CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 3 751–754
Communications Synchrotron Radiation Microbeam X-ray Analysis of Microstructures and the Polymorphic Transformation of Spherulite Crystals of Trilaurin Satoru Ueno,* Takefumi Nishida, and Kiyotaka Sato Graduate School of Biosphere Sciences, Hiroshima UniVersity, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan ReceiVed July 4, 2007; ReVised Manuscript ReceiVed December 22, 2007 嘷 w This paper contains enhanced objects available on the Internet at http://pubs.acs.org/crystal.
ABSTRACT: Using synchrotron radiation X-ray microbeam diffraction, we investigated microstructures and molecular arrangements during solid-state β′ f β polymorphic transformation in two-dimensional spherulites of trilaurin grown within thin spaces between polyethylene terephthalate (PET) films. The two-dimensional spherulites of β′ were composed of nanometer-sized crystals in which the lamellar planes were oriented parallel to the radial direction of the spherulites. It was verified that, following solid-state transformation from β′ to β, the orientations of the long-chain axes of the β form remained unchanged with respect to those of the β′ form. This suggests that the molecular arrangements of trilaurin during the β′ f β polymorphic transformation occurred through template effects of the lamellar structures of the mother phase of β′. The crystallization of triacylglycerols (TAGs) has high significance in applications related to the food, cosmetics, and the pharmaceuticals industries.1–3 In general, crystallization of TAG is followed by irreversible polymorphic transformations from metastable forms such as the R form with hexagonal (H) subcell packing or the β′ form with orthorhombic perpendicular (O⊥) subcell packing, to the most stable form of β form with triclinic parallel (T|) subcell packing. Two types of polymorphic transformations may occur: solid-state transformation and melt-mediated transformation (melt mediation). During the solid-state transformation, the TAG molecules in R or β′ form change their molecular orientations and subcell packings. In contrast, melt mediation is the crystallization of the more stable forms through the melting of the less stable forms.4,5 Particular interest has been focused on the solid-state transformation mechanisms, including the variations in molecular orientation of the long-chain axes with respect to the lamellar plane and the subcell axes between R or β′ and β.4,6 However, little information on molecular-level understanding of the transformation mechanisms has been available due to the difficulty in growing single crystals of TAGs, both for the metastable and the stable polymorphs, unlike the cases of saturated fatty acid crystals.7 Another interest concerning solid-state transformation may involve the formation of spherulites of fats, which are the main causes of the deterioration of texture in fat-based products, for example, granular crystal formation in margarine8 and chocolate (fat bloom).9 It has been believed that the polymorphic transformation from β′ to β is related to the * To whom correspondence should be addressed. E-mail: sueno@ hiroshima-u.ac.jp.
formation of spherulites, but no microscopic information has yet been obtained to verify this. In this study, we report on the microstructure of spherulites of trilaurin, which are formed by crystallization and solid-phase transformation and which were measured by the synchrotron radiation microbeam X-ray diffraction method (SR-µB-XRD). This is the first study involving the structural analysis of the texture of fat crystals using an X-ray microbeam method. Indeed, there are no studies using SR-µB-XRD concerning food science except for the study of the microstructure of starch.10–12 Trilaurin, of more than 99% purity, was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and used without further purification. The sample was melted at 60 °C, and a liquid droplet was dropped on glass and quickly covered with a cover-glass. This sample set was placed in a temperature-controlled furnace, Linkam LK-600, and kept at 60 °C. Thermal treatment of the sample was carried out as follows. The sample was maintained at 60 °C for 5 min, cooled to 5 °C at a rate of 5 °C/min, kept at 5 °C for 30 min, heated to 40 at 5 °C/min, and kept at 40 °C for 30 min. The first cooling process caused the crystallization of β′ at 5 °C and the solid-state β′ f β transformation occurred during the second heating from 5 to 40 °C. Using an Olympus CX31 microscope system, polarized optical-microscope observations and SR-µB-XRD measurements were carried out at 5 °C for the β′ form and 40 °C for the β form. The SR-µB-XRD measurement was performed at beam line 4A of the Photon Factory, the synchrotron radiation facility of the High-Energy Accelerator Research Organization (KEK), Tsukuba, Japan. A microbeam was prepared by reflecting a synchrotron X-ray beam onto a K-B mirror and focusing the reflected beam on the sample position.13 The wavelength of the X-ray microbeam
10.1021/cg0706159 CCC: $40.75 2008 American Chemical Society Published on Web 02/08/2008
752 Crystal Growth & Design, Vol. 8, No. 3, 2008
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Figure 1. Polarized microscopic images of the two-dimensional spherulites of trilairin; (a) β′ form and (b) β form. Figure 3. (a) The relationship between the direction of arcs in the XRD data and the orientation of a lamellar plane, (b) the azimuthal angle dependence of the SAXS patterns of β′ taken at the center and offcenter (80 µm) in the sphelulite of trilaurin, (c) the same kind of graph in (b) for the β form.
Figure 2. SAXS SR-µB-XRD patterns of the spherulites of trilaurin taken by scanning the microbeam with a 20 µm step from the center to the periphery; (a) β′ form, (b) β form.
was 0.11 nm, and the focused beam size was 5 × 5 µm. The position of the microbeam was detected by moving a wire (40 µm diameter) perpendicular to beam direction, and always pointed on a monitor image on which the sample subjected to microbeam diffraction was shown using an optical microscope (magnification 100 to 200×).13 The sample was moved by an x-y-z stepping motor (1 µm step) while being observed by the optical microscope. A sample with a thickness of less than 1 µm was placed between thin PET films; the length between the sample and the detector was around 110 cm. Because of the difficulty in simultaneous measurements of the small-angle and wide-angle X-ray scattering, we only observed the small-angle X-ray scattering (SAXS) in the SR-µB-XRD experiments. Figure 1 depicts the polarized microscopic figures of the twodimensional spherulites of trilaurin of β′ (Figure 1a) and β (Figure 1b) taken under the crossed-Nicol condition. The two images were taken at the same position before and after the transformation. The typical Maltese-cross figure of the spherulite of β′ indicates that tiny crystals with dimensions from 500 nm to 2 µm in length and 50 nm in thickness were nucleated at the center of the figure, and the crystallization was extended toward outer spheres of the spherulite, as directly observed by in situ observations (Figure S1, Supporting Information). Raising the temperature from 5 to 40 °C produced the spherulite crystals of β depicted in Figure 1b following the solid-state transformation from β′. The β′ f β transformation occurred through the nucleation and subsequent crystal growth of many tiny crystals at various positions in the β′ spherulite within 40 s (Figure S2, Supporting Information). Al, the area shown in Figure 1b, was surrounded by many tiny rod-shaped crystals of β. Although the typical Maltese-cross figure observed in Figure 1a for β′ disappeared in β, the tiny crystals in β are oriented along the radial direction. The same result was also reported by Blaurock.6 However, using optical observations, it was impossible to determine whether the directions of the lamellar planes of the tiny crystals of β′ were maintained or randomized during solid-state transformation
into β. To clarify this, we performed scanning SR-µB-XRD analysis by shifting the X-ray microbeam from the center to the periphery of the spherulites of β′ and β as illustrated in Figure 1. Figure 2 depicts the SR-µB-XRD patterns of the spherulites of β′ and β taken by scanning the microbeam with a 20 µm step from the center to the periphery, as illustrated by arrows in Figure 1. A strong, almost circular-shaped β′ diffraction pattern appeared at the center position (Figure 2a), whereas strong arc-diffraction patterns appeared at positions away from the center. The long spacing values of the SAXS SR-µB-XRD at the center and off-center positions were all 3.22 nm, which was in good agreement with the value reported in previous literature (3.2 nm).14 Basically, the two arcs appeared along meridian directions, and no peaks were detectable along the equator directions at all of the off-center positions. For β, the long spacing of 3.16 nm was obtained at all positions, which also agreed well with that reported in previous literature (3.1 nm).14 The sharpness of the arc peaks was largely lost in β compared with β′, as illustrated in Figure 2b. For example, the intensity of the diffraction patterns of β at 40 and 60 µm positions was lower than that of β′. This may be ascribed to either disturbance in the molecular arrangement during the β′ f β transformation, or to radiation damage of the strong SR-µB during long-time exposure of the X-ray microbeam. However, it is clear that the directions of the strong arc peaks of β were almost the same as those of β′ when we compared the two patterns at the same SR-µB positions. Two movies (Movies 1 and 2) in mpg format of the crystallization and polymorphic transformation process are available. The dependence of the diffraction intensity of SAXS peaks on the azimuthal angle indicates the distribution of the lamellar plane direction of the trilaurin crystals present in the area of the spherulite that was subjected to SR-µB-XRD. As illustrated in Figure 3a, we can discern that the lamellar plane is directed normal to the connecting line of the two arcs, when sharp arc patterns were observed. The same result should be observed for β′ and β, although the angle of the chain inclination with respect to the lamellar plane differs by 4° between the two forms. Taking this into account, we calculated the azimuthal angle dependence of the intensities of the SAXS patterns of β′ and β taken at the center and off-center (80 µm) in the spherulites of β′ (Figure 3b) and β (Figure 3c). The intensity of the SAXS patterns taken at the center of β′ was not equal at all azimuthal angles, yet the diffraction peaks appeared in almost all directions. The same property was observed for β at the center position of the spherulite, although sharp peaks were detectable at the angles of 50° (strong), 120° (medium), and 260°
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Crystal Growth & Design, Vol. 8, No. 3, 2008 753 into two types: two sharp arcs like those at the 80 and 100 µm positions, and spot peaks like those at the 20, 40, and 60 µm positions in Figure 2b. Out of 120 patterns (as shown in Figure S4 of the Supporting Information), we counted that the sharp arcs amounted to 13, whereas the spot peaks amounted to 107. For arc peaks, the line connecting the two arcs at every position also makes a right angle with respect to the radial axis from the center, similar to the β′ spherulite for the spot peaks; the peaks are distributed along the direction roughly normal to the radial axis of the spherulite. We interpreted this result by taking the transformation mechanisms into account as follows. Solid-state β′ f β transformation occurs through nucleation and the subsequent crystal growth processes. The nucleation of β within the crystal of β′ occurs in accordance with thermodynamic stability, including the molecular processes of chain inclination with respect to the lamellar plane and conversion in the subcell packing from O⊥ to T|. Once the whole crystal of β′ is transformed to β, grain growth develops through volume diffusion of trilaurin molecules moving from the other β′ crystal particles. The direction of the lamellar plane of the transformed β may be the same as that of β′ when the β′ f β polymorphic transformation forming the β nucleus in a crystal may occur through template effects of the lamellar structures of the mother phase of β′. The above results, illustrating the same distribution directions of the SR-µB-XRD patterns of β and β′, were caused by this transformation mechanism. The differences between the SR-µB-XRD patterns of β two arcs or spot peaks, may be ascribed to the crystal sizes of β following transformation; small crystals whose lamellar directions are the same as those of β′ might exhibit the two-arc pattern, and large crystals formed by volume-diffusion controlled grain growth might reveal the spot peaks.
Figure 4. SAXS SR-µB-XRD patterns of the spherulites of trilaurin shown by a 40 µm step from the center to the periphery; (a) β′ form, (b) β form.
(medium) in Figure 3c. This may be caused by accidental crystallization of large single β crystals during the β′ f β transformation. In contrast, the crystals present at the 80 µm offcentered positions exhibited strongly anisotropic angular dependence both for β′ and β. In particular, two strong peaks from the β′ spherulite appeared at 87° and 267°, and the diffraction intensity at the other angles was quite low. Since the two patterns were taken for the same spherulite crystals before and after the β′ f β transformation, this result suggests that the molecular arrangements of trilaurin occurred in a well-ordered manner and that the lamellar planes of the β′ and β forms remained unchanged. Figure 4 depicts the SAXS SR-µB-XRD patterns taken at the 24 off-centered positions of the spherulite of β′ and β shown in Figure 1. Except for the pattern from the center position (red box), the SR-µB-XRD patterns of β′ are distributed in a perfectly radial manner so that the line connecting the two arcs at every position makes a right angle with respect to the radial axis from the center, as illustrated in Figure 4a. This regularity was confirmed in the SR-µB-XRD patterns of β′ taken at the 120 off-centered positions by scanning the microbeam with a 20 µm step (as shown in Figure S3 of the Supporting Information). For β (Figure 4b), although the SR-µB-XRD patterns of β were deteriorated compared to those of the β′ crystals in Figure 4b, the SR-µB-XRD patterns are classified
Acknowledgment. This work is partially supported by the following two project; the Ministry of Education, Culture, Sports, Science, and Technology (Grant-in-Aid, 18540406), and Food Nanotechnology Project, the Ministry of Agriculture, Forestry and Fisheries of Japan. The experiment has been performed under the approval of the Photon Factory Program Advisory Committee (Proposal No. 06G303). The authors (S.U. and T.N.) acknowledge Dr. Y. Nozue, Sumitomo Chemical Co., and Mr. Y. Shinohara, University of Tokyo, for their useful suggestion and kind help of the microbeam SAXS experiment. The authors also appreciate Prof. A. Iida, the station manager of beam line 4A at Photon Factory, KEK Institute, Tsukuba, Japan. Supporting Information Available: Two XRD figures are available free of charge via the Internet at http://pubs.acs.org.
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