a) “imperfect” R form, (b) “perfect” R form.

the single peaks for the long and short spacing spectra. This phenomenon can be interpreted as an ordering process from “imperfect” to “perfect” R forms, as illustrated in Figure 12. The “imperfect” R form may consist of the SOS molecules which are stacked and packed in several domains: two typical ones are illustrated in Figure 12a. Domains A contain the stearoyl chains in the one leaflet and the oleoyl chains in the other leaflet in a separate manner. This domain gives rise to two short spacing spectra in which the stearoyl leaflet may have smaller values than the oleoyl leaflet. By contrast, extended molecules like in Figure 11b are involved in domains B, exhibiting larger long spacing values than domains A. In the “perfect” R form (Figure 12b), the stearoyl and oleoyl moieties are packed at equal concentrations in the two leaflets of the double chain length structure, showing the single peaks of the long and short spacings. We infer that the isotropic liquid of SOS involves randomly oriented molecules in both the fatty acid chains and glycerol groups. Quenching of the isotropic liquid may first cause the formation of the “imperfect” R form. The “imperfect” R form did not convert to the “perfect” R form even after 30 min at 10 ( 1 °C. The “imperfect” R form, however, may transform to the “perfect” R form within 15 min during annealing at 22 °C, as observed in Figure 6, because of higher thermal activation energy. In this connection, one may note that the R form of the molecular compound of POP-PPO showed the splitting of the short spacing spectra, as revealed by the time-resolved SRXRD experiment.31 This splitting was also interpreted by the segregation of the palmitoyl and oleoyl leaflets in the double chain length structure. The SR-XRD study of tripalmitin showed that no splitting in the short spacing spectra of R was observed by quenching the isotropic liquid at 30 °C, which is far below the melting point of R (46 °C).27 This indicates that the “imperfect” R form may be caused solely in SOS, because of steric hindrance between the stearoyl and oleoyl moieties. Finally, there is an indication that the occurrence of the LC1 and LC2 liquid crystals is related to the presence of the “imperfect” R form, since no liquid crystals were detectable by the R-melt mediation after the “perfect” R form was formed. Further elucidation is needed to resolve this interesting problem. As a concluding remark, the differences between the previous SR-XRD experiments of TAGs,26-28 which did not observe the liquid crystal formation and the anomalous structure of R, should be commented on. Two interpretations are possible. The rates of temperature change of previous work, 1.25 °C/min, were low compared to the present work (12-50 °C/min) (Table 2). The

6854 J. Phys. Chem. B, Vol. 101, No. 35, 1997 thermal annealing actually occurred during the slow heating and may have veiled the occurrence of the unstable structures. The other possibility is that the anomalies observed in SOS are solely due to the steric hindrance between the stearoyl and oleoyl chains and, in turn, specific attractive interactions among the oleoyl chains, as revealed in the formation of molecular compounds of the mixed systems of POP-PPO,31 SOS-OSO,37 and SOS-SSO.38 References and Notes (1) Bailey’s Industrial Oil and Fat Products, 5th ed.; Hui, Y. H., Ed.; Wiley-Interscience Pub.: New York, 1996; Vol. 3. (2) Small, D. M. In The Physical Chemistry of Lipids; Plenum: New York, 1986; Chapter 10, pp 345-394. (3) Hagemann, J. W. In Crystallization and Polymorphism of Fats and Fatty Acids; Garti, N., Sato, K., Eds.; Marcel Dekker: New York, 1988; pp 9-95. (4) Fatty Acids in Foods and Their Health Implications; Chow, C. K., Ed.; Marcel Dekker: New York, 1992. (5) Formo, M. W. In Bailey’s Industrial Oil and Fat Products, 4th ed.; Swern, D., Ed.; John Wiley & Sons: New York, 1979; Vol. 1, pp 177-232. (6) Sato, K.; Arishima, T.; Wang, Z. H.; Ojima, K.; Sagi, N.; Mori, H. J. Am. Oil Chem. Soc. 1989, 66, 664-674. (7) Arishima, T.; Sagi, N.; Mori, H.; Sato, K. J. Am. Oil Chem. Soc. 1991, 68, 710-715. (8) Yano, J.; Ueno, S.; Sato, K.; Arishima, T.; Sagi, N.; Kaneko, F.; Kobayashi, M. J. Phys. Chem. 1993, 97, 12967-12973. (9) Sato, K. In AdVances in Applied Lipid Research; Padley, F., Ed.; JAI Press Inc.: Greenwich, 1996; Vol. 2, pp 213-268. (10) Koyano, T.; Hachiya, I.; Arishima, T.; Sato, K.; Sagi, N. J. Am. Oil Chem. Soc. 1989, 66, 675-679. (11) Koyano, T.; Hachiya, I.; Arishima, T.; Sagi, N.; Sato, K. J. Am. Oil Chem. Soc. 1991, 68, 716-720. (12) Rousset, P.; Rappaz, M. J. Am. Oil Chem. Soc. 1996, 73, 10511057. (13) Rousset, P.; Rappaz, M. J. Am. Oil Chem. Soc. 1997, 74, 693698. (14) Kobayashi, M.; Kaneko, F.; Sato, K.; Suzuki, M. J. Phys. Chem. 1986, 90, 6371-6378. (15) Kaneko, F.; Yamazaki, K.; Kobayashi, M.; Kitagawa, Y.; Matsuura, Y.; Sato, K.; Suzuki, M. J. Phys. Chem. 1996, 100, 9138-9148.

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