The binary phase behavior of 1, 3-dipalmitoyl-2-stearoyl-sn-glycerol and 1, 2-dipalmitoyl-3-stearoyl-sn-glycerol

Chemistry and Physics of Lipids 160 (2009) 11–32

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The binary phase behavior of 1, 3-dipalmitoyl-2-stearoyl-sn-glycerol and 1, 2-dipalmitoyl-3-stearoyl-sn-glycerol M.V. Boodhoo, L. Bouzidi, S.S. Narine ∗ Alberta Lipid Utilization Program, Department of Agricultural Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada

a r t i c l e

i n f o

Article history: Received 19 January 2009 Received in revised form 16 February 2009 Accepted 17 February 2009 Available online 4 March 2009 Keywords: Binary phase behavior Symmetrical and asymmetrical triacylglycerols Polymorphism Crystallization Microstructure Crystal network Rheological properties Powder X-ray diffraction Differential scanning calorimetry

a b s t r a c t The binary phase behavior of purified 1, 3-dipalmitoyl-2-stearoyl-sn-glycerol (PSP) and 1, 2-dipalmitoyl3-stearoyl-sn-glycerol (PPS) was investigated at a very slow (0.1 ◦ C/min) and a relatively fast (3.0 ◦ C/min) cooling rate. Mixtures with molar fractions of 0.1 increments were studied in terms of melting and crystallization, polymorphism, solid fat content (SFC), hardness and microstructure. Only the ␣-form of a double chain length (DCL) structure was detected for all mixtures in both experiments. The kinetic phase diagram, constructed using heating DSC thermograms, displayed two distinct behaviors separated by a singularity at the 0.5PSP composition: a eutectic in the XPSP ≤ 0.5 and a monotectic in the XPSP ≤ 0.5 concentration region. The singularity was attributed to the formation of a 1:1 (mol:mol) molecular compound. Apart from the segment from 0.0PSP to the eutectic point, XE , the simulation of the liquidus line using a model based on the Hildebrand equation suggested that the molecular interactions are strong and tend to favor the formation of unlike pairs in the liquid state and that the miscibility is not significantly dependent on cooling rate. The kinetic effects are manifest in all measured properties, particularly dramatically in the XPSP ≤ XE concentration region. An analysis of induction time as measured by pulse nuclear magnetic resonance (pNMR) showed that PPS retards crystal growth, an effect which can explain the peculiarity of this concentration region. At both cooling rates, fit of the SFC (%) versus time curves to a modified form of the Avrami model revealed two common growth modes for all the mixtures. The polarized light microscope (PLM) of the PSP–PPS mixtures revealed networks made of spherulitic crystallites of size, growth direction and boundaries that are varied and sensitive to composition and cooling rate. The change in the microstructure and final SFC (%), particularly noticeable at compositions close to the eutectic, explain in part the differences seen in relative hardness. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Triacylglycerols (TAGs) form a large and important group of compounds and are the most widely occurring form of lipids stored in plant and animal tissues (O’Brien, 2004). They are used extensively in human nutrition and are increasingly used in the formulation of pharmaceuticals and cosmetics (Gunstone and Padley, 1997; O’Brien, 2004). The study of the chemical and physical properties of the individual constituent TAGs and their mixtures is an effective method to gain insight into the thermal, structural and rheological properties of natural and modified oils and fats (Rossell, 1967; Timms, 1984; Humphrey and Narine, 2004b; Himawan et al., 2006; Zhang et al., 2007). The comprehensive phase diagrams describing the transformation behavior of TAGs, and more generally of lipids,

∗ Corresponding author at: 4-10 Agricultural/Forestry Centre, University of Alberta, Edmonton, Alberta T6G 2P5, Canada. Tel.: +1 780 492 9081; fax: +1 780 492 7174. E-mail address: [email protected] (S.S. Narine). 0009-3084/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2009.02.008

accumulated over the years (Timms, 1984; Koynova and Caffrey, 2002), are used to understand and help monitor the crystallization, fractionation, phase development and stabilization of TAG mixtures (Gibon et al., 1986). TAGs are made up of a glycerol backbone esterified to three fatty acid (FA) moieties which allow for many different combinations of arranging the FA moieties and therefore for a wide diversity in natural systems (Sreenivasan, 1978). The complex structure, packing characteristics, and physical and thermodynamic properties of TAGS have been the subject of numerous publications and reviews (Malkin and Meara, 1939; Chapman, 1962; Timms, 1984; Small, 1986; Larsson, 1986; Hagemann, 1988; Ghotra et al., 2002; Sato and Ueno, 2005; Himawan et al., 2006). However, detailed conformational and packing information from crystallography and molecular modeling studies have only been published for some pure TAGs (Jensen and Mabis, 1966; van Soest et al., 1990; Birker et al., 1991; van Langevelde et al., 2000; Sato et al., 2001; Chandrasekhar and van Gunsteren, 2002). According to their hydrocarbon subcell packing, the polymorphs of TAGs are classified into three main crystallographic types: ␣,

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␤ and ␤ (Larsson, 1986). The chain packing of the ␣-polymorph is hexagonal with nonspecific chain-chain interactions. The common subcell packing of the ␤ -polymorph is orthorhombic, with the alternate acyl chains packing in planes perpendicular to each other (O⊥ ). The hydrocarbon chains of the ␤-form are commonly packed parallel to each other in a triclinic parallel subcell (T// ). TAGs generally stack in either a double chain length (DCL) or a triple chain length (TCL) structure, but can display more complicated layering (Fahey et al., 1985). In general, the ␤-polymorph is the most stable crystal form with the highest melting temperature of the three polymorphic states and the ␣-polymorph, is the least stable crystal form with the lowest melting form (Ghotra et al., 2002; Timms, 2003). The elaborate structural hierarchy in a typical lipid network is arranged in a fractal manner (Narine and Marangoni, 1999a). As the fat crystallizes from the melt, the TAGs form domains of particular polymorphism/polytypism which grow into larger microstructural elements (single crystallites then clusters). These microstructural elements then aggregate into larger microstructures until a continuous three-dimensional network is formed by the collection of microstructures (Narine and Marangoni, 1999b). The structure–function relationships in these materials is still a difficult and open field of study (Sato, 1996; Narine and Marangoni, 1999b; Walstra et al., 2001; Marangoni and Narine, 2002). The study of the 1, 3-dipalmitoyl-2-stearoyl-sn-glycerol (PSP)/1, 2-dipalmitoyl-3-stearoyl-sn-glycerol (PPS) system, presented here, is part of a systematic examination, in our laboratory, of a series of binary symmetrical (BAB)/asymmetrical (BBA) TAG systems. The TAG series consists of compounds with the naturally occurring saturated FAs with neighboring chain length mismatch (CLM). All of the TAGs contain A = stearic acid (S, C18) and another fatty acid. Here, B = palmitic acid (P, C16), whereas in our earlier studies B = myristic acid (M, C14; Boodhoo et al., 2008), lauric acid (L, C12; submitted to CPL), or capric acid (C, C10; Boodhoo et al., 2009a) resulting in CLM of 2, 4, 6 and 8, respectively. Samples were processed at two very different constant cooling rates, namely 0.1 and 3.0 ◦ C/min, in order to investigate the kinetic effects. The TAG mixtures were studied in terms of polymorphism, crystallization and melting, solid fat content (SFC), microstructure and rheology, as a function of composition (mole fraction), with an emphasis on the thermodynamics, characteristic phase structures, and phase transition kinetics. Studies of binary mixtures of symmetrical and asymmetrical mixed-acid TAGs, which would compare to the systems investigated in our laboratory, are few, but are the subject of increasing interest (Timms, 1984). It is known that the ␤-polymorph of asymmetrical TAGs is not readily formed and that the stability of the ␤ -phase in symmetrical TAGs is increased compared to asymmetrical TAGs, a phenomenon which has been attributed to a closer and more homogenous packing of the glycerol groups (Elisabettini et al., 1998). The existence of different sub-forms of ␤ in the symmetrical and asymmetrical TAGs is well documented. The polymorphism of pure symmetrical saturated TAGs of the series n.n + 2.n, to which PSP belongs, and asymmetrical n.n.n + 2-TAG series have been comprehensively investigated (Birker et al., 1991; van de Streek et al., 1999; van Langevelde et al., 2000). Two ␤ -forms, with a DCL pack  ing (coined ␤1 -2 and ␤2 -2, higher and lower melting point phases, respectively) have been solved from single-crystal XRD data for 10.12.10 (CLC) (van Langevelde et al., 2000) and 16.16.14 (PPM) (Sato et al., 2001), and from high-resolution X-ray powder diffraction (XRPD) data for 14.16.14 (MPM) (van Langevelde et al., 2000) and 12.14.12 (LML) (Birker et al., 1991). Both the ␤ -2 modifications of these TAGs crystallize in a chair conformation in which the fatty acid chains on glycerol positions sn-1 and sn-2 are adjacent and the chain on the sn-3 position form the back rest of the chair (Birker et al., 1991; van de Streek et al., 1999). From the detailed crystallographic data collected by Birker et al. (1991) for LML, van de

Streek et al. (1999) assembled and optimized by molecular dynamics a crystalline structure consistent with experimental data of the ␤ -2 polymorph observed in compounds of the n.n + 2.n series. van Mechelen et al. (2008b) using DSC and time-resolved XRPD have determined the crystalline structure, polymorphic stability and phase transition behavior of PSP, PPS, PEP and PPE (E, elaidic acid). They have found evidence for the existence of the lower melt ing ␤2 -2 for PSS and discovered a novel polymorph of PSS, coined   ␤0 -2, that melts at a higher temperature than the ␤1 -2 polymorph. Note that we use here the common nomenclature (n.p.r) in which each TAG molecule is denoted by the lengths of the three fatty acids making up the TAG molecule (for example, 16.16.16 for tripalmitin). More recently, interest has been focused on symmetrical and asymmetrical TAGs containing oleic acid (O: cis-9-octadecenoic acid), one of the most abundant unsaturated fatty-acid chains in natural fats and oils. One can cite the detailed structural and phase behavior studies performed by Sato’s group (Yano et al., 1999; Zhang et al., 2007) and Shenk’s groups (van Mechelen et al., 2006a,b). Yano et al. (1999), for example, investigating the polymorphic transformations in SOS and OSO by XRD and Fourier transform infrared (FT-IR) spectroscopy, have reported for both TAGs a reversible phase transition between a hexagonal and a pseudo-hexagonal packings (␣ and sub-␣, respectively), and a series of irreversible transitions (␣ → ␤ → ␤). Thermodynamic and kinetic studies by DSC and XRD with a conventional generator and synchrotron radiation of OOP and its mixtures with POP showed that POP/OOP mixtures exhibited immiscible eutectic natures in both their metastable ␣and their most stable ␤ -states (Zhang et al., 2007). Shenk’s group has solved from high-resolution (HR) laboratory and synchrotron powder diffraction (SPD) data the crystalline structures of the ␤-1 polymorphs of pure MOM, POP and SOS compounds, and of the 1:1 molar mixture of SOS and POP (van Mechelen et al., 2006a) and of the ␤-2 polymorphs of POP and SOS and have established a crystal structure model for the ␤-2 polymorph of SOS (van Mechelen et al., 2006b). Literature reporting specifically on PSP and PPS is presented and contrasted with our findings in the Discussion section. The thermo-physical properties of TAGs, and more generally of organic compounds, depend strongly on molecular symmetry (Wei, 1999; Pinal, 2004) and other factors such as the conformational degrees of freedom of the molecules, inter- and intramolecular forces (Dearden, 1991), crystal structure, and crystal packing (Katritzky et al., 2001; Chickos and Nichols, 2001). For example, with few exceptions which can be explained, crystals of symmetrical molecules always possess higher melting temperatures (Gavezzotti, 1995) and are less soluble than the crystals of less symmetrical molecules with similar structures (Gilbert, 2007). In fact, symmetrical molecules are less soluble because of the higher melting temperature of their crystals (Yalkowsky and Valvani, 1980). Polymorphism and intersolubility phenomena of TAG mixtures are closely linked and many of the important properties of TAG mixtures are controlled by the liquid–solid phase transitions (Braipson-Danthine and Gibon, 2007). Note that the relationships between the different parameters that describe the molecule or group of molecules and physical property is often non-linear and require sophisticated non-linear regression methods to model them (Bhat et al., 2008). Polymorphism and CLM between the TAGs play key roles in determining the phase behavior of binary TAG mixtures. In mixtures of two tri-monosaturated TAGs, different phase behavior is frequently observed for different polymorphs. For example, PPP/SSS shows complete miscibility of the less stable forms (␣ and ␤ ) but a eutectic system for the ␤-form (MacNaughtan et al., 2006). Eutectic and monotectic behavior are observed in the ␤-form for the LLL/PPP and LLL/SSS systems, respectively, with the ␣-form of SSS co-existing with the ␤-form of LLL (Takeuchi et al., 2003). After studying the phase diagrams of LLL/MMM, LLL/PPP, and LLL/SSS and

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also considering that of PPP/SSS, Takeuchi et al. (2003) concluded that for binary monosaturated TAG mixtures, the metastable ␣- and ␤ -forms are miscible when CLM between two monosaturated TAGs is 2 or less, and that immiscibility of the metastable phases appears when CLM of 4 or 6 are present. The behavior of TAG mixtures is strongly influenced by the CLM between FA moieties as observed in our studies of mixed chain symmetrical and asymmetrical TAGs but its relative importance compared to other contributions is difficult to predict. The asymmetrical TAG demonstrated lower melting and crystallization points, whether the sample is cooled at 0.1 ◦ C/min or 3 ◦ C/min. The observed differences in melting temperature between the symmetrical and asymmetrical TAGs show an increase of approximately 2.5 ◦ C per 2 carbon atoms increase in CLM between the fatty acid moieties (∼7.8 ◦ C, 10.6 ◦ C and 13.1 ◦ C in the 0.1 ◦ C/min cooling experiment for MSM–MMS (Boodhoo et al., 2008), LSL–LLS (Boodhoo et al., submitted for publication) and CSC–CCS (Boodhoo et al., 2009a), respectively). In both the solid and liquid states of MSM–MMS (CLM = 4) binary mixtures, unlike pair interactions are favored over like pair interactions resulting in a monotectic behavior (Boodhoo et al., 2008) whereas, in the case of LSL–LLS (CLM = 6) and CSC–CCS (CLM = 8) binary mixtures, like-pair interactions are favored over unlike-pair interactions and result in limited miscibility and a eutectic behavior (Boodhoo et al., 2009a). In both experiments (0.1 ◦ C/min and 3.0 ◦ C/min cooling rates), the eutectic composition, XE , increased with CLM. For MSM/MMS, LSL/LLS and CSC/CCS systems, XE is 0, 0.16 and 0.50, respectively in the slow cooling experiment and shifted to the smaller values, i.e. 0, 0.10 and 0.25, respectively in the fast one. LSL–LLS mixtures, with CLM = 6, form so-called “molecular compounds” with a 1:1 molar ratio of the two components and display two distinct behaviors: a eutectic in the XLSL ≤ 0.5 and a monotectic in the XLSL ≥ 0.5 concentration region (Boodhoo et al., submitted for publication), whereas, the MSM–MMS (Boodhoo et al., 2008) or CSC–CCS (Boodhoo et al., 2009a) mixtures, where CLM is 4 and 8, respectively do not. PSP–PPS mixtures, with CLM = 2 (this study), form also such molecular compounds. The formation of 1:1 (mol:mol) compounds has been suggested in both SPS–PSS and PPP–PPS (Knoester et al., 1972) and observed in systems of two TAGs which both contain an unsaturated fatty acid such as SOS/OSO (Koyano et al., 1992), SOS/SSO (Takeuchi et al., 2002; Engstrom, 1992), POP/PPO, and POP/OPO (Minato et al., 1997a). Note that it is reported that molecular compounds crystallize faster than the pure components of the same polymorph and consistently form DCL structures in the metastable and stable phases (Sato and Ueno, 2001; Takeuchi et al., 2002). Kinetic factors are important in determining the amount, composition and properties of the crystalline phase and which polymorph will form from the melt and should be considered in order to describe properly the behavior of fats (Los and Floter, 1999; Hollander et al., 2002). Los’s group showed, via simulation using thermodynamic parameters, that the effect of kinetics is substantial (Los and Floter, 1999; Los et al., 2002a,b,c). The strong influence of kinetics on polymorphic occurrence in fats is illustrated in the kinetic phase diagrams of our binary TAG systems. Similarly to systems found in the literature, such as PPP/SSS (Himawan et al., 2007), either ␣- or ␤ -forms crystallize in MSM/MMS (Boodhoo et al., 2008) and LSL/LLS (Boodhoo et al., submitted for publication) mixtures, depending on the cooling rates applied. In CSC/CCS (Boodhoo et al., 2009a), the mixtures present related forms of the ␤ polymorph for all composition as well as a ␤-form for mixtures with CSC molar fraction ≥0.7 whether cooled at 0.1 ◦ C/min or 3.0 ◦ C/min (Boodhoo et al., 2009a). XE shifts to lower values when the cooling rate is increased, as reported above. Note that the asymmetrical TAG-rich phases observed in the mixtures studied in our laboratory, are more dramatically affected by the kinetics than the symmetri-

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cal one and that this effect is stronger for larger molecular weights and larger CLMs. We have also observed that crystals obtained with a cooling rate of 0.1 ◦ C/min were more homogeneous and packed more closely than those obtained with a cooling rate of 3.0 ◦ C/min. This is not unusual as rapid crystallization is known to result in poorly packed crystals (Gibon et al., 1986; Dafler, 1977; Timms, 1991). The thermal properties of such crystals depend on their “degree of imperfection” and can deviate significantly from those of well-ordered ones (Hagemann, 1988). Imperfect crystals may persist for years in the absence of a liquid phase but can easily recrystallize into well packed crystals via the liquid phase, if a liquid phase is present (Hernqvist, 1988). Most vegetable oils, used in consumer products where harder fats are needed, are modified via fractionation (Timms, 2005; Kellens et al., 2007) or interesterification or hydrogenation (Sreenivasan, 1978). The TAGs under investigation in our laboratory are relevant to numerous industrial applications involving positional isomers, particularly those involving inter-esterification (List et al., 1995). Note that palm oil and its fractions which contain large amounts of PPO may be a starting source for the production of PSP, PPS and PSS (Kellens et al., 2007). One can mention that PSP and PPS, subject of this study, when present in a shortening type system containing a wide variety of TAGs, even in small quantities, influence key functional properties such as melting behavior and hardness (Humphrey and Narine, 2004a; Narine et al., 2007). 2. Materials and methods 2.1. Sample preparation The purified PSP and PPS TAGs were synthesized according to known procedures (Bentley and McCrae, 1970). Purity of all samples exceeded 98.0%. It was determined using a gas chromatograph (GC) equipped with a universal flame ionization detector (FID) having a range of 0–10 V. The sample was run in chloroform, using the CP-TAP (Chromo Pack-Triglycerides Analysis Phase, Varian, USA) column, specially designed for TAG analysis. The mixtures were prepared by mixing the purified TAGs in 0.1-PSP molar fraction increments, melted at 100 ◦ C, held there for 5 min and stirred for a further 5 min using a mechanical stirrer to obtain homogeneous mixtures. 2.2. Thermal processing A Linkam LS 350 temperature chamber (Linkam Scientific Instruments, Tadworth, Surrey, United Kingdom) was used to process the samples for XRD, microscopy and relative hardness measurements. For DSC analysis, the samples were processed in the cell of the instrument. All samples were subjected to thermal profiles which allow for comparison. For DSC, XRD and microscopy the samples were heated to 100 ◦ C and held for 5 min to erase crystal memory, cooled at a constant rate down to 15 ◦ C where the sample was held isothermally for 1 h and also down to −25 ◦ C then held isothermally for 10 min at this temperature. The holding time was designed to ensure that the crystallization is complete and that no further thermal event is ongoing. Note that in these cases, similar DSC, XRD and microscopy results have been recorded. The experiments in which the sample is cooled to −25 ◦ C are presented in this study as they allow for the best defined baselines and hence more precise determination of the parameters. For relative hardness and solid fat content measurements, the samples were processed as in the DSC, microscopy and XRD experiments except that, they were only cooled down to 15 ◦ C where the sample was held isothermally for 1 h. The crystallization was considered to be complete at the measurement temperature as confirmed by the large flat baseline observed in the DSC cooling thermograms and in the final SFC ver-

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sus time curve. Two very different cooling rates (0.1 ◦ C/min and 3.0 ◦ C/min) have been used in order to investigate the influence of processing conditions on the materials’ properties. Samples in the DSC were reheated immediately after the cooling process at a constant rate of 5.0 ◦ C/min to 100 ◦ C to obtain the melting profile. All measurement temperatures are reported to a certainty of better than ±0.5 ◦ C. 2.3. Experimental techniques 2.3.1. X-ray diffraction A Bruker AXS X-ray diffractometer equipped with a filtered Cu K␣ radiation source ( = 0.1542 nm) and a 2D detector was used to obtain XRD patterns. The procedure was automated and controlled by Bruker AXS’s “General Area Detector Diffraction System” (GADDs V 4.1.08) software. The samples were processed in the temperature controlled chamber described in Section 2.2. The samples processed as indicated in Section 2.2, were quickly transferred for measurement to the XRD stage where the temperature was already set up and maintained at the holding temperature, −25 or 15 (±0.5 ◦ C), by an air jet cooling system (Kinetics-Thermal Systems, USA). The frames were processed using the Bruker AXS’s GADDS software and the resulting spectra were analyzed using Bruker AXS’s “Topas V 2.1” software. Note that the low- and wide-angle spacing regions accessible to our XRD system are 2 = [1.6–15◦ ] and [15–32◦ ]), respectively. 2.3.2. Differential scanning calorimetry Approximately 5.0–10.0 (±0.1) mg of fully melted and homogenously mixed sample was placed in an aluminum DSC pan and hermetically sealed. The DSC measurements were carried out using a DSC Q100 model (TA Instruments, New Castle, DA). An empty aluminum pan was used as a reference and the experiments were performed under a nitrogen flow of 50 mL/min. The samples were processed as reported in Section 2.2. The “TA Universal Analysis” software coupled with a method developed by our group (Bouzidi et al., 2005) was used to analyze the data and extract the main characteristics (onset temperature, TOn , offset temperature, TOff , temperature of peak maximum, Tm , enthalpy, H and full width at half maximum, FWHM) of the peaks. The temperature window over which a thermal event occurs is defined as the difference between the offset and the onset temperatures of that event. It is labeled TC for crystallization and TM for melting. The characteristics of the non-resolved individual peaks and shoulder signals were estimated using the first and second derivatives of the signal and a simple decomposition of the signal into its obvious main components. The kinetic binary phase diagrams of the PSP/PPS system were constructed from the heating runs of the DSC. TOn , TOff and Tm were used to determine the boundaries in the phase diagram as typically done in the study of binary lipid mixtures (Höhne et al., 2003; Inoue et al., 2004b; MacNaughtan et al., 2006; Costa et al., 2007; Abes et al., 2007, 2008; Boodhoo et al., 2008, 2009a). 2.3.3. Solid fat content determination A Bruker Minispec mq 20 pulse nuclear magnetic resonance (pNMR) spectrometer (Milton, Ontario, Canada) with a retro-fitted temperature controlled chamber was used to monitor SFC evolution with time. Thermal processing for the pNMR experiment, described in Section 2.2, was controlled and maintained by a series of external circulating chillers as described by Narine and Humphrey (2004). Data acquisition was started immediately upon transfer of the sample into the NMR chamber. The SFC values are reported as the ratio of the intensity of the NMR signal of the solid part to the total detected NMR signal in percent (labeled as SFC (%)). Uncertainties are the calculated standard deviations of at least two runs.

2.3.4. Relative hardness A TA.XT. plus texture analyzer (Stable Microsystems, Surrey, U.K.) fitted with a 1.0 kg load cell and standard needle (cone 8◦ , 55 in.; ASTM: D 1321-65) was used for hardness measurements. Testing was carried out in open DSC pans containing approximately 50 mg of sample. The samples were processed in the temperature controlled chamber described in Section 2.2 then quickly transferred to the temperature controlled chamber (Autotune CAL 9300, CAL Controls Ltd., Herts, U.K.) fitted to the texture analyzer for testing. The chamber was already equilibrated at 15 ◦ C prior to the transfer of the sample. Penetration was performed at a constant speed of 0.5 mm/s to a fixed depth of 1.5 mm according to an optimization procedure developed by the Alberta Lipid Utilization Research Program (Boodhoo et al., 2009b). Penetration and data acquisition were controlled by the Texture Exponent 32 software (Version 2.0.0.7 www.SaxSoft.com, Eugene, OR). Force versus displacement graphs were plotted and the maximum force detected within the penetration depth of 1.5 mm was chosen as a measure of the relative hardness. 2.3.5. Microscopy A Leica DMRX polarized light microscope, PLM (Leica Microsystems, Wetzlar, Germany) fitted with a Hamamatsu (C4742-95) digital camera was used for image capture. The samples were processed in the “Linkam LTS 350” temperature-controlled stage fitted to the PLM. They were as described in Section 2.2. The micrographs presented here were taken at magnification of 100× and 500×. 2.4. Data analysis and modeling 2.4.1. X-ray data analysis and polymorphism of triacylglycerols In the solid state, the arrangement of two TAGs determines the tilt of the hydrocarbon chains with respect to the plane through the methyl end groups, and this tilt determines the low-angle (long-spacing) reflections in a XRPD diagram. The subcell of the hydrocarbon chains is responsible for the characteristic wide-angle (short-spacing) reflections. The ␣-polymorph is characterized by one strong wide-angle line in the XRD pattern at a lattice spacing of ∼4.2 Å and the ␤ -polymorph is characterized by two strong wide-angle lines at lattice spacings of ∼4.2–4.3 Å and 3.7–4.0 Å, respectively. The ␤-polymorph is characterized in the wide-angle region by a lattice spacing of ∼4.6 Å and a number of other strong lines around 3.6–3.9 Å. 2.4.2. The phase diagrams and thermodynamic analysis of their boundaries A thermodynamic model based on the Hildebrand equation (Hildebrand, 1929) coupled with the Bragg–William approximation for non-ideality of mixing (Bragg and Williams, 1934) was used to simulate the phase boundaries in the temperature–molar fraction (T–X) phase diagram and investigate the miscibility of the components. This model is a powerful tool commonly used to study lipid mixtures (Lee, 1977a,b; Inoue et al., 2004b; Abes et al., 2007, 2008; Boodhoo et al., 2008, 2009a), and reported in some detail in a previous study (Abes et al., 2007). In a system composed of components A (e.g. PSP) and B (e.g. PPS), the equilibrium liquidus line can be described by either of the following two equations depending on whether the composition is smaller or larger than the eutectic composition, XE (Lee, 1977b; Tenchov, 1985): ln XA = −

HA R

ln XB = −

HB R

1 T

1 T

−

1 TA

−

1 TB



(1)

 (2)

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where R is the gas constant. XA represents the mole fraction of A, HA and TA are the molar heat of fusion and the melting point of component A, and XB , HB and TB are those of component B. Eq. (1) models the liquidus in the XE ≤ XA ≤ 1 range and Eq. (2) models it in the 0 ≤ XA ≤ XE range. The deviation from an ideal behavior, as described by the Bragg–Williams approximation, is characterized by a non-ideality parameter , given by:



uAA + uBB  = z uAB − 2



(3)

where z is the first coordination number and uAA , uBB and uAB are the interaction energies for A–A, B–B, and A–B pairs, respectively. According to this approximation, when assuming non-ideality of mixing in the liquid state, the liquidus line in the composition range between XE ≤ XA ≤ 1, is given by: ln XA +

HA (1 − XA )2 =− RT R

1 T

−

1 TA

 (4)

and in the composition range 0 ≤ XA ≤ XE is given by: ln XB +

(1 − XB )2 HB =− RT R

1 T

−

1 TB

 (5)

The parameter  (Eq. (3)) is a measure of non-ideality of mixing, and can be interpreted in terms of the difference in the energy of mixed-pairs (A–B) and the average pair interaction energy between like pairs (A–A and B–B) formed in the mixture. For ideal mixing, the intermolecular interaction of like-pairs is equal to that of a mixedpair and consequently  = 0. A positive  means that a mixed-pair formation is energetically less favorable than the like-pair formation whereas negative  values indicates that mixed-pair formation is energetically more favorable (Lee, 1977b). Thus, a large positive  tends to lead to a phase separation, while a large negative  tends to result in a regular arrangement of the two components in the mixture.

15

on a linear regression protocol (Narine et al., 2006). The process defines the segments to be treated individually. 3. Results 3.1. X-ray diffraction There was no detectable influence of the cooling rate on the final crystalline phase observed in solid mixtures of the PSP and PPS TAGs. Similar XRD profiles were obtained for samples cooled at 0.1 ◦ C/min and at 3.0 ◦ C/min. All of the mixtures presented XRD patterns with similar overall shape. Fig. 1 shows selected XRD patterns of the different PSP/PPS compositions, obtained at −25 ◦ C after the 3.0 ◦ C/min cooling experiment. The inset shows an expansion of both the X- and Y-scales of the XRD profile obtained from the 0.8PSP sample in order to highlight the weakest reflections. Each XRD pattern shows eight resolved peaks (P1–7 and P␣ from the lowest to the highest-diffraction angle in Fig. 1). The corresponding d-spacings d1–7 and d␣ are shown in Fig. 2. The relevant XRD data is provided in Table 1. In the wide-angle region, the line P␣ , appears at the same position in all the XRD patterns at a diffraction angle of 21.63◦ . This reflection line corresponds to a d-spacing d␣ = 4.12 ± 0.01 Å originating from the {1 0 0} family of planes of a hexagonal subcell and is characteristic of an ␣-polymorph. In the low-angle region, the diffraction peaks P1–6 appear at the same positions for the mixtures with concentrations in the range between XPSP = 0.0 and 0.5, whereas, their positions shifted continuously to slightly higher

2.4.3. Fit of the SFC versus time curves The SFC (%) versus time were fitted to a modified form of the Avrami model that takes into consideration the variances within the growth curve (Narine et al., 2006). In this model, the crystallization of a lipid system is regarded as a succession of p different crystallization events, occurring in steps with different incubation times,  i . Each step i (i = 1, 2, 3, . . ., p) is characterized by a constant growth rate Gi , and is described by the Avrami equation: Fi (t) = Fi∞ (1 − exp(Ai (t − ti )ni ))

(6)

where Fi (t) is the absolute crystallinity at time t, Fi∞ , is the crystallinity at some time when either the growth rate or the nucleation conditions change, ti is the induction time for crystallization of the sample, and Ai and ni are the Avrami constant and exponent applicable to the nucleation, growth, and dimensionality of the crystallizing lipid over that segment of time. The total absolute crystallinity is the sum of p individual absolute crystallinities (i.e. segments in the SFC (%) versus time plot). The number of segments in the experimentally determined SFC versus time curves which represent different crystallization mechanisms, and values of incubation times are determined semiempirically. Each segment illustrates a different crystallization mechanism or step but does not necessarily indicate an intermediate phase. The time domains for each segment are defined by plotting ln(−ln(1 − F)) versus ln(t) from experimentally determined SFC versus time data. A sequence of straight lines is produced for each data set, emphasizing the changes in increasing percent SFC. The boundaries of each line segment in this linearized data are determined unambiguously following a rigorous algorithm based

Fig. 1. Representative XRD spectra obtained for PSP/PPS mixtures cooled at a rate of 0.1 ◦ C/min from 100 ◦ C to −25 ◦ C. Inset zooms in the angle region 2 = [1–17◦ ] of the XRD spectrum of the 0.8PSP mixture. The samples were measured at −25 ◦ C. Molar fractions are reported at the right-hand side of each curve.

16

M.V. Boodhoo et al. / Chemistry and Physics of Lipids 160 (2009) 11–32

ment of crystal packing and crystal perfection with the increase of PSP content. 3.2. DSC results 3.2.1. Crystallization behavior The crystallization thermograms obtained at cooling rates of 0.1 ◦ C/min and 3.0 ◦ C/min are shown in Fig. 3a and b, respectively. The corresponding phase transition temperatures, onset, offset and maximum of the peaks (TOn , TOff and Tm ) are displayed in Fig. 3c and d, respectively. TC is shown in Fig. 3e. The corresponding thermodynamic data is listed in Table 2. In the 0.1 ◦ C/min cooling experiment, a single peak is observed for the 0.0PSP mixture, but as PSP content is increased, a second thermal event appears as a shoulder at a higher temperature for the 0.2PSP mixture, and as a well resolved exotherm thereafter as shown in the cooling thermogram obtained from the 0.4PSP mixture for which the Y-axis scale is expanded (dashed curve in Fig. 3a). The peaks are labeled P1 and P2 , respectively, in Fig. 3a. Note that Tm of P1 follows the same trend as its onset (filled triangles and open circles, respectively, in Fig. 3c), indicating that either can represent the liquid–solid transition line. In this experiment, the transition liquid–solid line exhibits an obvious eutectic point at approximately XE = 0.1PSP and TE = 45 ◦ C and a prominent singularity formed by the intersection, close to the 0.5PSP concentration, of two segments having different slopes of the liquid–solid transition line (indicated by an arrow in Fig. 3c). A kinetic phase diagram is constructed using the cooling thermograms. The liquidus line is represented by TOn (open circles in Fig. 3c) and the solidus line is represented by the transition line constructed using Tm of P1 (open triangles in Fig. 3c). One can observe two distinct regions separated at the 0.5PSP singularity: an obvious eutectic region with XE ∼ 0.1 and a following monotectic region (Fig. 3c). Note that the eutectic line, which extends from the purified PPS to the 0.5PSP concentration (dashed line passing through the open triangles in Fig. 3c), decreases with increasing PSP content. The cooling thermograms obtained at 3.0 ◦ C/min for the PSP/PPS mixtures show a broad asymmetrical exotherm which probably consists of at least two component peaks (Fig. 3b). The 0.2PSP to 0.8PSP mixtures present similar thermograms with similar TOn (∼42.5 ◦ C) and Tm (∼43.7 ◦ C) displayed as large flat minima in Fig. 3d. Note that in this experiment, the difference between the lowest and the highest value of Tm is less than 1.5 ◦ C and that TC is almost constant for all mixtures, with an mean value of 3.4 ± 0.1 ◦ C (Fig. 3e) suggesting an overall similar crystal packing and phase homogeneity. In the 0.1 ◦ C/min cooling experiment, the total enthalpy of crystallization, HC (filled circles in Fig. 4a) shows a minimum at XPSP = 0.2 which can be directly related to the eutectic observed at this concentration. The increase of HC thereafter is an indication of an overall increase in the crystal perfection and crystalline homogeneity. In the 3.0 ◦ C/min cooling experiment, HC (filled triangles in Fig. 4a) also shows a minimum at XPSP = 0.2, albeit less promi-

Fig. 2. Variations of the X-ray diffraction structural parameters for PSP–PPS mixtures cooled at a rate of 0.1 ◦ C/min from 100 ◦ C to −25 ◦ C. The samples were measured at −25 ◦ C. The dashed lines are linear fits.

diffraction angles with increasing PSP content for XPSP > 0.5 (Fig. 2). For XPSP > 0.5, the d1–6 versus PSP molar fraction were satisfactorily fitted with linear functions with correlation coefficients R2 higher than 0.99 for all the lines. All the lines were parallel with an average slope, corresponding to a lateral expansion coefficient as a function of TAG composition, of −2.58 ± 0.14 Å/mol. For all mixtures, the first six peaks P1–6 (Fig. 1 inset for the 0.8PSP mixture), form a series (S) of ratio (d1–6 ) 1:1/2:1/3:1/4:1/5:1/6 characteristic of a lamellar periodicity (Fontell, 1974; Inoue et al., 2004a). P1 is the first order reflection and P2–6 are the following orders of reflection. P1–6 originate from the {0 0 1}–{0 0 6} families of planes of the ␣-polymorph and represent the straight lamellar packing of the hydrocarbon chains. d1 -value represents the lamellar periodicity length, LP , and also the c-axis length of the ␣ subcell. The value of LP for the purified PPS and for the mixtures with XPSP ≤ 0.5 was 44.6 ± 1.2 Å. As PSP content increases, LP values decrease linearly, as discussed above for the d1 -spacing, to reach a value of 43.0 ± 1.1 Å for the purified PSP. This linear reduction in the lateral repeating unit length suggests a direct influence of the PSP hydrocarbon chains on the molecular arrangements at the “terrace” level via methyl-end groups interactions and indicate a steady improve-

Table 1 Average X-ray diffraction spacings (cell lattice parameters d, Å) observed for the PSP–PPS mixtures. XPSP is the PSP molar fraction. Temperature of measurement is −25 ◦ C. Uncertainty (in parenthesis) is the standard deviation of the mean of the observed parameter (Å). XPSP P1 P2 P3 P4 P5 P6 P7 P␣

(±1.47) (±0.63) (±0.16) (±0.33) (±0.17) (±0.16) (±0.18) (±0.03)

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

45.00 23.48 16.03 12.01 9.64 7.97 5.96 4.10

44.50 24.10 15.98 11.96 9.54 7.97 5.99 4.11

44.04 23.90 15.87 11.87 9.47 7.98 5.95 4.09

44.16 24.15 15.96 11.94 9.58 7.94 6.08 4.10

45.00 23.80 15.90 12.01 9.60 7.99 6.01 4.11

45.09 23.31 15.83 11.79 9.56 7.98 5.93 4.10

44.63 23.41 15.35 11.35 9.33 7.81 5.78 4.09

43.96 22.80 15.26 11.49 9.13 7.64 5.78 4.09

43.65 23.15 15.44 11.26 9.15 7.61 5.67 4.11

43.37 22.27 15.12 11.10 8.99 7.37 5.54 4.12

43.00 21.94 14.58 10.78 8.74 7.16 5.35 4.14

M.V. Boodhoo et al. / Chemistry and Physics of Lipids 160 (2009) 11–32

17

Table 2 Thermodynamic data from the cooling runs of the PSP/PPS mixtures. Tmax , temperature at the maximum of the peak (◦ C); TC = TOff-C − TOn-C , difference between the offset (TOff-C ) and onset (TOn-C ) temperature of crystallization (◦ C); HC , enthalpy of crystallization (kJ/mol). Uncertainty (in parenthesis) is the mean of the standard deviations calculated for each sample from 3 replicates. XPSP 0.1 ◦ C/min P1 Tmax HC P2 Tmax HC

0.00

0.10

0.20

0.30

(±0.10) (±6)

44.68 116

44.52 114

44.47 101

44.41 102

44.41 92

44.32 81

44.50 53

44.79 48

45.72 45

47.92 25

50.11 0

(±0.20) (±5.00)

–

–

45.08 5.03

45.35 12.00

45.58 18.69

45.66 44.02

45.50 69.40

46.55 85.06

48.25 90.63

49.50 114.50

50.11 138.80

45.09 0.8 116

44.93 0.8 114

45.82 1.7 106

46.20 2.1 114

46.43 2.4 111

46.60 2.6 121

47.10 3.1 122

48.30 4.1 133

49.70 4.8 136

50.59 3.8 138

51.30 2.6 139

42.89 44.12 3.44 120

42.76 43.86 3.27 117

42.62 43.72 3.52 112

42.35 43.66 3.43 112

42.64 43.55 3.00 114

42.54 43.77 3.88 111

42.61 43.62 3.20 113

42.54 43.86 3.50 114

42.55 43.85 3.48 114

43.13 44.27 3.48 117

43.41 44.46 3.25 120

Whole exotherm (±0.15) TOn-C (±0.2) TC (±5) HC 3.0 ◦ C/min Tmax TOn-C TC HC

(±0.15) (±0.20) (±0.25) (±4)

0.40

nent, but remains almost constant for all other concentrations. Obviously, the ordering effect of PSP on the overall crystallinity is hindered by the fast crystallization. The competition between the ordering effect of the symmetrical PSP and the kinetic effects are discussed in detail in the Discussion section. In the 0.1 ◦ C/min cooling experiment, the estimated enthalpy of P1 , HC1 (open triangles in Fig. 4b), decreases as a function of XPSP concomitantly with the increase of HC2 for P2 (filled triangles in Fig. 4b). Because only the ␣-polymorph has been detected by XRD in all the mixtures at the end of the cooling process, P1 and P2 can be attributed to different ␣-phases, a PSP-rich and PPS-rich phase, respectively. The increase of the PSP-rich phase content is obviously occurring at the detriment of that of the PPS-rich phase. One can note that the curves cross at approximately 0.6PSP , close to 0.5PSP where the two phases would have the same amounts, and that both HC1 and HC2 versus XPSP are not a straight line suggesting that this is not the result of a simple dilution effect. This indicates that even if the two phases have the same polymorphic form, the crystal perfection and homogeneity of the two phases are not the same. PSP strongly influences the crystallization behavior of the mixtures as will be discussed later.

0.50

0.60

0.70

0.80

0.90

1.00

3.2.2. Melting behavior The melting curves obtained using a heating rate of 5 ◦ C/min for samples cooled at 0.1 ◦ C/min and 3.0 ◦ C/min are represented in Fig. 5a and b, respectively. The corresponding PSP/PPS binary kinetic phase diagrams constructed from the heating runs are shown in Fig. 6a and b, respectively. The corresponding thermodynamic data is listed in Table 3. The pattern of thermal behavior is relatively complex and is influenced by the rate at which the samples were cooled, but not to the extent of changing the sequence of the phase transitions observed. Each thermogram shows several thermal events with at least two or three prominent endotherms, depending on PSP content. However, the peaks were much less resolved for the samples cooled at 3.0 ◦ C/min than for those cooled at 1.0 ◦ C/min. The endotherms appearing close to the first and second peaks of the purified PPS are labeled P1 and P2 , respectively. The endotherm appearing close to the peak of the purified PSP is labeled P3 . To simplify the analysis of the thermograms, dashed lines are passed through P2 and P3 to guide the eye. Note that P3 appears as a prominent shoulder for the 0.3PSP mixture and grows with increasing PSP content into a well resolved peak for the pure PSP, whereas, P2 is

Table 3 Thermodynamic data from the heating runs of the PSP–PPS mixtures. Tm , Temperature at the maximum of the peak (◦ C); TOn , onset temperature of melting (◦ C); TOff , offset temperature of melting (◦ C); H, enthalpy of melting (kJ/mol). Indices: 1 for peak P1 , 2 for peak P2 and R for the exotherm. Uncertainty (in parenthesis) is the mean of the standard deviations calculated for each sample for 3 replicates. XPSP

0.00

0.1 ◦ C/min TOn TOff Tm1 Tm2 H1 H2 HR

(±0.30) (±0.40) (±0.30) (±0.30) (±5) (±5) (±4)

46.52 59.19 48.43 55.83 105 9 1

3.0 ◦ C/min TOn TOff Tm1 Tm2 TmR H1 H2 HR

(±0.30) (±0.30) (±0.20) (±0.50) (±0.30) (±5) (±5) (±4)

46.16 57.00 48.18 55.30 52.87 112 5 1

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

46.33 57.23 48.07 55.34 96 21 1

46.63 57.54 47.84 55.94 74 35 11

46.27 59.38 47.61 56.04 53 53 22

46.73 61.07 47.67 58.57 32 90 20

45.79 62.90 47.35 60.41 10 120 6

65.01 47.31 62.44 4 125 0

66.73 47.85 63.51 3 126 0

68.69 49.97 65.32 3 127 0

69.78 54.50 66.85 2 135 4

70.78 61.27 69.50 1 131 3

46.15 57.07 47.83 55.24 52.09 102 19 4

46.09 57.79 47.81 55.89 51.77 77 34 8

46.00 59.50 47.72 57.05 51.35 62 65 17

45.93 60.79 47.25 58.36 50.28 62 80 28

46.16 62.74 47.43 59.98 50.57 45 101 30

45.65 64.66 47.00 62.25 49.26 36 117 39

45.72 67.18 46.98 64.70 48.90 19 120 26

44.75 68.25 46.45 66.07 48.18 9 124 24

47.18 69.22

47.30 69.81

67.70 48.51 4 134 23

68.11 48.84 1 135 22

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M.V. Boodhoo et al. / Chemistry and Physics of Lipids 160 (2009) 11–32

Fig. 4. (a) Total enthalpy of crystallization (HC , kJ/mol) obtained at cooling rates of 0.1 ◦ C/min (䊉) and 3.0 ◦ C/min (). (b) Estimated area (HC , kJ/mol) of P1 ( ) and of P2 () obtained in the 0.1 ◦ C/min cooling experiment.

Fig. 3. Crystallization thermograms of the different PSP–PPS mixtures obtained at (a) 0.1 ◦ C/min and (b) at 3.0 ◦ C/min cooling rate. The Y-axis scale is expanded in the cooling thermogram obtained from the 0.4PSP mixture to show the crystallization peak P2 . Molar fractions are reported above each curve at the left-hand side of the figure. Thermodynamic parameters obtained from DSC measurements of PSP/PPS mixtures cooled at (c) 0.1 ◦ C/min and (d) 3.0 ◦ C/min cooling rate. Onset temperature of crystallization ; temperature at maximum heat flow of peak P1 (Tm1 ), ; temperature at maximum heat flow of peak P2 (Tm2 ), ; and offset temperature of crystallization (TOff-C ), . The dashed lines are linear fits and the dotted lines are guide for the eye. (e) Absolute value of the difference between the offset and onset temperature of the crystallization event (TC ), obtained at cooling rates of 0.1 ◦ C/min (䊉) and 3.0 ◦ C/min (). The dashed lines are guide for the eye. All temperatures are in ◦ C.

well resolved for mixtures with less than 0.3PSP and its FWHM gradually increases becoming a broad shoulder in the thermogram of the 0.8PSP sample. The temperature at maximum heat flow of the endotherm P1 , Tm1 , shifts slightly linearly to lower temperature with PSP mole fraction (open triangles in Fig. 6a and b). Note that TOn (filled squares in Fig. 6a and b) runs parallel to Tm1 indicating that no extra thermal event is taking place before P1 . Except for the 0.9PSP and 1.0PSP mixtures, P1 is present for all mixtures in both experiments, albeit faint in the heating thermograms of the 0.6PSP to 0.8PSP mixtures in the 0.1 ◦ C/min cooling rate experiment. P1 indicates the melting of the ␣-phase which was detected after cooling. It is followed by a prominent exotherm indicating a recrystallization mediated by melt of a higher stability polymorphic phase (␤ , as will be shown later). An exotherm is the first thermal event that is visible in the heating thermograms of the 0.9PSP and 1.0PSP mixtures, probably due to the direct recrystallization of a more stabile polymorphic phase (␤ ) from the pre-existing ␣-phase. One can observe that the heating thermograms of the 0.0PSP to 0.2PSP mixtures present only two well resolved endotherms (P1 and P2 ) and that all the transformation features in the heating thermograms of the 0.3PSP to 0.7PSP mixtures are common to reheated samples, whether they were cooled quickly or slowly. Fig. 5c shows typical sequences of phase transitions, as shown by the heating thermograms obtained using a heating rate of 5 ◦ C/min after the samples are cooled at a rate of 3.0 ◦ C/min, illustrated by representative mixtures of the different concentration ranges discussed above, i.e. the 0.1PSP mixture for the [0.0PSP –0.2PSP ] range, 0.5PSP mixture for the [0.3PSP –0.8PSP ]

M.V. Boodhoo et al. / Chemistry and Physics of Lipids 160 (2009) 11–32

19

Table 4 Result of the linear fit of the temperature at maximum heat flow of the endotherm P1 , Tm1 , and of the exotherm, TR , in the regions where a linear behavior with PSP mole fraction (y = y0 + ax) is observed. 0.1 ◦ C/min

Tm1 TR

3.0 ◦ C/min

R2

XPSP ≤ 0.5

XPSP ≥ 0.5

XPSP ≤ 0.8

XPSP ≥ 0.8

−1.78 + 48.29XPSP −8.39 + 53.30XPSP

– 20.17 + 39.27XPSP

−1.89 + 48.16XPSP −5.69 + 52.86XPSP

– 3.28 + 45.56XPSP

concentration range and the 0.9PSP mixture for the PSP-rich mixtures. The polymorphic development in the PSP–PPS mixtures and the details of Fig. 5c are further detailed in the Discussion section. Due to the decrease of the area of P1 , the apparent temperature at maximum heat flow of the exotherm, TR , shifts linearly to lower temperature with increasing PSP content up to 0.5PSP molar fraction in the case of the 0.1 ◦ C/min experiment and up to 0.8PSP in the case of the 3.0 ◦ C/min experiment where it increases linearly (open lozenges in Fig. 6a and b, respectively). This indicates that the two sides of TR versus molar ratio curve represent the transformation of qualitatively different phases; one representing the recrystallization of the ␤ -phase in PPS which contributes to the total heat flow decreases for 0.0PSP to 0.5PSP and the other the recrystallization of the ␤ -phase in PSP which contributes to decreases from 1.0PSP to 0.5PSP . Tm1 and TR have been very well fit with straight lines in each case, suggesting a proportional contribution of TAG content to the transformations (linear fit parameters are listed in Table 4). The enthalpy of P1 , HM1 , of the combined P2 and P3 signals, HM23 , and of the exotherm, HMR , was accurately measured for all mixtures (Fig. 7 and Table 3). As can be seen, HM1 decreased almost exponentially with increasing PSP content while HM23 increased accordingly, in both experiments (open and filled triangles, respectively in Fig. 7a and b). HMR presents a maximum at the 0.3PSP concentration in the 0.1 ◦ C/min cooling experiment and at the 0.5PSP concentration in the 3.0 ◦ C/min cooling experiment (open lozenges in Fig. 7a and b, respectively) outlining the effect of the kinetics on phase transformations. In order to use the heating temperature profile of the mixtures to identify the polymorphic transformations that occur in the PSP/PPS system, as is customary for lipid systems (Lutton, 1955; Himawan et al., 2007), we have isolated the main polymorphic phases showing in PSP and PPS mixtures. The same thermal protocol was used in the DSC and XRD experiments to stimulate the crystallization of the phases. The diffraction patterns were recorded after the cooling process and subsequent heating was used to record the DCS melting profile of the phases. The least stable polymorphic form was singled out by quenching (25 ◦ C/min) a seed-free melt, well above the melting temperature of the most stable forms of the TAGs (100 ◦ C) to −25 ◦ C, and subsequent melting of the crystallized solid at different heating rates. The XRD patterns of the resulting solid, recorded at −25 ◦ C present each a single reflection for both PSP and PPS (at 22.3◦ for PPS and 22.1◦ for PSP) characteristic of the ␣-polymorph

0.928 0.988

(P␣ in Fig. 8a). The heating thermograms (15 ◦ C/min) of the ␣-phase in PSP and that of PPS are shown in Fig. 8b. Unlike in PPS, where for heating rates as low as 10 ◦ C/min, only the melting peak of the ␣phase (P1PPS in Fig. 8b) is showing, the melting peak of the ␣-phase in PSP (P1PSP in Fig. 8b) is followed by the recrystallization exotherm of the ␤ -phase and its subsequent peak of melting (P2PSP in Fig. 8b) even when the fastest heating rate available in our DSC (45 ◦ C/min) is used. However, the melting temperature of the ␣-phase in PPS as well as in PSP was accurately determined for all heating rates. HM of the ␣-phase in PPS was ∼105 ± 2 kJ/mol and ∼6 ± 2 kJ/mol in PSP, a low value due the recrystallization of most of the phase into a ␤ -phase. The relevant data are reported in Table 5. We have been able also to stimulate the formation of the most stable ␤ -phase in both PSP and PPS. The ␤ -polymorph was stimulated by a seeded melt at the nucleation temperature of the phase in the TAG. The sample was cooled first from 100 ◦ C (3 ◦ C/min) to −25 ◦ C to crystallize the ␣-phase then quickly heated (5 ◦ C/min) to a temperature close to the recrystallization point, TR (54 ◦ C for PPS and 60 ◦ C for PSP) where it was left for 1 h, then cooled (3 ◦ C/min) to −10 ◦ C to crystallize the ␤ -phase. At this point, the sample was analyzed with XRD for structure determination. Melting temperature of the isolated phase was determined by subsequently heating the sample (5 ◦ C/min) to 100 ◦ C. Note that the nucleation temperature and time were optimized and that the cooling and heating rates used in this procedure, similar to those of our study to allow for comparison, were sufficient to fully isolate the ␤ -phases. As can be seen in Fig. 8c, the wide-angle region of the XRD patterns displays the (2 0 0) and (1 1 0) reflections characteristic of the ␤ polymorph (1 P␤ and 2 P␤ in Fig. 8c at 23.9◦ and 21.4◦ for PPS, respectively, and 24.0◦ and 21.7◦ for PSP, respectively). The DSC heating thermograms of the isolated ␤ -phases in both PSP and PPS present each a single endotherm (P3PSP at 67.6 ◦ C and P2PPS at 58.2 ◦ C, respectively in Fig. 8d). HM of the ␤ -phase is practically the same (∼157 ± 3 kJ/mol) in both TAGs, outlining their very close nature. Note that, as expected, it is higher than that of the ␣-phase in PPS. The relevant data is reported in Table 5. These results are sufficient to completely identify the polymorphic transformations as depicted by the heating thermograms of the PSP–PPS mixtures. One may determine the equilibrium melting temperature of the ␤ -phase in PPS and in PSP by varying the heating rate and extrapolating the TM versus heating rate to zero, but this would be a different topic of study and would not add any critical insight to

Table 5 Structural and thermodynamic data of the forms encountered in the PSP and PPS TAGs. T (measurement temperature, ◦ C); c (subcell parameter, Å); Tm (melting temperature, C); and HM (enthalpy of melting, kJ/mol).

◦



Form

␣ (this work)

␤ (this work)

PPS T (◦ C) c (Å) Tm (◦ C) HM (kJ/mol)

−25 44.60 50.5 105 ± 2

−25 86.64 58.5 157 ± 3

−23 86.746 59 –

−23 41.93 65 –

PSP T (◦ C) c (Å) Tm (◦ C) HM (kJ/mol)

−23 41.60 50.5 6±2

−25 84.44 67.6 157 ± 3

−23 85.263 66.5 –

22 41.60 70 –

␤1 -2 (van Mechelen et al., 2008b)

␤-2 (van Mechelen et al., 2008a)

20

M.V. Boodhoo et al. / Chemistry and Physics of Lipids 160 (2009) 11–32

Fig. 6. PSP/PPS binary phase diagram constructed from the DSC heating runs of the samples cooled at (a) 0.1 ◦ C/min and (b) at 3.0 ◦ C/min. Offset temperature of melting (TOff ), ; onset temperature of melting (TOn2 ) of peak P2 , 䊉; temperature at maximum heat flow of the exotherm (TR ), ♦; temperature at maximum heat flow (Tm ) of peak P1 , ; onset temperature of melting (TOn ), . All temperatures are in ◦ C. The solid lines are obtained from the simulation of the experimental liquidus line ( ) using the non-ideality of mixing parameter  (kJ/mol) listed in Table 5. The dashed lines are linear fits of the experimental points through which they pass. The dotted lines are guides for the eye.

our understanding of the polymorphic development in the PSP–PPS mixtures.

Fig. 5. Heating curves of the different PSP–PPS mixtures obtained using a heating rate of 5 ◦ C/min for samples cooled at rates of (a) 0.1 ◦ C/min and (b) at 3.0 ◦ C/min. Molar fractions are reported above each curve at the left-hand side of the figure. The lines are guide for the eye. (c) Sequence of the most likely phase transitions as shown by the heating thermograms of the 0.9PSP , 0.5PSP and 0.1PSP mixtures obtained using a heating rate of 5 ◦ C/min after the samples were cooled at a rate of 3.0 ◦ C/min.

3.2.3. Phase diagram of the PSP/PPS binary system The binary kinetic phase diagrams for the slow (Fig. 6a) and fast cooling experiment (Fig. 6b) were constructed using the heating thermograms. The liquidus line is constructed from TOff of melt (open circles in Fig. 6a and b) and the solidus line is constructed from TOn of the last melting peak, i.e. the highest temperature endotherm, P3 (filled circles in Fig. 6a and b). TOn of P1 (filled squares in Fig. 6a and b) and its Tm (open triangles in Fig. 6a and b), which run parallel to it, are used construct the solid–solid transition line. In both experiments, the PSP/PPS kinetic phase diagram exhibits a singularity in the liquidus line at the 0.5PSP concentration which divides the experimental kinetic phase diagram into two regions. One can distinguish a eutectic region from 0.0PSP to 0.5PSP and a monotectic region afterwards. The eutectic concentration, XE , is located at approximately 0.15PSP in both experiments. The simulation of the phase boundaries (presented in Section 3.2.4 below) confirms this analysis. This type of phase boundary is indicative of the formation of a 1:1 (mol:mol) compound (Minato et al., 1997b) as will be discussed later. Note that the eutectic behavior is more marked in the 0.1 ◦ C/min than in the 3.0 ◦ C/min cooling experiment. In the case of the 0.1 ◦ C/min cooling experiment, the solidus line is horizontal in the XPSP = [0.1–0.5] concentration range and the

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21

solid–solid transition line extends linearly to the XPSP = 0.6 composition with a slight shift to lower temperature with increasing PSP molar fraction (dashed lines passing through the experimental data points 䊉 and , respectively, in Fig. 6a) . The above lines show the same qualitative characteristics in the case of the 3.0 ◦ C/min cooling experiment where both extend to XPSP = 0.7 (dashed lines passing through the experimental data points 䊉 and , respectively, in Fig. 6b).

Fig. 7. Estimated enthalpies of melting (HM , kJ/mol) as a function of the PSP molar fraction for the PSP–PPS binary mixtures for (a) the 0.1 ◦ C/min and (b) the 3.0 ◦ C/min cooling rate (peak P1 , ; combined P2 and P3 endotherms, ; and exotherm, ♦).

3.2.4. Thermodynamic analysis of the boundaries in the phase diagram The liquidus line of the binary system was simulated using the thermodynamic model described in Section 2.4.2 with values of (HA , TA ), and (HB , TB ) as determined from the DSC heating curves of the purified PSP (A) and PPS (B) samples (Table 6). The nonideality of mixing parameter, , was adjusted first manually in small steps to obtain a liquidus line which lies closest to the experimental boundaries. This line was then refined to calculate the curve that has the least sum of squares of the difference between experimental and calculated temperatures over the whole experimental compositions. The calculated liquidus line of the binary system assuming an ideal fluid phase using Eqs. (1) and (2) did not reproduce the experimental phase boundary for either crystallization rate and is not shown. It was also impossible to reproduce the phase boundaries by the introduction of  when simply considering a eutectic behavior and using Eqs. (3) and (4) for the two sides of the eutectic. The fit of the experimental liquidus line has been possible only by considering the singularity at 0.5PSP and applying Eqs. (3) and (4) to both the eutectic and monotectic regions which it delimits. The experimental liquidus line has been very satisfactorily reproduced by applying the model to the two segments of the eutectic and to the segment of the monotectic region. Moreover, the singularity has been successfully confirmed at 0.5PSP in both experiments. The simulation yielded negative values of  for all segments, except for

Fig. 8. (a) XRD patterns of the least stable polymorphic form in PPS and PSP cooled at a rate of 25 ◦ C/min from 100 ◦ C to −25 ◦ C. (b) Heating curves of purified PSP and PPS obtained using a heating rate of 15 ◦ C/min for samples cooled at a rate of 25 ◦ C/min. (c) XRD patterns of the most stable polymorphic form in PPS and PSP cooled at a rate of 3 ◦ C/min from the nucleation temperature (54 ◦ C for PPS and 60 ◦ C for PSP) to −25 ◦ C. (d) Heating curves of purified PSP and PPS obtained using a heating rate of 5 ◦ C/min for samples cooled at a rate of 3 ◦ C/min from the nucleation temperature (54 ◦ C for PPS and 60 ◦ C for PSP).

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Table 6 Thermodynamic parameters H (kJ/mol), the molar heat of fusion and Tm (◦ C), the melting point of purified PSP (A), purified PPS (B) and 0.50PSP mixture (C) used to model the liquidus line in the phase diagram for the 0.1 ◦ C/min and 3.0 C/min cooling rates. TE , eutectic temperature, (◦ C); and XE , eutectic composition (PSP molar fraction) determined by the intersection of the calculated segments of the liquidus line. The non-ideality parameter  (kJ/mol) is derived from the simulation of the phase diagram boundaries in the 0 ≤ XPSP ≤ XE range, the XE ≤ XPSP ≤ 0.50PSP range and the 0.50PSP ≤ XPSP ≤ 1 range. Tm (◦ C)

0.1 ◦ C/min 3.0 ◦ C/min

B

C

A

B

C

70.8 70.6

59.2 57

62.4 62.6

130 114

113 116

124 115

Singularity

◦

0.1 C/min 3.0 ◦ C/min

H (kJ/mol)

A

0.50PSP 0.47PSP

XE

0.14 0.15

TE (◦ C)

55.8 56.8

 (kJ/mol) 0.0PSP − XE

XE − 0.50PSP

0.50PSP − 1.0PSP

−35.0 +14.0

−8.0 −4.0

−5.0 −3.5

Fig. 9. Selected SFC (%) versus time curves for (a) the 0.1 ◦ C/min and (b) the 3.0 ◦ C/min cooling rate. (1, 0.0PSP ; 2, 0.1PSP ; 3, 0.2PSP ; 4, 0.4PSP ; 5, 0.6PSP ; 6, 0.8PSP ; 7, 1.0PSP . The curves are shifted to the right to show the successive individual curves.) Induction time of the PSP–PPS mixtures, ti (min), relative to the induction time of the 1.0PSP mixture, as a function of the PSP molar fraction in the (c) 0.1 ◦ C/min and (d) 3.0 ◦ C/min cooling rate experiment. (e) Final solid fat content (SFC, in %) of the PSP–PPS binary mixtures as a function of the PSP molar fraction obtained at 0.1 ◦ C/min (䊉) and 3.0 ◦ C/min () cooling rate. The dashed lines in c–e are linear fits of the experimental points through which they pass and the dotted lines a visual indication of the zero of ti .

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Table 7 Avrami constant, A, and Avrami exponent, n, determined applying the modified form of the Avrami model (Narine et al., 2006) to the SFC (%) versus time curves of the PSP–PPS mixtures cooled at 0.1 ◦ C/min and 3.0 ◦ C/min. 0.1 ◦ C/min

Segment 1 A, 1 × 10−5

Segment 2 A, 1 × 10−3

n

n

0.1 ◦ C/min 0.0 0.1 0.2 0.4 0.6 0.8 1.0

1.10 0.78 1.84 1.08 2.60 3.40 2.64

± ± ± ± ± ± ±

1.70 0.25 0.40 0.50 0.60 0.60 1.11

1.2 1.3 1.3 1.3 1.2 1.2 1.5

± ± ± ± ± ± ±

0.1 0.2 0.3 0.1 0.2 0.1 0.3

1.03 1.01 0.71 0.90 0.81 1.25 1.56

± ± ± ± ± ± ±

0.03 0.17 0.10 0.10 0.14 0.05 0.06

1.2 1.2 1.2 1.2 1.1 1.1 1.1

± ± ± ± ± ± ±

0.1 0.2 0.2 0.1 0.2 0.1 0.1

3.0 ◦ C/min 0.0 0.1 0.2 0.4 0.6 0.8 1.0

1.10 0.80 1.84 1.10 2.56 3.40 5.24

± ± ± ± ± ± ±

0.35 0.25 0.40 0.40 0.45 0.06 0.89

1.2 1.4 1.3 1.3 1.3 1.2 1.8

± ± ± ± ± ± ±

0.1 0.1 0.2 0.2 0.2 0.3 0.1

1.03 1.01 0.71 0.89 0.81 1.25 1.56

± ± ± ± ± ± ±

0.03 0.17 0.10 0.10 0.14 0.05 0.06

1.2 1.2 1.2 1.1 1.2 1.1 1.1

± ± ± ± ± ± ±

0.1 0.1 0.2 0.1 0.1 0.2 0.1

the XPSP ≤ XE segment in the 3.0 ◦ C/min cooling experiment where it is positive. The simulated three segments of the liquidus line are represented by the solid lines in Fig. 6a and b for the 0.1 ◦ C/min and 3.0 ◦ C/min cooling experiments, respectively. The calculated values of , XE and TE are listed in Table 6.

Fig. 10. Relative hardness (maximum force, equivalent kg) of the PSP/PPS binary mixtures as a function of the PSP molar fraction obtained at 0.1 ◦ C/min (䊉) and 3.0 ◦ C/min () cooling rate.

3.3. Solid fat content measurements and analysis Selected SFC (%) versus time curves are shown in Fig. 9a and b for the 0.1 ◦ C/min and the 3.0 ◦ C/min cooling experiments, respectively.

Fig. 11. Polarized light micrographs taken at −25 ◦ C of the purified TAGs. Magnification is 100× and 500× for the micrographs obtained in the 0.1 ◦ C/min and the 3.0 ◦ C/min cooling rate experiment, respectively. (a) Purified PPS cooled at 0.1 ◦ C/min (bar = 100 ␮m), (b) purified PPS cooled at 3.0 ◦ C/min (bar = 20 ␮m), (c) purified PSP cooled at 0.1 ◦ C/min (bar = 100 ␮m), and (d) purified PSP cooled at 3.0 ◦ C/min (bar = 20 ␮m).

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Fig. 12. Selected polarized light micrographs taken at −25 ◦ C of samples cooled at 0.1 ◦ C/min. (Magnification = 100×, bar = 100 ␮m.) XPSP = (a) 0.1, (b) 0.2, (c) 0.4, (d) 0.6, (e) 0.8, and (f) 1.0.

Note that they are not represented in their actual time axis; they are shifted to the right to show the successive individual curves. The corresponding relative induction time, ti , versus PSP molar fraction is shown in Fig. 9c and d, respectively. The value of ti is measured relative to that of the 1.0PSP mixture (dotted lines in Fig. 9c and d). The final SFC (%) for both cooling rates is shown in Fig. 9e. All SFC curves display non-sigmoidal shapes, as can be seen more markedly in the SFC curve of the 1.0PSP sample (curve 7 in Fig. 9a and b). The effect of cooling rate on ti is noticeable, ti (0.0PSP ) is comparable to ti (1.0PSP ) when the mixtures are cooled at 3.0 ◦ C/min (Fig. 9d), whereas it is 38 min larger when cooled at 0.1 ◦ C/min (Fig. 9c). As can be seen, ti displays a maximum in both experiments. The effect of PSP content on the induction time is most dramatic when the samples are cooled at the slow rate. At this cooling rate, and up to 40 mol% PSP, the addition of PSP delayed crystallization. The crystallization of 1.0PSP was delayed by approximately 8 min compared to PPS. Up to 0.4PSP , crystallization was further delayed by approximately 0.1 min/1 mol% PSP. For mixtures with more than 0.4PSP , ti decreased linearly by approximately 0.8 min/1 mol% PSP. This trend is also seen when the samples were cooled at the high cooling rate. In this case, ti increased linearly (4 s/1 mol% PSP) for molar fractions between 0.0 and 0.2 and decreased linearly thereafter (0.7 s/1 mol% PSP). These are small but still significant changes which outline the effect of PSP on the crystallization behavior of the mixtures on both sides of the eutectic point. The SFC versus time curves were well fitted with the modified Avrami model using two distinct segments. The calculated Avrami parameters A and n are listed in Table 7. The first segment yielded almost the same values for A (∼1.9 × 10−5 ) and for n (∼1.3) for all mixtures in the 0.1 ◦ C/min cooling experiment. In the 3.0 ◦ C/min cooling experiment, it yielded the values of A ∼ 2.0 × 10−5 and n ∼ 1.3 for all mixtures except the purified PSP which yielded A = 5.24 × 10−5 and n = 1.8. Note that a visual inspection of the SFC

curves singles out this last mixture. The second segment yielded the same values for A (∼1 × 10−3 ) and n (∼1) for all the mixtures and in both experiments. All the PSP–PPS mixtures achieved a greater final SFC (%) in the slow cooling experiment compared to the fast cooling experiment (Fig. 9e). The measured final SFC (%) values lie between 99% and 99.3%. Apart from the purified PPS, the measured SFC (%) is the same for all mixtures in the 3.0 ◦ C/min cooling experiment. In the 0.1 ◦ C/min cooling experiment, the final SFC (%) versus XPSP shows a linear decrease up to 0.8PSP where a marked minimum is experienced suggesting that the microstructure, particularly PSP–PPS phase boundaries, achieved by the mixtures is the primary responsible for oil trapping and that PSP phase is less able to trap liquid in its network. 3.4. Relative hardness Notably, all samples were harder at the higher cooling rate (Fig. 10). For both cooling rates, the addition of small amounts of PSP (10 mol%) increased somehow the relative hardness which then decreased to a minimum at around 0.5PSP . Note that the depression is more pronounced for the mixtures cooled at 0.1 ◦ C/min. 3.5. Microscopy The microstructure as revealed by PLM shows striking differences in the shape of the crystalline network of the purified PPS and PSP (Fig. 11a–d). To show the microstructural details seen in the purified samples cooled at the 3.0 ◦ C/min cooling, the micrographs taken with 500× magnification, are presented (Fig. 11b and d). When cooled at 0.1 ◦ C/min, PPS displays well defined and very tightly packed Maltese cross shaped crystallites with an average radius of 200 ␮m (Fig. 11a) whereas PSP grow very large dendritic

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25

Fig. 13. Selected polarized light micrographs taken at −25 ◦ C of samples cooled at 3.0 ◦ C/min. (Magnification = 100×, bar = 100 ␮m.) XPSP = (a) 0.1, (b) 0.2, (c) 0.4, (d) 0.6, (e) 0.8, and (f) 1.0.

crystallites of approximately 400–450-␮m radius (Fig. 11c). The wavy radial growth of this TAG is noticeable and contrasts with the straight radial growth of PPS indicating a rapid growth through selected facets of the PSP crystals compared to an isotropic growth for the PPS crystals. The straight and clean boundaries seen in PPS microstructure also contrast with the apparent relative interlocking of the PSP crystallites. When cooled at 3.0 ◦ C/min, PPS crystallizes in a network-like microstructure constituted of a large number of interlocked small crystallites with an average estimated diameter of 5–10 ␮m (Fig. 11b), whereas, PSP has well defined larger crystallites with an average estimated diameter of 80 ␮m (Fig. 11d). Both microstructures show smooth Maltese crosses indicative of spherulitic growth. In the 0.1 ◦ C/min cooling experiment, the addition of up to 20 mol% PSP to PPS dramatically increases the number of crystallites. For example, the number of crystallites seen in the micrographs of 0.2PSP is 5 times that seen for the purified PPS (Fig. 12a). Moreover, as PSP is added, the PLM images of the mixtures present a more defined microstructure with better defined Maltese crosses. The microstructure changes markedly for mixtures with larger PSP content suggesting a direct relationship with the eutectic behavior observed by DSC. The fewer spherulites shown in the micrographs of the 0.3PSP to 0.6PSP mixtures have fuzzy centers of growth and uneven radiuses of growth reminiscent of the pure PSP microstructure. The crystal network looks as an intricate but separate combination of different phases which can be related to the close separate peaks detected in the DSC cooling thermogram and which are assigned to PPS-rich, PSP-rich and ␣C phases. The fuzzy network is therefore, the result of an inhomogeneous distribution of the phases in the solid. The boundaries between the grains are relatively large and undefined (Fig. 12b–d). However, as PSP content is increased, the microstructures and the boundaries sharpen, producing a microstructure with very well defined Maltese cross-

shaped crystallites which impinge and define clear, thin boundaries (Fig. 12e). The microstructure of the 0.9PSP mixture is constituted of a dendritic-like network similar to that of the purified PSP. The formation of dendrites indicates a rapid growth of the crystals through selected facets. In the 3.0 ◦ C/min experiment, only Maltese cross-shaped crystallites are seen in the micrographs (Fig. 13). The 0.0PSP and the 0.1PSP mixtures presents small and interlocked crystallites without defined boundaries (Fig. 13a and b). Noticeably larger crystallites with clearer Maltese crosses and very well defined boundaries appear for the 0.2PSP concentration. This is clearly related to the eutectic composition. Their size increases further with increasing XPSP up to the 0.8PSP concentration. The crystallites are very tightly packed, probably due to strong impingement which also induced long, thin boundaries between the crystallites, giving them square or lozenge-like shapes. This indicates a homogeneous distribution of the phases. Notice that smaller Maltese cross-like crystallites are also present and evenly distributed in the mixtures (Fig. 13c–e). These could be crystallites of PPS as they nucleate later than PSP. There is no other evidence to distinguish the different phases or relate features of the micrographs to specific phases. The 0.9PSP shows smaller crystallites and an overall microstructure similar to that of the purified PSP. 4. Discussion 4.1. Crystallization behavior, polymorphism and phase development The two peaks, P1 and P2 , which are resolved for the mixtures with XPSP ≥ 0.2 in the slow cooling experiment, represent two different thermal events that indicate a phase separation or de-mixing during the crystallization process (MacNaughtan et al., 2006). The

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Fig. 14. Example of a double chain length (DCL) packing of a TAG molecule in a chair-shaped conformation (van Mechelen et al., 2008a,b).

close alignment of P1 and P2 with the crystallization temperature of PPS and PSP, respectively allows for a tentative attribution of P1 to the crystallization of a PPS-rich component and P2 to the crystallization of a PSP-rich component. Given the very narrow range of temperature where P1 and P2 appear and the fact that XRD detected only an ␣-polymorph for all mixtures, both peaks are attributable to the crystallization of similar ␣-forms which cannot be resolved with our XRD system. In this experiment, the singularity appearing at XPSP = 0.5 in the liquidus line constructed using the DSC cooling thermograms (indicated by an arrow in Fig. 3c) is an indication of the formation of a 1:1 (mol:mol) compound, ␣C . If one assumes that all available PSP and PPS molecules in a mixture form ␣C , the relative content of the ␣C phase would decrease equally away from the 0.5PSP molar ratio and induce corresponding effect on the physical properties governed by this phase. The 0.5PSP mixture displaying a central role for all measured thermal properties, particularly with its closest mixtures in concentration, i.e. the 0.4PSP to 0.6PSP mixtures in the case of the 0.1 ◦ C/min cooling experiment and 0.2PSP to 0.7PSP mixtures in the case of the 3.0 ◦ C/min cooling experiment, is strong support of the coexistence of ␣C with PPS in one side of the phase diagram and with PSP in the other. The symmetricity about XPSP = 0.5 of HC1 and HC2 versus XPSP curves (Fig. 4b) confirms the presence of qualitatively different phases in the two concentration regions separated by 0.5PSP ; a PSP- and a PPS-rich phases which would form a monotectic and a eutectic, respectively with ␣C . The linear decrease of the eutectic line with increasing LSL content (dashed line passing through the experimental data points  in Fig. 3c) indicates that even at this very slow cooling rate, kinetics still affect crystal growth. The existence of different molecular interactions in the two concentration regions manifests itself by the presence of an “interchange coupling” revealed by the enthalpy of crystallization of the PSP-rich and PPS-rich components. In the absence of such a coupling, the enthalpies of melting of the PSP-rich component would increase linearly and that of PPS-rich component would decrease linearly with increasing PSP content. The magnitude of the coupling can be derived from the difference between the measured (open and filled triangles in Fig. 4b) and the extrapolated (dashed lines in Fig. 4b) values of enthalpy in the absence of coupling. One also can notice the symmetry about XPSP = 0.5 of the coupling. The presence of ␣C , which coexists with PPS in one concentration region and with PSP in the other, may largely explain these differences. The cooling rate did not affect the XRD results. The d-spacings as well as the intensities were essentially the same in both experiments. Moreover, in another experiment which is not detailed here, the purified PSP and PPS cooled down from 100 ◦ C to 0 ◦ C using a cooling rate of 1 ◦ C/min and measured regularly during a period of one month storage at 0 ◦ C yielded the same XRD results. The single subcell reflection d␣ which appears for all measured mixtures is due to the {1 0 0} plane of a hexagonal subcell and shows that only

the metastable ␣-polymorph, the least stable of all possible polymorphs, can grow under the processing conditions of this study. All the measured d-spacings correspond to a DCL structure and reveals an isotropic packing of the hydrocarbon chains (Ghotra et al., 2002; Timms, 2003). The measured lamellar periodicity length, LP , for the pure PSP and for pure PPS are in agreement with the calculated PSP–PSP and PPS–PPS bilayers’ length, respectively, using a CH2 CH2 unit length of 2.54 Å (Birker et al., 1991). The ␣polymorph consists of loosely packed vertical chains and may exist as a DCL and/or TCL structure (Timms, 2003), but there is no experimental evidence for the existence of a TCL structure for any of the PSP–PPS mixtures. The very close similarity of cell parameters, including the c-axis length (i.e. LP value) of the ␣-form obtained in this study and those reported for the ␤-2 and ␤ -2 forms of PPS and PSP (van Mechelen et al., 2008a,b) suggests that the TAGs in the PSP–PPS mixtures adopt the same chair-shaped conformation. In this conformation, the TAGs are packed in symmetry-related pairs with the seats of the chairs facing each other, with an inversion point in-between the seats (Fig. 14) similarly to the trisaturated monoacid TAGs such as ␤-2 SSS and ␤-2 MMM (van Langevelde et al., 1999). The pairs of chairs form layers with a double chainlength thickness, referred to as ‘two-packs’; the ‘two-packs’ facing each other at the methyl end-plane. One would then think that a CLM of 2 between the FA moieties, while being a hindrance to the promotion of more stable crystalline forms, does not affect the overall lateral packing of the TAGs. The conjunction of the strong effect of CLM with symmetry and molecular weight on the polymorphism and hence on the physical properties of the mixtures is discussed in later paragraphs. The miscibility of PSP and PPS TAGs in the solid state and the formation of a compound cannot be fully resolved using our XRD data. Because PSP and PPS, mixed or not, would pack in similar size structures, the XRD lines originating from them would be very similar and would not be resolved by the XRD system used in this study. The position of the XRD lines in the small diffraction-angle region show that the packing morphology did not change, but the line shapes are obviously not those of single lines. However, fitting accurately the XRD lines with two separate contributions was not possible. Phase separation and compound formation will be further discussed in light of the DSC results. The fact that only the ␣-polymorph is detected in our study outlines the major role of thermal history on the polymorphism of the PSP–PPS mixtures and the difficulty to grow the stable forms from the melt using constant cooling rates even as low as 0.1 ◦ C/min. This is not surprising. Using temperature controlled XRPD and DSC, Gibon et al. (1985, 1986) have found that after melting and quenching (25 ◦ C/min), pure PSP crystallizes in an ␣-form and changes,  when heated at constant rate (5 ◦ C/min), into a ␤2 -2 form which in turn recrystallizes with the occurrence of an exothermic peak into a  more perfect form, labeled ␤1 -2. They reported that PSP stabilizes

M.V. Boodhoo et al. / Chemistry and Physics of Lipids 160 (2009) 11–32 

into the less stable ␤2 -2 polymorphic form only when tempered for a few months at relatively high temperature (50 ◦ C) due to its high melting point and lower transformation rate. This incidentally explains why our samples did not transform into a more stable form when tempered at 0 ◦ C. The pure tempered PSP has been found to melt at the same temperature and in the same polymorphic form as the non-tempered one. Note that it has been known for some time, now, that PSP and PPS can be stabilized in the ␤ -form (Lutton et al., 1948; Lutton, 1950, 1972). More recently, the ␤ -forms of PSP and PPS have been solved and their melting points determined from HRPSD, time resolved XRD data and DSC, and discussed in great detail (van Mechelen et al., 2008b). Using a thermal protocol consisting of quenching a seed-free melt from ∼10 ◦ C above the melting point of the most stable ␤ -polymorph to −20 ◦ C and subsequent heating of the sample at 0.5 ◦ C/min, they characterized fully the development    of the polymorphs: ␣ transforming into ␤2 , ␤2 transforming into ␤1  and, finally, ending in a melt of the ␤1 . The resolved ␤ -forms of PSP and PPS have been found to pack in a DCL structure with different molecular conformations for the symmetrical and the asymmetri  cal TAGs. Although the ␤2 to ␤1 transition process is clearly visible with their time-resolved XRD equipment, it was not easy for them to trace with DSC using the same temperature profile. The melting temperatures of the ␤ -forms of PSP and PPS agree very well with the peak temperatures of the endotherms obtained from our isolated ␤ -phases (Table 5). Their reported transition temperatures also agree with the TR -values obtained for PSP and PPS in this study. The ␤-polymorphs of PSP and PPS exist, but they are difficult to obtain and even impossible to form from the melt (van Mechelen et al., 2008a). Nevertheless, their structures have been solved and their melting points determined from HR-PSD, time resolved XRD data and DSC (van Mechelen et al., 2008a, b). The resolved ␤-forms of PSP and PPS have been found to pack in a DCL structure with the same molecular conformations for the symmetrical and the asymmetrical TAGs. The authors acknowledged that they have used the samples obtained as powders from a supplier, as is, and could not retrieve the methods used to obtain the ␤-phase and suggested that it was probably achieved via crystallization from a solvent. For the mixtures studied here, it is very likely that the perturbation which takes place at the “terraces” level is increased by the small CLM between the long stearic and palmitic hydrocarbon chains. The interpenetration of the chains that would stabilize the ␤ -form is obviously not favored by the thermal protocol used to crystallize the mixtures. This seems to be achievable only with careful tempering at high temperature, and even then, PPS does not  easily transform. A ␤1 -2 to ␤-2 transformation is even more difficult to achieve as the longer C18 stearic chains prevent the occurrence of any rotations and translations transformation as suggested by Lutton (1971). There is a large melting temperature difference (∼18 ◦ C) between the two phases which may be explained by differences in crystalline symmetry. CLM between the FA moieties being the same in PSP and PPS, the packing at the methyl end-plane and the position of the step plane, must be the determining factors. The complexity of the heating thermograms results from the co-transformation and melt of PSP- and PPS-rich phases; the low temperature features of the thermogram representing the transformation of PPS-rich components and the high temperature features those of the PSP-rich components. Very small differences in total chain length, or a simple rearrangement of the chains in PSP with PPS, are so critical in this regard. Based on the XRD results, only the ␣-form pre-existed in all the mixtures before reheating. It is therefore safe to attribute the first endotherm P1 , which aligns close to the temperature of the first endotherm appearing for the purified PPS, to the direct melting from the ␣-phase (thermogram of the 0.1PSP mixture in Fig. 5c). P2 of PPS and P3 of PSP have been undoubtedly shown as the melting

27

of their respective ␤ -phases. P2 (either as resolved peaks or shoulders), the endotherm which aligns close to the temperature of the second endotherm appearing for the purified PPS, can be assigned to the melting of a ␤ -polymorph. In the 0.3PSP to 0.7PSP concentration region, P2 is visible at approximately 56 ◦ C directly followed by the endotherm P3 without an intermediate exotherm. This is probably due to a concomitant recrystallization of a higher melting  temperature ␤ -phase, probably the ␤1 -form according to the literature, and melt of a lower melting temperature ␤ -phase, probably   the ␤2 -form. P2 can be assigned to the melting of the ␤1 -form of  the PPS-rich phase and P3 to the melting of the ␤1 -form of the PSPrich phase (thermogram of the 0.5PSP mixture in Fig. 5c). In the case of the PSP-rich mixtures, 0.9PSP and 1.0PSP , the endotherm which would align with P1 and characterize the melting of the ␣-phase is not detected. This is probably due to a direct recrystallization of a ␤ form of PSP as suggested by our experiments conducted to isolate this phase in PSP (see Fig. 8b and comments in Section 3.2.2), which produces an exotherm which compensates for the endotherm due to the melting of the pre-existing ␣-phase. The exotherm appearing in the heating thermograms of these mixtures can be attributed to the crystallization mediated by melt of the most ordered ␤ -form, which in turn melts (thermogram of the 0.9PSP mixture in Fig. 5c). The polymorphic transitions were influenced by the kinetic effects of the cooling rate, but not at the extent of changing the sequence of the transitions. The exact nature of the polymorphs involved in the different transformations cannot be directly inferred from the heating thermograms alone. However, based on our polymorphic phase determination and from other detailed studies (Gibon et al., 1986; van Mechelen et al., 2008b), it is safe to assume the following concomitant polymorphic development for both PSPand PPS-rich phases: the ␣-phase melts and recrystallizes into a low  temperature ␤ -phase, i.e. ␤2 , which finally transforms to a higher   temperature ␤ -phase, i.e ␤1 . 4.2. Analysis of the kinetic phase diagrams The data in Table 3 supports a kinetic phase diagram of the PSP/PPS system cooled at 0.1 ◦ C/min (Fig. 6a) and 3.0 ◦ C/min (Fig. 6b), in which the liquidus line suggests the formation of a compound, ␣C , having the 1:1 (mol:mol) composition. It displays two regions separated at the 0.5PSP mixture: a region of PPS/␣C with a typical eutectic behavior spanning from 0.0PSP to 0.5PSP and a monotectic region of PSP/␣C from 0.5PSP to 1.0PSP . The simulation of the phase diagram boundaries confirms the position of the singularity in the liquidus line. Note that the reported position of the eutectic point as well as of the transformation lines depends on the thermal procedure used to identify phase transformation and development. The thermal protocol (cool and heat at constant rates) used to construct the phase diagrams of our binary systems do not produce equilibrium states. However, they allow the study of each TAG solubility and may be extrapolated to describe equilibrium states. They are also interesting from an applied view point as the thermal protocol closely similar to that/those used in industry. The PSP/PPS binary phase diagram obtained a cooling rate of 0.1 ◦ C/min is comparable to that obtained earlier using differential thermal analysis (DTA) measurements of PSP/PPS samples heated and cooled at a constant rate of 1.2 ◦ C/min (Perron et al., 1971). Their phase diagram is less detailed than our own, but shows a singularity at the 0.5PSP mixture as well as a eutectic point at ∼0.1PSP . The same group established later an equilibrium phase diagram of the system using an extensive and complex thermal procedure so-called “thermal conditioning” involving oscillating the temperature of the sample around TM and slow heating rate (0.1 ◦ C/min) (Ollivon and Perron, 1979). Their second phase diagram is more detailed than their previous work and show clearly a singularity at ∼0.5PSP and a eutectic point at ∼0.38PSP with a eutectic transformation at ∼61 ◦ C spanning

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from 0.15PSP to 0.6PSP . The authors suggested a probable peritectic transformation at ∼63 ◦ C spanning from 0.55PSP to 0.85PSP , but could not obtain it directly from their experimental data. The equilibrium solid–liquid phase behavior of 15 binary systems of six TAGs with palmitic and stearic chains including the PSP/PSS system was investigated with a microcalorimeter using an extensive cycling stabilization procedure (Knoester et al., 1972). The cycle consisted of heating the sample to the clear point using a rate of 0.1 ◦ C/min and chilling to low temperature. A eutectic type phase diagram (XE = 0.3 and TE ∼ 61 ◦ C) for the PSP/PPS binary system was obtained, and interpreted in terms of ideal miscibility in the liquid phase and immiscibility in the solid-state. Little work has been reported on the molecular structures and kinetic properties of systems which form molecular compounds such as SPS/PSS and PPP/PPS (Knoester et al., 1972). The formation of a 1:1 molecular compound has been suggested in both SPS–PSS and PPP–PPS to explain the singularity of the clear-point of the 1:1 mixture which is well above the liquidus curve (Knoester et al., 1972). The phase diagram shows clearly a eutectic behavior in each side of the 1:1 (mol:mol) composition for both systems. The formation of a 1:1 molecular compound in these systems was justified by conformational considerations. The authors suggested that the shape of the TAG molecules is such that a very dense packing becomes possible with equal amounts of both molecules, though the crystals of each of the pure components can accommodate only a small amount of the other component. The formation of a 1:1 molecular compound is also observed in systems of two TAGs which both contain an unsaturated fatty acid such as POP/OPO (Rossell, 1967), SOS/OSO (Koyano et al., 1992), POP/PPO and POP/OPO (Minato et al., 1997a,b), and SOS/SSO (Engstrom, 1992; Takeuchi et al., 2002). The formation of such compound in SOS/SSO (Engstrom, 1992) and SOS/OSO (Koyano et al., 1992) for example, is explained by specific molecular interactions through the acyl chain moieties. It has been suggested that the arrangement in these systems is less problematic, as like chains from either TAG can arrange themselves together, than in mixtures where monosaturated and mixed-acid saturated/unsaturated TAGs are combined, such as the PPP/POP system (Minato, 1996), where there is a pronounced steric effect. Such a mechanical interlocking of the TAGs cannot fully explain the formation of the compound in the case of our samples. It is however, possible that due to specific interactions (molecular interactions of acyl chain packing, glycerol conformation, and methyl end stacking) as is generally accepted (Timms, 1984), symmetrical and asymmetrical TAGs can display a synergistic compatibility and pack more easily together than on their own to form a molecular compound. Note that as reported by Zhang et al. (2007), the formation of a molecular compounds of a DCL structure in POP/PPO and POP/OPO mixtures appears to be most influenced by the contribution of the glycerol conformation. More experimental and modeling work is needed to understand this behavior. The effect of the cooling rate on mixing is quite interesting. The sensitivity to the processing conditions of PPS-rich mixtures was noticeable in the liquidus line simulation results. The kinetic effects are clearly seen, particularly dramatically in the XPPS ≤ XE side of the phase diagram. The experimental kinetic phase diagram of the PSP/PPS binary system was well described by the introduction of negative values of  for all the segments considered, apart from the XPSP ≤ XE region in the 3.0 ◦ C/min cooling experiment (Table 6). In this region, a large positive value was necessary to fit the data, and the calculated line has a relatively flat minimum centered at 0.14PSP . In this case, however, given the weakness of the slope of the liquidus segment and the small number of experimental points which were fitted, the calculated -value carries a large uncertainty of around 5 kJ/mol compared the uncertainty attached to the other calculated -values which is less than 0.3 kJ/mol. However, even if the uncertainty attached to the calculated  for the XPPS ≤ XE con-

centration region is much larger than in the others, the positive value of  calculated in the 3.0 ◦ C/min cooling experiment, contrast strikingly with the large negative value calculated in the 0.1 ◦ C/min cooling experiment. Recall that the Bragg–Williams approximation attributes the origin of the non-ideality of mixing to the enthalpy term of the free energy of mixing and assumes the same entropy term as in the ideal mixing case (Moore, 1972). The non-ideality of the mixing parameter, , is the energy difference between (A–B) pair and the average of (A–A) pair and (B–B) pair. For ideal mixing,  is zero. Positive  reflects a tendency of like molecules to cluster, which beyond some critical value, c, leads to a phase separation. A negative  reflects a tendency for order, i.e. the formation of AB pairs is energetically more favorable compared with AA or BB pair formation (Lee, 1977b). The molecular interactions, as depicted by the negative -values, are strong and tend to favor the formation of unlike pairs in the liquid state. These values are comparable to published values for binary lipid systems such as binary mixtures of diacylphosphatidylethanolamines (Nibu and Inoue, 1995), fatty acids (Inoue et al., 2004b), propanediol diacetates (Abes et al., 2007, 2008) and TAGs (Boodhoo et al., 2008, submitted for publication, 2009a). Although the time available during the 3.0 ◦ C/min cooling experiment for crystal growth is far smaller relative to the 0.1 ◦ C/min experiment and a difference in behavior is not to be excluded (Chen et al., 2005), the miscibility in the XPPS ≥ XE concentration region as inferred from the model in both experiments did not change dramatically with the processing condition. Other key parameters obtained from the phase diagram of this lipid system, such as the solid–solid transformation lines, did not show any dependence on the kinetics. The effect of cooling rate is, however, obviously revealed in the shift of the maximum of the estimated HR versus XPSP from 0.3PSP to 0.5PSP (Fig. 7a and b) and in the blurring of the eutectic composition when the cooling rate is increased (Fig. 6a and b). The marked kinetic effects seen in the XPSP ≤ XE concentration region, as reflected by the flattening of the liquidus line at the eutectic composition in the fast cooling experiment, is probably due to the halting effect of the asymmetrical PPS which combined to a rapid crystallization prevented the formation of the most stable packing and conformation possible. This is supported by SFC data, where a net increase in the relative induction time is seen for this concentration region, followed by a steep linear decrease (Fig. 9c and d). In the solid-state, the lipid molecules with rigid hydrocarbon chains are forced to arrange within a defined crystalline structure resulting in greatly reduced miscibility in the solid phase. The solid phase which is growing at conditions well away from equilibrium is determined by different crystallization growth modes at different crystallization times. This is supported by the SFC (%) versus time analysis and PLM micrographs as discussed below. The solid phase depends on the surface kinetics at the solidification front and on the transport of mass and heat away from that front, as is seen for the asymmetrical TAG sample as manifested in the thermal behavior of the mixtures of the XPSP ≤ XE concentration region. The simultaneous simulation of the liquidus and solidus lines, for the whole phase diagram as well as for the eutectic region and monotectic region alone, have been inconclusive due to the simplicity of the model, which does not take into account the dependence of the non-ideality parameter with molar fraction. 4.3. Solid fat content, relative hardness and microscopy The application of the modified Avrami model confirms what can already be qualitatively deduced from the shape of the SFC versus time curves. Two growth modes characterized each by an Avrami constant, A, and an Avrami exponent, n, are detected. In both

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experiments, the A values of the second segment are two orders of magnitude larger than those of the first segment and its n values are only slightly higher. This indicates that as the crystallization proceeds, the rate of crystallization increases without altering significantly the mode of growth. A is dependent upon the nucleation rate, suggesting a similar nucleation rate for all samples. The exponent n is a function of the number of dimensions in which growth takes place. These higher A and n values for pure PSP implies a higher crystallization rate, as well as a different mode of growth (n ∼ 2). Note that the growth characteristics of the eutectic composition as revealed by A and n are similar to the other mixtures. Except for different A values for the various segments, the Avrami index n was ∼1 at both cooling rates, indicating a common mode of growth. There were, however, subtle differences in these non-integer values for n. Possible explanations put forward for such small n-values include the changing rate of radial growth, geometric restrictions to domain growth, or differences in underlying geometries which influence growth (Yang and Nagle, 1988). Except for the pure PSP, the crystallization rate and the mode of growth were not sensitive to the cooling rate. This could be explained, if one considers that the overall crystallization kinetic is driven by the formation of the compound, by the interactions of PPS with the compound in the eutectic region and the interaction of PSP with the compound in the monotectic region and their relative kinetics. The change with molar fraction in image sharpness, size of the crystallites and overall crystallinity shown by PLM seem to indicate that the symmetrical TAG (i.e. PSP) is the component which is most effective in driving crystal growth at both cooling rates. The eutectic composition delimits clearly different microstructure in both experiments. As observed in the micrographs of the 0.3PSP to 0.6PSP mixtures when cooled at 0.1 ◦ C/min, the microstructure may be constituted of an intricate combination of the ␣C , PPSand PSP-rich phases which have been detected in the DSC cooling thermogram. The vague boundaries between the crystallites and fuzzy network seen in these mixtures suggest inhomogeneous distribution of these phases in the solid. The ordering effect of PSP is illustrated in increased sharpness of the boundaries as PSP content increases. The dendritic-like growth of the crystal in PSP-rich mixtures indicates that growth tends to occur along definite directions rather than random directions. When cooled at 3.0 ◦ C/min, the crystallites are smaller and evenly distributed, due to more nucleation sites and rapid growth, allowing for a homogeneous distribution of the phases. There is no microstructural evidence to distinguish between different phases. This can be related to the large and convoluted exotherm seen in the cooling thermogram for each mixture at this cooling rate. However, the smaller crystallites which show in the micrographs (Fig. 13c–e) could be tentatively related to crystallites of PPS-phase as they nucleate later than PSP phase. The change of ti versus composition (Fig. 9c) clearly indicates that there are strong interactions between PPS and PSP. The induction time of the asymmetrical PPS was higher than that of the symmetrical PSP which indicates the latter’s propensity to pack more readily. Even at lower concentrations, the asymmetrical TAG dramatically slows the crystallization, i.e. the inception of the PSPrich phase. This shows that the asymmetrical PPS TAG puts a “break” on the crystallization process similar to that noted for the asymmetrical POS in POP- and POS-TAGs (Rousset and Rappaz, 1997; Rousset et al., 1998). At concentrations up to 0.5PSP and 0.2PSP in the 0.1 ◦ C/min and the 3.0 ◦ C/min cooling experiments, respectively, the nucleation of the PSP phase is delayed by the presence of PPS molecules. One can correlate the changes in ti with the presence of the compound as detected by the cooling thermograms in the 0.1 ◦ C/min experiment and with the broad minimum starting with the 0.2PSP mixture seen in the DSC crystallization peaks characteristics.

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All the mixtures attained significantly the same final SFC (%) of 99.12 ± 0.02% when cooled at 3.0 ◦ C/min and experienced a small, but significant, depression around 0.8PSP when cooled at 0.1 ◦ C/min (Fig. 9e). The depression in SFC (%) may be due to the microstructure of the compound, which would allow for more liquid component than the microstructure of adjacent PSP or PPS (depending on the concentration region). Note that the difference in final SFC (%) between any two compositions is less than 2%. This explanation would be valid if one assumes that the liquid part is trapped preferably between adjacent different domains and not between the same adjacent ones. The depression in relative hardness would not be surprising as it is entirely conceivable that the boundaries, in which the liquid part is entrapped, would weaken the grain–grain bonds in the fat network and present less resistance to the probe than the others. The small, but significant, dip seen in relative hardness for the 3.0 ◦ C/min indicates that the contribution of such effects are pronounced enough to be finite even for differences in final SFC that could not be accurately measured. If one can correlate the depressions in SFC and relative hardness using such an explanation, the larger hardness values for the 3.0 ◦ C/min cooling experiment compared to those for the 0.1 ◦ C/min cooling experiment would clearly mean that the SFC is not the only explanation for the changes in hardness. It is likely that the relatively smaller crystallites which were formed when the samples were cooled at 3.0 ◦ C/min compared to when cooled at 0.1 ◦ C/min, play a significant role in this regard. A pinning effect due to the small grains, which may be composed of a PPS-phase, formed when the mixtures are cooled at 3.0 ◦ C/min could play a role in the difference in hardness measured in the two experiments. One may see the almost parallel trends and directly link them to the relative sizes of the crystallites formed. However, there is no other direct evidence to relate a specific phase to the relative hardness of the mixtures. 5. Conclusion The binary phase behavior of purified PSP and PPS was investigated at a very slow (0.1 ◦ C/min) and a relatively fast (3.0 ◦ C/min) cooling rate in terms of melting and crystallization, polymorphism, solid fat content, hardness and microstructure. Only the ␣-form of a DCL structure was detected for all mixtures in both experiments. The kinetic phase diagram constructed using the heating DSC thermograms displayed a singularity at the 0.5PSP composition which was attributed to the formation of a molecular compound, ␣C , at the 1:1 (mol:mol) concentration. This analysis is supported by the simulation results of the liquidus line in the kinetic phase diagram, with a thermodynamic model based on the Hildebrand equation. The central position of the 0.5PSP mixture for all measured properties corroborates the formation of ␣C . Two distinct behaviors in the kinetic phase diagram are seen: a eutectic behavior of PPS/␣C in the XPSP ≤ 0.5PSP and a monotectic behavior of PSP/␣C in the XPSP ≥ 0.5PSP concentration region. The apparent eutectic point, is well defined in the 0.1 ◦ C/min cooling experiment at approximately XE = 0.15PSP and TE of 55.8 ◦ C, but in the 3.0 ◦ C/min cooling experiment, it is fuzzy and located on a flat minimum centered at the 0.14PSP composition and TE of 56.8 ◦ C. The simulation of the liquidus line yielded negative values of the non-ideality parameter, , for all its segments except for the 0.0PSP ≤ TE concentration region in the 3.0 ◦ C/min cooling experiment, where it yielded a positive value. The molecular interactions, as depicted by the negative -values, were found to be strong and to favor the formation of unlike pairs in the liquid state. Apart from the XPSP ≤ XE side of the phase diagram, the miscibility as inferred from the simulation of the liquidus line using a model based on the Hildebrand equation did not change dramatically with the processing conditions. The kinetic effects are manifest in all other measured properties, particularly dramatically in the XPSP ≤ XE concentration

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region. The shift and blurring of XE in the fast cooling experiment and the simulation results of the corresponding segment of the liquidus line highlights the importance of the kinetic effects. Even if the uncertainty attached to the calculated  for this concentration region is much larger than in the others, the positive value of  calculated in the 3.0 ◦ C/min cooling experiment contrasts strikingly with the large negative value calculated in the 0.1 ◦ C/min cooling experiment. The analysis of the induction time as measured by SFC showed that PPS is dramatically “slowing down” crystal growth, an effect which can explain the peculiarity of the PPS-rich concentration region. In both experiments, the SFC (%) versus time curves show two distinct segments of growth for each mixture. The fit of the curves to a modified form of the Avrami model revealed that the growth modes are similar for all the mixtures. The PLM of the PSP–PPS mixtures showed networks made of spherulitic crystallites of size, growth direction and boundaries that are varied and sensitive to composition and cooling rate. The change in the microstructure is particularly noticeable at compositions close to the eutectic. PSP component was found to be instrumental in promoting tighter packing, more homogeneous crystallization and more organized networks. The measured final SFC (%) and the effect of the cooling rate on the microstructure explained in part the differences seen in relative hardness. The mixtures of the PSP–PPS system with CLM between FA moieties of 2, under the thermal condition used in our studies pack in lower stability forms than their asymmetrical/symmetrical counterparts with higher CLM. This is understandable if one takes into account the disturbances introduced at the methyl-end level and the higher probability that a TAG with a larger CLM, such as MSM or MMS, packs in a higher stability form. The disturbance being more pronounced for PPS than PSP, could explain also the relative ordering effect of PSP on crystal perfection and homogeneity. However, the higher temperature difference between the melting temperature of the most stable forms of the symmetrical and asymmetrical TAGs of ∼18 ◦ C as well as the value of XE (0.15 in both the slow and fast cooling experiments), does not fit in the trends observed for the asymmetrical/symmetrical binary system studied in our laboratory. This suggests that even if melting temperature and other thermophysical properties, which are bulk properties in many respects, strongly reflect molecular features of symmetry (Wei, 1999), they are also determined largely, if not predominantly, by other parameters, such as molecular weight and specific bonding (Godavarthy et al., 2006, 2008). The competition between the strong effect of molecular weight and CLM between FA moieties on the behavior of symmetrical/asymmetrical mixtures is also illustrated by the formation of a “molecular compound” in PSP–PPS (CLM = 2) and LSL–LLS (CLM = 6) mixtures contrary to the MSM–MMS (Boodhoo et al., 2008) or CSC–CCS (Boodhoo et al., 2009a) mixtures, where CLM is 4 and 8. Our work highlights the critical effect of the conformation of the molecules on the phase behavior of TAG mixtures and shows that even small differences in total chain length, or a simple rearrangement of the chains as in PSP with PPS, have dramatic effect on the mutual solubility of long-chain saturated TAGs. The fact that using simple thermal protocol such as those applied in our studies, PSP–PPS mixture can only be stabilized in lower polymorphic forms is useful for food application in particular, as fat crystals in these forms provides a smooth feel in the mouth (Wiedermann, 1978; Ghotra et al., 2002) whereas transformation into the ␤-form produces textural changes. For example, the problem of graininess in margarines due to the occurrence of ␤-phase crystallites is well known (Watanabe et al., 1992). The formation of molecular compounds impacts upon the performance of fractionation processes, as only limited separation is thus experienced, but this can be useful for blending purposes (Sato and Ueno, 2001; Koyano et al., 1992).

Acknowledgments We would like to thank Bunge Oils, Alberta Agricultural Research Institute, Alberta Canola Producers Commission, Alberta Crop Industry Development Fund, Alberta Agriculture Food and Rural Development and NSERC for financial support. The authors would also like to acknowledge Ereddad Kharraz for his technical assistance and Vanessa Incani and Mildred Koranteng for their assistance in the synthesis of PPS and PSP.

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