Original papers
Phase Behavior of Binary Mixtures of Tripalmitin, Triolein, and Trilinolein
2020 Volume 26 Issue 5 Pages 589-595
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2020 Volume 26 Issue 5 Pages 589-595
The phase behaviors of tripalmitin (PPP), triolein (OOO), and trilinolein (LLL) mixtures were investigated using differential scanning calorimetry (DSC). Samples were cooled from 85 to −80 °C for the PPP/OOO and PPP/LLL systems and from 50 to −100 °C for the OOO/LLL system and heated to the initial temperature. With respect to the PPP/OOO and PPP/LLL mixtures, the crystallization and melting temperatures of PPP decreased with decreasing fraction of PPP while those of OOO and LLL were almost constant at any fraction. It was presumed that pure or nearly pure PPP and OOO crystallized in an unstable form during cooling and transformed into the stable form during heating. In the OOO/LLL mixture, the crystallization and melting temperatures of OOO and LLL depended on their respective fractions. It was suggested that the two components crystallized almost independently in the PPP/OOO and PPP/LLL mixtures and interacted with each other in the OOO/LLL mixture.
Oils and fats are essential nutrients for humans and are consumed through diet. Vegetable oils produced by pressing plant seeds are used for cooking, food preservation, and as raw materials for food. More than 90% of vegetable oil components consist of triacylglycerols (TAGs), in which three fatty acids are ester-bonded to a glycerol skeleton (Gordon and Rahman, 1991). TAGs exist in polymorphs with different thermodynamic stabilities (Sato, 2001). They typically exhibit three polymorphs, called the α, β′, and β forms, which in turn have higher thermodynamic stability and melting points (Sato, 2001; Larsson, 1966). The melting point of TAG also depends on the number of carbon atoms and the degree of unsaturation of the constituent fatty acids (Himawan et al., 2006). For example, the melting points of tripalmitin in the α- and β-forms are approximately 45 °C and 66 °C, respectively (Wang, 2011; Hageman and Tallent, 1972). The melting point of the tristearin β-form is about 70 to 73 °C, and that of the trilinolein β-form crystal is −10 to −13 °C (Wang, 2011; Hageman and Tallent, 1972). Many aspects of the crystallization and melting behavior of a single TAG have been clarified. However, it is difficult to understand and predict the behavior of vegetable oil, which consists of many types of TAG. Minor components present in vegetable oils affect nucleation, crystal growth, and polymorphic phase changes (Himawan et al., 2006; Smith et al., 2011). The effect becomes more significant when the chemical structure of the minor component resembles that of the main component (Smith et al., 2011). To understand the crystallization behavior of vegetable oil in detail, it is important to understand the interaction among constituent TAGs and their crystallization behavior.
The crystallization behavior of a binary mixture of high-purity tristearin (SSS) and tripalmitin (PPP) has been studied (Himawan et al., 2007; MacNaughtan et al., 2006). The crystallization rate and polymorphism that obtained for pure TAG or nearly pure TAG were different from those for a mixture containing equal amounts of two TAGs (Himawan et al., 2007; MacNaughtan et al., 2006). The crystallization behavior of binary TAG mixtures containing saturated (S, P) and unsaturated fatty acids (O), such as SOS/OSO, SOS/SSO, POP/OPO, and POP/PPO mixtures, was also studied (Sato, 2001). When two TAGs were contained in equal amounts in these systems, a state called molecular compounds was formed (Sato, 2001). The phase behavior of a mixture of TAG and fatty acids was also reported (Costa et al., 2010). We previously studied the crystallization behavior of rapeseed oil during a constant cooling process using DSC (Miyagawa and Adachi, 2019). The peak observed during cooling showed a complex shape with multiple overlapping peaks. Therefore, the peaks were deconvoluted and the influences of the cooling rate on the crystallization temperature and peak area examined for each peak. The results suggested that TAGs of similar chemical structures crystallized simultaneously (Miyagawa and Adachi, 2019). We also investigated the crystallization behavior of some vegetable oils kept isothermally at low temperatures (Miyagawa et al., 2020, 2018; Yoshida et al., 2019). For soybean and rice bran oils, it was observed that TAGs with high melting points and those with low melting points crystallized independently (Miayagwa et al., 2020). On the other hand, TAGs of rapeseed, safflower, and olive oil crystallized together (Miyagawa et al., 2018; Yoshida et al., 2019).
Studies on the crystallization behavior of TAGs have been performed exclusively for TAGs containing palmitic and stearic acids, which have high melting points. There are few studies on the crystallization of mixtures containing triolein and trilinolein, which are contained in rapeseed oil, soybean oil, etc. In this context, the crystallization temperature, crystallization enthalpy, melting temperature, and melting enthalpy at various mixing ratios of two TAGs in a binary system of high-purity tripalmitin, triolein, or trilinolein were measured using DSC. The effect of the TAG chemical structure on the crystallization behavior is also discussed.
Materials Tripalmitin, 1,2,3-trihexadecanoyl-glycerol (PPP, purity ≥ 99%) was purchased from Sigma-Aldrich Japan, Tokyo. Triolein, 1,2,3-tri(cis-9-octadecenoyl)glycerol (OOO, purity ≥ 99%) and trilinolein, 1,2,3-tri (cis,cis-9,12-octadecadienoyl) glycerol (LLL, purity ≥ 99%) were purchased from Funakoshi, Tokyo.
Measurement of the DSC curves A sample (5 to 13 mg) was accurately weighed into an aluminum seal cell (Al6f1.5, Shimadzu, Kyoto, Japan), and the DSC curve was measured using a DSC-50 (Shimadzu). An empty cell was used as reference. The measuring section was cooled with liquid nitrogen using a LTS-50 (Shimadzu). Samples were prepared by mixing two of the three TAGs at various molar fractions. Each TAG in the reagent bottle was thawed and then stirred well. A certain amount was put in the cell using a syringe. The mixture of two TAGs was prepared by placing each TAG in a cell using a syringe and then mixing well. The DSC curves were measured for two samples with approximately the same molar fractions. The mixture of PPP and OOO or PPP and LLL was maintained at 85 °C for 10 min, and then cooled to −80 °C at a rate of 4 °C/min. Immediately after the temperature reached −80 °C, the sample was heated to 80 °C at a rate of 2 °C/min. After a mixture of OOO and LLL was kept at 50 °C for 10 min, it was cooled down to −100 °C at 4 °C/min and heated again to 50 °C at 2 °C/min immediately after the temperature reached −100 °C. Data were recorded at 1 second intervals. The heat flux during the cooling and heating processes was recorded to obtain the DSC curves. The peak area of the DSC curve was calculated using the peak area calculation function of OriginPro 2019 (Light Stone, Tokyo, Japan). A peak with two tops was split at the baseline into two peaks and the peak top temperature and enthalpy calculated. A peak with a shoulder was regarded as one peak.
Figures 1 to 3 show examples of DSC curves for PPP/OOO, PPP/LLL, and OOO/LLL mixtures, respectively. Figure 4 presents the peak top temperatures observed during cooling and heating for mixtures of various OOO or LLL fractions. The enthalpies were calculated from these peak areas (Fig. 5).
DSC curves for binary mixtures of PPP and OOO during (A) cooling and (B) heating. The letters a to k indicate the molar fraction of OOO: a: 0, b: 0.1, c: 0.2, d: 0.3, e: 0.4, f: 0.5, g: 0.6, h: 0.7, i: 0.8, j: 0.9, k: 1.
DSC curves for binary mixtures of PPP and LLL during (A) cooling and (B) heating. The letters a to k indicate the molar fraction of LLL, which are the same as those in Fig. 1.
DSC curves for binary mixtures of OOO and LLL during (A) cooling and (B) heating (B). The letters a to k indicate the molar fraction of LLL, which are the same as those in Fig. 1.
Peak top temperatures for exotherms (open symbols) and endotherms (closed symbols) observed during (A) cooling and (B) heating for the mixtures of (i) PPP and OOO, (ii) PPP and LLL, and (iii) OOO and LLL. The symbols, ◊, △, ○ and □, indicate different peaks.
Crystallization (open symbols) and melting (closed ones) enthalpies calculated from the peak areas during (A) cooling and (B) heating for mixtures of (i) PPP and OOO, (ii) PPP and LLL, and (iii) OOO and LLL. The symbols correspond to the respective peaks in Fig. 4.
Crystallization and melting behavior of PPP in the PPP/OOO or PPP/LLL mixture Exothermic and endothermic peaks with different heights, depending on the OOO or LLL fraction, were observed in both the PPP/OOO and PPP/LLL mixtures (Figs. 1 and 2). The peaks detected at higher temperatures are derived from PPP, and those noted at lower temperatures correspond to OOO or LLL. The peak top temperature Tp and enthalpy during the cooling and heating of PPP were similar in both PPP/OOO and PPP/LLL mixtures, except at the molar LLL fraction of xL = 0.9. Peaks with two tops were observed during cooling in the range of xO (molar fraction of OOO) or xL of 0 to 0.2. Although the enthalpies of these peaks were calculated separately, the total enthalpies of the two peaks were approximately 350 kJ/mol (xO = 0.09), 330 kJ/mol (xL = 0.12), and 320 kJ/mol (xO = xL = 0.16), respectively. Peaks with a shoulder were observed at xO or xL of 0.2 to 0.5 and a peak with two tops was observed at xL = 0.9. During heating, two endothermic peaks (Tp = 45.3 and 65.7 °C) and two exothermic peaks (Tp = 47.7 and 48.9 °C) were observed at xO or xL of 0 to 0.1 (Figs. 1B-a and 2B-a). Because the melting points of pure PPP in α- and β-form are 44.7 °C and 65.6 to 66.4 °C, respectively (Wang, 2011; Hagemann and Tallent, 1972), these endotherms and exotherms were caused by melting of the α-form, transitioning to the β-form, and melting of the β-form. No polymorphic transition occurred at higher xO and xL values.
Crystallization and melting behavior of OOO in the PPP/OOO mixture Pure OOO crystallized at a low temperature (Tp = −35 °C) during cooling and exhibited exothermic (Tp = −22.1 °C) and endothermic (Tp = 4.3 °C) peaks during heating. As the melting point of OOO in the β-form is 4.9 °C (Hagemann and Tallent, 1972), this endotherm is due to the melting of OOO in the β-form. Therefore, it is presumed that OOO produced an unstable crystal during cooling and then transformed to the β-form during heating. Except for pure OOO, the OOO fraction did not affect the Tp of the crystallization and melting peaks derived from OOO (Tp = −10 and 4 °C), and no polymorphic transition was observed.
Crystallization and melting behavior of LLL in the PPP/LLL mixture Pure LLL (xL = 1) did not show a distinct crystallization peak during cooling. It exhibited two exothermic peaks (Tp = −59.9 and −47.7 °C) and one endothermic peak (Tp = −11.9 °C) during cooling and heating, respectively. The exothermic Tp is not shown in Fig. 4. The enthalpy was also not calculated because it was difficult to determine the baseline. Since the melting point of LLL in the β-form is between −13.1 to −10.5 °C (Wang, 2011; Hagemann and Tallent, 1972), pure LLL did not crystallize or form an unstable crystal during cooling. However, LLL produced the β-form during heating. In the mixture, the LLL crystallization peak Tp decreased with decreasing xL from −22 to −28 °C. Between xL of 1 to 0.4, the Tp for the melting of LLL during heating was constant, independent of xL (Tp = −12 °C). A shouldered peak and peaks with two tops were observed at xL = 0.3 and between the xL = 0.2 to 0.1, respectively.
Crystallization and melting behavior of OOO and LLL in their mixture A major peak was observed for mixtures of xL = 0 to 0.8 in the Tp range of −40 to −65 °C during cooling. The peaks at Tp = −68 to −94 °C for the mixtures of xL = 0 to 0.3, and those at Tp = −95 °C for xL mixtures between 0.6 to 1 were very small (Fig. 5A-iii). During heating of pure OOO, once the exotherm was observed (Tp = −33.3 °C), the baseline shifted, and the endotherm started at about −20 °C (Tp = 4.3 °C). A similar exotherm was observed in the xL range of 0 to 0.5, while the Tp did not depend on the fraction. During the heating of pure LLL, two exothermic peaks (Tp = −80.3 and −46.8 °C) and two endothermic peaks (Tp = −52.6 and −11.9 °C) were observed. The LLL melting points of the α- and β-form are −49 °C and −13.1 to −10.5 °C, respectively (Hagemann and Tallent, 1972). Therefore, it can be concluded that the α-form transformed to the β-form via melting, and the β-form melted during heating. Similar polymorphic transitions were observed at xL = 0.6 to 1. In the xL range of 0 to 0.6, several small and complex changes in the DSC curves were observed at temperatures lower than −40 °C, but it was difficult to determine whether an exotherm or an endotherm occurred. The crystallization and melting behavior during heating of pure OOO and pure LLL were different from those in their mixtures. This difference was ascribed to the variation in the initial temperature increase.
Interaction between TAGs and their crystal structures In the PPP/OOO and PPP/LLL mixtures, the melting point of PPP in the β-form decreased with increasing xO or xL, while the melting point of OOO in the β-form did not depend on its fraction. The melting point of LLL in the β-form also did not depend on its fraction at xL = 0.3 or higher. Therefore, it is possible that the PPP crystal contained OOO or LLL, and its proportion depended on xO or xL. At xL below 0.3, the peak had two tops, that is, two melting points, suggesting that the crystal contained PPP. In the PPP/OOO mixture, the transition from the α- to the β-form was observed for nearly pure PPP or OOO. In other words, PPP or OOO directly crystallized in the β-form during cooling when its composition was not pure or nearly pure.
The melting point of OOO in the β-form decreased and that of LLL in the β-form increased for higher xL in the OOO/LLL mixture. The OOO in the β-form was formed during the heating process (xL = 0 to 0.5). The formation of LLL in the α-form and its transition to the β-form was observed at the xL of 0.6 to 1. These facts indicate that OOO and LLL did not crystalize separately, but that crystals consisting mainly of OOO and LLL were produced. The composition depended on their fraction. Palmitic acid is a straight-chain fatty acid with 16 carbon atoms, and oleic acid is an unsaturated fatty acid with 18 carbon atoms having a double bond at the 9-position (cis-type). Linoleic acid is an unsaturated fatty acid with 18 carbon atoms having cis-type double bonds at both 9 and 12 position. The structure of oleic acid is similar to that of linoleic acid, while those of palmitic and linoleic acids differ. Therefore, the above-mentioned phase behaviors are reasonable. The reason why only LLL crystals contain PPP remained unclear.
The relationship between the melting enthalpy of the β-form and the fraction of OOO or LLL was examined. When TAGs are independently crystallized in the same polymorphs, the enthalpy is proportional to their molar fractions. The melting enthalpy for each peak (Fig. 5B) was divided by the molar fraction of TAG (Fig. 6). Peaks with two tops in the PPP/LLL mixture for xL of 0.3 or lower were excluded from the examination. For PPP/LLL mixtures, the melting enthalpy of PPP decreased slightly with decreasing molar PPP fraction. The OOO enthalpy of PPP/OOO mixtures was identical at xO of 0.4 to 1 and seemed to decrease slightly at xO = 0.4 or lower. However, due to the large variation in the data, it was reasonable that the enthalpy did not depend on the fraction. The melting enthalpy of LLL for PPP/LLL mixtures did not depend on its fraction. For OOO/LLL mixtures, the OOO melting enthalpy was similar for xL of 0 to 0.4, and became smaller at xL = 0.4 or higher. The melting enthalpy of LLL was constant for the xL between 0.7 to 1 and decreased at xL lower than 0.7. Hence, the enthalpy study confirmed that OOO and LLL did not crystallize independently. It is noteworthy that the phase behavior and melting enthalpy in OOO/LLL mixtures changed around xL = 0.6 and not at the equimolar mixture (xL = 0.5). The position of the double bond might affect the packing structure of the crystal.
Melting enthalpies of PPP (◊), OOO (△), and LLL (○) divided by their molar fractions for the mixtures of (A) PPP and OOO, (B) PPP and LLL, and (C) OOO and LLL.
The phase behavior of binary mixtures of tripalmitin (PPP), triolein (OOO), and trilinolein (LLL) was investigated using differential scanning calorimetry. Regarding the PPP/OOO and PPP/LLL mixtures, the crystallization and melting temperatures and the enthalpy of OOO and LLL did not depend on the fractions. However, the values of the mentioned properties for PPP decreased slightly with decreasing molar PPP fraction. It was observed that OOO and LLL did not crystallize independently in OOO/LLL mixtures, and the phase behavior changed at the molar LLL fraction of 0.6. Under all conditions, melting of the β-form of PPP, OOO, or LLL was observed during heating. The polymorph produced during cooling was dependent on the system and the fraction.
