In-situ Synchrotron Radiation X-Ray Diffraction Study of Crystallization Kinetics of Polymorphs of 1,3-dioleoyl-2-palmitoyl glycerol (OPO)

and 0.5oC·min), and the dynamic polymorphic transformations were characterized on heating by simultaneously using Differential Scanning Calorimetry (DSC) and Synchrotron Radiation X-Ray Diffraction (SR-XRD) with smallangle X-ray diffraction (SAXD) and wide-angle X-ray diffraction (WAXD). Thermo-optical Microscopy (TOM) was also used in order to observe the polymorphic transitions. Polymorphic forms of OPO were identified and characterized. As the cooling rate decreased, more stable forms crystallized, not following the Ostwald step rule, 15


Introduction
Many crystals exhibit polymorphism, in which structural determination and thermodynamic stabilization of the polymorphic modifications are of primary significance to 20 determining the overall polymorphic nature of every substance. 1 In addition, the kinetic properties of crystallization and structural transformation are important, particularly for the application of polymorphic crystal systems in pharmaceutical, biomedical, food technology, and other 25 applications 2 . As for the crystallization of different polymorphic forms, the Ostwald step rule [3][4][5][6] has been used for macroscopic estimation of the behavior of multiple polymorphic forms when they are crystallized from vapor, solution, and melt phases. This rule dictates that less stable 30 polymorphic forms crystallize much faster than more stable forms when the driving force for crystallization takes place under certain values of supersaturation and supercooling and less stables forms transform to more stable forms after the crystallization. Actually, the kinetics of polymorphic 35 nucleation of various systems often follows this rule. For example, a metastable vaterite form crystallizes faster than the more stable calcite in CaCO3. 7, 8 However, more detailed studies are needed to examine the effects of supercooling, additives, etc. and becomes necessary to establish some rules 40 of behavior of the influence of kinetics on the polymorphic crystallization and study the situations where the Ostwald step rule may be complemented. Triacylglycerols (TAGs) are the main components of alimentary fats and oils. Fat structures and compositions 45 determine their physical properties 9, 10 (e.g., rheology, morphology, and texture), where polymorphism exerts a strong influence. Many studies have focused on the polymorphism of pure TAGs and binary mixtures [11][12][13][14][15][16] and the influence of kinetics on polymorphic behavior. [17][18][19][20][21] The 50 kinetics of fat crystallization is important to produce the desired product characteristics. Chong et al. 22 studied the influence of very low cooling rates on the crude palm oil crystallization in order to understand effects of cooling rates on its fractionation. Roelands et al. 23 compared theoretical and 55 experimentally determined nucleation rates in the precipitation processes of ionic and molecular compounds, and developed a standard procedure to measure them. Referring to isothermal crystallization kinetics, Foubert et al. 24 describes a stop-and-return DSC method used in fat 60 samples, which consists of stopping the crystallization at different moments during the isothermal crystallization and raising the temperature of the sample. However, Marangoni et al. 25 focused on nonisothermal nucleation of TAGs and developed a model to estimate activation energy for 65 nucleation in palm oil, milk fat, and other palm oil-based fats, defining the new parameter of supercooling-time exposure. Bouzidi et al. 26 used a model-free analysis of the kinetics of liquid-solid transformations for TAGs, and demonstrated the existence of critical rates of cooling and specific growth 70 modes. Moreover, Lam et al. 27 analyzed the influence of cooling rate on the formation of self-assembled fibrillar networks of 12 hydroxystearic acid (12HSA). The present study analyzes a TAG (1,3-dioleoyl-2-palmitoyl glycerol, OPO) that is important in palm oil fractionation, 28-30 75 as it belongs to low-melting temperature fractions. Palm oil is often modified to improve its applicability as an edible oil. Due to its heterogeneous composition, well-defined fractions with a high added value are obtained through fractionation. Basically, dry fractionation consists of separating successively 80 different crystal fractions obtained by controlled cooling from the melt. As the temperature decreases during fractionation, fractions richer in high levels of monounsaturated TAGs are separated. Minato et al. 12 studied the thermodynamic and polymorphic behavior of OPO and 1,3-dipalmitoyl-2-oleoyl 85 glycerol (POP) and their binary mixtures, showing the occurrence of α, β' and two β forms of OPO. However, Figure 1. DSC thermograms of OPO obtained by changing the cooling rate and keeping a heating rate of 15º C·min -1 . a) Cooling profile at a rate of 15º C·min -1 and heating profile; b) Cooling profile at a rate of 2º C·min -1 and heating profile; c) Cooling profile at a rate of 1º C·min -1 and heating profile; d) Cooling profile at a rate of 0.5º C·min -1 and heating profile. 5 kinetic properties regarding to the crystallization rates of four forms of OPO at different cooling rates were not studied. The present work is focused on the influence of a kinetic parameter (cooling rate) on the polymorphic crystallization of (OPO). In particular, the relationship between this behavior 10 and the Ostwald step rule is examined. Synchrotron Radiation X-Ray Diffraction (SR-XRD) has been applied to the polymorphic transformations of TAGs. 17,31,32 Using this technique and Differential Scanning Calorimetry (DSC) enables rapid thermal programs to provide highly 15 accurate structural information. However, few studies using SR-XRD have been conducted to determine the relative occurrence of the polymorphic forms of TAGs, since most work has been performed with an optical microscope, DSC, -0.4 ± 0.5 6.9 ± 0.4 and ex-situ (not on-line) XRD measurements with laboratory equipment after crystallization ceased. SR-XRD can facilitate fast cooling rate measurements because of its high-intensity X-ray beam; thus, it is interesting and valuable to examine the polymorphic crystallization of multiple forms of OPO. 10 This study characterized the polymorphic crystallization behavior of OPO by DSC, Thermo-optical Microscopy (TOM) and SR-XRD as a function of kinetics (variation of the cooling rates). 15

Experimental
The 1,3-dioleoyl-2-palmitoyl glycerol (OPO) used for thermal analysis (DSC and TOM) (99% pure without further purification) was purchased from Larodan Fine Chemicals (Malmö, Sweden). The 1,3-dioleoyl-2-palmitoyl glycerol used for SR-XRD was also 99% pure and it was obtained from 5 Tsukishima Foods Industry (Tokyo, Japan). The DSC profiles for both compounds were completely identical. DSC was performed at atmospheric pressure using a Perkin-Elmer DSC-7 calorimeter. Samples (9.0 to 9.4mg) were weighed into 50µl aluminum pans, and covers were sealed 10 into place. The instrument was calibrated by reference to the enthalpy and melting point of indium (melting temperature: 156.6ºC; ∆Hf: 28.45J· g -1 ) and decane (melting temperature: -29.7ºC; ∆Hf: 202.1J· g -1 ) standards. An empty pan was used as reference. Dry nitrogen was used as the purge gas in the DSC 15 cell at 23cm 3 · min -1 . Thermograms were analyzed with Pyris Series Software to obtain enthalpy (J· g -1 ) (integration of the DSC signals), Tonset (ºC), and Tend (ºC) of the transitions (intersection of the baseline and the initial and final tangent at the transition). 20 Samples were cooled at different cooling rates (15ºC· min -1 , 2ºC· min -1 , 1ºC· min -1 and 0.5ºC·min -1 ) from 35ºC to -80ºC and heated at 15ºC· min -1 . A minimum of three independent measurements was made for each experiment (n = 3). Random uncertainty was estimated with a 95% threshold of reliability 25 using the Student's Method, which enables estimating the mean of a normally distributed population when the population is small. For analysis that did not take place at a rate of 2ºC· min -1 , a correction had to be made, as the DSC-7 was calibrated at the before-mentioned rate. The following 30 expression was applied at each value: 33  Figure 1 depicts the OPO DSC thermograms (cooling and heating) obtained by changing the cooling rate and keeping the same heating rate of 15ºC·min -1 . Table 1 specifies Tonset for each transition. Crystal forms were identified by the SR-70 XRD patterns of simultaneous SAXD and WAXD measurements following the same DSC programs (Fig. 2).

Results and discussion
Long spacing values of α, β', β1, and β2 forms are known from the work of Minato et al. 12 Cooling rate: 15ºC· min -1 . On cooling, the polymorphic form 75 that crystallizes is α (less stable). Afterwards, a solid-solid transition takes place at -50.8 ± 1.6ºC (Table 1) to obtain another α form (α*), characterized by an appreciable double peak in the SAXD pattern (5.4 and 5.2nm) and a broad peak in the WAXD pattern (0.42nm). Ueno et al. 32 reported an α form 80 for SOS, characterized by a double peak in the SAXD pattern and they explained the transformation from the double SAXD peak into a single peak (characteristic of α) as a relaxation process of the α form. In the present work, we have called this During the heating process, the α * form transforms into α and melts. β' then crystallizes (identified by two peaks in the 5 WAXD pattern, with d-spacings of 0.43 and 0.40nm) and is transformed into the β1 form (exothermic transition, see Fig.  1). Finally, the β1 form, which is the most stable one, melts. These results are in agreement with the work of Minato et al., 12 who studied the polymorphism of POP:OPO binary 10 mixtures and observed the same behavior for pure OPO.
Cooling rate: 2ºC· min -1 . In the SAXD pattern, concurrent crystallization of α and β' forms is observed. However, on heating, the intensity of α peaks decreases, while that of β' peaks increases, possibly due to an α  β' transition. 15 Nevertheless, in the WAXD pattern, only β' crystallization is clearly noticeable. However, the first β' peak (0.43nm) is broader than when the cooling rate is 15ºC· min -1 , possibly due to an overlapping of the β' first peak and a smooth α peak. According to the DSC thermogram, on heating, a transition 20 from β' to β1 and β2 must take place simultaneously, as two melting endothermic peaks are present. Nevertheless, in both the SAXD and the WAXD patterns, only the β1 polymorphic form could be observed. Figure 3 illustrates a thermo-optical polarized microscopy 25 experiment using the same DSC program. In the temperature range of the solid-solid transition β'  β1 + β2, the sample, crystallized as spherulites, exhibited clear color changes, due to the solid-solid transition: before the transition it is possible to appreciate some brown and green colors ( Figure 3a) and 30 there are some changes to obtain some blue, yellow and brown tonalities (Figure 3b). However, it is not possible to distinguish between the newly formed β1 and β2 forms using this technique. Cooling rate: 1ºC· min -1 . On the one hand, the SAXD pattern 35 exhibits concurrent crystallization of α and β' forms in the cooling process, although the α peak does not become important and disappears immediately. On the other hand, the crystallization of β' is also present in the WAXD pattern, but is accompanied by some β1 crystallization. Therefore, both 40 SAXD and WAXD results indicate concurrent crystallization of α, β' and β1 forms. When the sample is heated, all β' is transformed into β1, but before this transition, some peaks of possible intermediate forms can be seen in the SAXD profile (7.1 and 6.1nm). We 45 must point out that only one endothermic peak with a shoulder is observed in the DSC heating thermogram, whereas the β'  β1 transition should be exothermic. This may be because the β1 melting process becomes so energetic that the convolution of both phenomena (β'  β1 transition and β1 50 melting) becomes a single endothermic peak in the DSC. profiles were similar to the one obtained by the cooling at 2ºC· min -1 whose details are explained in Figure 1b.
Overall, as the cooling rate decreases, the DSC profiles become simpler. This fact is represented in the triangular shape of Table 1. Also, Tend and enthalpy values were 65 determined for each transition, and some comparisons can be established. When the cooling rate is 15ºC· min -1 , α form crystallizes, giving an energetic peak; however, the α  α* transition is less energetic and much broader. Afterwards, the energy of the α melting is low when heating, and the 70 correspondent peak becomes very narrow. The energy for the following β' crystallization and β' β1 transition are about the same magnitude as for the α crystallization. Finally, the β1 melting is more energetic than all the previous phenomena, and the peaks become broader than that of the α 75 crystallization. With cooling rates of 2º, 1º, and 0.5ºC· min -1 , concurrent crystallizations take place, so the processes are more complex and energies are higher (100J· g -1 ), due to the overlapping of some phenomena, although the correspondent peaks are not so broad. The same happens during the heating 80 step with 1 and 0.5ºC· min -1 : as the endothermic peak is, in fact, a convolution of the β'β1 transition and the β1 melting, and energies become high. When the cooling rate is 2ºC· min -1 , the energy value for the transition β' β1 + β2 (heating step) is similar to that for the β' β1 transition with a cooling 85 rate of 15ºC· min -1 . Comparison of the two experiments also indicates that the energy value of the convolution of β1 and β2 melting (it was not possible to separate the two peaks) is the same as for β1 melting. Therefore, it can be concluded that β2 crystallization and melting are not as energetic as that of β1, 90 and the amount of β2 crystallized is not as high. The Tonset of β1 melting when the cooling rates are 15º and 2ºC· min -1 are  slightly different due to the fact that, at 2ºC· min -1 , the β1 melting peak is close to the β2 melting peak, so the β2 melting process makes some influence to change somehow the β1 melting value. However, at 15ºC· min -1 , the β1 melting peak is 10 completely isolated. All the crystallographic data obtained in order to characterize each polymorph are summarized in  20 According to the Ostwald step rule for polymorphic nucleation, the solid first formed on crystallization of a melt or a solution would be the least stable polymorph, which is first produced by spontaneous crystallization. As Ostwald stated: "When leaving a given state and in transforming to 25 another state, the state which is sought out is not the thermodynamically stable one, but the nearest in stability to the original state" and this must be the next least stable (the one with the smallest energy barrier expressed in kinetic/thermodynamic terms) 4 . However, some factors (e.g., 30 cooling rate) affect crystallization 26,27 and polymorphic nucleation. In this work, we have shown the influence of kinetics, so that as the cooling rate decreases, more stable forms crystallize, not following the Ostwald step rule. In most cases, concurrent crystallizations of different polymorphs 35 occur, but the tendency becomes clear. Therefore, this becomes an example where kinetics, thermodynamics and structural factors are competing. However, the Ostwald's Rule also states that the polymorph first formed is followed successively by forms of increasing stability. This fact has 40 been observed on the heating profiles of the experiments performed in the present work, as the phase transitions follow a sequence by continuously increasing the stability of the forms obtained. Nevertheless, further work would be required to determine the effects of changing heating rates on 45 polymorphism. Figure 4 summarizes the influence of cooling rate variation on the OPO polymorphism. The proposed diagram includes all the polymorphic sequences experimentally observed. As DSCs show, a more simplified polymorphic behavior is obtained with a decreased cooling 50 rate because the transformation sequence becomes shorter. Moreover, we studied the relation between the cooling and the crystallization rates of the β' form (Fig. 5). The variation of the SAXD peaks intensity of β' (at 4.5 or 4.4 nm), present during the cooling process (at three rates of cooling), is 5 represented in front of the supercooling (∆T = Tm-Tc), in which Tm is the melting temperature of β' form and Tc becomes each experimental temperature. It was evident that the SAXD peak intensity increased more rapidly as the cooling rate decreased above the ∆T values of 22°C. Although 10 the degree of perfection of crystals or crystallinity may vary the corresponding XRD peaks in that less perfect crystal diffract less X-ray beams, the intensity of the XRD peaks basically depends on the concentration of crystals present in the growth medium that were subjected to XRD measurement. 15 Therefore, the results (Fig. 5) may be related to the amount of β' crystals formed during different rates of cooling. It appeared that the rate of crystallization decreased with increasing cooling rate, since the peak intensity of the SAXD pattern decreased with increasing ∆T and cooling rate. 20 However, this was not the case. The amount of crystals is the summation of the crystallized materials in the supercooled liquid, resulting from both the rate of nucleation and subsequent crystal growth. The rate of nucleation is defined as the increase in crystal numbers per unit of time and unit of 25 volume of supercooled liquid, and the rate of crystal growth is defined as the increase in volume of crystals per unit time. As the rate of cooling decreases, the amount of time required for the growth medium to allow crystal nucleation and crystal growth to occur increases; therefore, the integrated amount of

Conclusions
DSC and SR-XRD clarified the polymorphic behavior of OPO as a function of kinetics. As the cooling rate decreases, the polymorphic crystallization is directed to more stable forms, 45 not following the Ostwald step rule. Nevertheless, concurrent crystallization often occurs in a complex phenomena. In more detail, when the cooling rate was 15ºC· min -1 , α form crystallized; at 2ºC· min -1 , α and β' formed; at 1ºC· min -1 concurrent crystallization of α, β' and β1 occurred; and when 50 the controlled cooling rate was 0.5ºC· min -1 , β' and β1 crystallized. Using SR-XRD coupled with DSC, rapid dynamic polymorphic transformations on the heating step could be identified. Also, intensities of the SAXD peaks have been related to supercooling for β' form, by comparing the 55 slopes of the graphs obtained at each cooling rate. Results indicate that the nucleation rate of β' tends to increase as the cooling rate decreases.