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New Insights into the β Polymorphism of 1,3-Palmitoyl-stearoyl-2oleoyl Glycerol Saeed M Ghazani and Alejandro G Marangoni*

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Department of Food Science, University of Guelph, Guelph, Ontario, N1G 2W1, Canada ABSTRACT: In this communication, we report on the discovery of two new triclinic crystal polymorphic forms (β3 and β2) of 1,3-palmitoylstearoyl-2-oleoyl glycerol (POS), the most abundant triacylglycerol molecular species in cocoa butter. Three distinct triclinic crystal forms were crystallized from acetone and were found to have melting points of 32.9 °C (β3), 33.8 °C (β2), and 38.7 °C (β1), and enthalpies of fusion of 141.5 kJ/mol (β3), 147.9 kJ/mol (β2), and 158.5 kJ/mol (β1). All crystal forms had a similar long spacing (d001 = 64.1, 63.5, and 63.9 Å for β3, β2, and β1, respectively) but displayed distinctly different wide-angle scattering patterns, i.e., short-spacings. In this communication, we clarify the 60-yearold confusion related to the polymorphism of POS, a key component responsible for the mechanical and thermal properties, and ultimately quality, of chocolate.

T

WAXS reflections at 5.4 Å (m), 5.11 Å (w), 4.57 Å (s), 4.38 Å (m), 4.22 Å (m), 4.02 Å (m), 3.89 Å (w), 3.84 Å (w), and 3.67 Å (m). Thereafter, Lavery6 studied the melting behavior and polymorphism of seven pure mono-oleyl disaturated TAGs (SOS, POP, OSS, OPP, POS, OPS, and OSP). He showed that slow crystallization of POS from the melt (during 12 h) from 50 to 18 °C, followed by further cooling to 0 °C, yielded a β′-3 form with a melting point of 30.8 °C, a long spacing of 67.5 Å, and WAXS reflections at 4.13 Å (vs), 3.82 (Å s), 4.38 Å (s), and 4.56 Å (m). Moreover, he obtained β-3 POS either by solvent crystallization from acetone or by holding the dry molten POS at 32 °C for 2 days. This procedure yielded crystals with a melting point of 35.5 °C, long spacing of 63.7 Å, and WAXS reflections at 4.59 Å (vs), 4.05 Å (m), 3.87 Å (m), and 3.68 Å (s). A slightly different thermal behavior for the different POS polymorphs was identified by Landmann et al.7 in 1960. They found that POS in different polymorphic forms exhibited four main melting points, 18.2, 25.5, 33.0, and 37.4 °C. They called these crystal forms I to IV, respectively. Crystal form II (melting point of 33 °C) was obtained by tempering quickly chilled POS at 22 °C, while form I (most stable crystal form with a melting point of 37.4 °C) was crystallized from acetone. In 1966, a comprehensive study of the polymorphism of cocoa butter and phase behavior of binary mixtures of SOS:POS was carried out by Wille and Lutton.8 In this study, the β-3 crystal form of POS was obtained by melting POS followed by chilling to 21 °C, storing at this temperature for a day, followed by incubation for 2 days at 27 °C. They reported the melting point of β-3 POS as 33.5 °C, with WAXS reflections at 5.41 Å

he asymmetric disaturated triacid triacylglycerol (TAG), 1,3-palmitoyl-stearoyl-2-oleoyl glycerol (POS), where S represents stearic acid, P represents palmitic acid, and O represents oleic acid, is the most abundant TAG in cocoa butter. POS has been shown to display several different crystal polymorphic forms.1 These have characteristic powder X-ray diffraction patterns and melting points. For the sake of this discussion, it is important to define the nomenclature used in the classification of TAG crystal forms. Beta (β) refers to the triclinic subcell packing of the fatty acid chains of a TAG crystal, while “2” or “3” refers to the number of fatty acid chain lengths within the long axis of the TAG unit cell.2,3 Polymorphism usually refers to the fatty acid chain packing while polytypism usually refers to the number of fatty acid chain lengths within the unit cell. In 1951, Lutton4 showed that POS had a melting point of 33 °C and called this crystal form β′-3, for orthorhombic perpendicular fatty acid chain packing of polytype 3. He showed that incubation of β′-3 POS for a week at 32 °C led to a polymorphic transition to the β-3 form with a melting point of 39 °C. In this study, the “long spacing”, the reflection corresponding to the (001) plane of the β′-3 crystal, was reported at 66.8 Å, while for the β-3 form, 63.1 Å were reported (see Table 1 for long and short spacing data). Moreover, wide angle reflections for POS in the β′-3 crystal forms were reported at (5.56 Å (w), 4.56 Å (m), 4.36 (Å (m), 4.11 Å (s), 3.84 Å (s), and 3.63 Å (w)), while for the β-3 form at (5.42 Å (m), 4.58 Å (vs), 4.24 Å (vw), 4.05 Å (m), 3.86 Å (m), 3.72 Å (s), and 3.51 Å (w)). In this field, vs, s, m, and w represent “very strong”, “strong”, “medium”, and “weak” peak intensities. These reflections remain, to date, unindexed. In 1957, Chapman et al.5 showed that POS crystallized in the β-3 crystal form from acetone had a melting point of 37.5− 38 °C. Moreover, he also reported a long spacing of 64.1 Å and © XXXX American Chemical Society

Received: April 20, 2018 Revised: July 18, 2018 Published: July 23, 2018 A

DOI: 10.1021/acs.cgd.8b00598 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Communication

Table 1. Small- and Wide-Angle Powder X-ray Diffraction Data for the Three β Polymorphic forms of POS Obtained in This Study polymorph

long-spacing (Å)

β3 β2 β1

64.1 63.5 63.9

short-spacing (Å)a 4.61 (vs) 4.60 (vs) 4.60 (vs)

3.98 (m) 3.97 (m) 4.04 (m)

3.87 (m) 3.85 (m) 3.93 (m)

3.75 (m) 3.74 (w) 3.86 (m)

3.66 (m) 3.69 (w) 3.70 (s)

3.65 (w)

vs, s, m, and w represent “very strong”, “strong”, “medium”, and “weak” peak intensities.

a

polymorphic forms was examined by X-ray diffraction (Multiflex Powder XRD spectrometer, Rigaku, Tokyo, Japan). The copper X-ray tube (wavelength of 1.54 Å) was operated at 40 kV and 44 mA. The measurement scan rate was set at 0.1°/min in the range 2θ = 1−30° at 20 °C. Peak positions were determined using MDI Jade 9 (MDI, Livermore, CA, USA) software. Melting points (peak-top) and enthalpies of fusion for the three β polymorphic forms of POS was obtained using a differential scanning calorimeter (DSC) model Q2000 (TA Instruments, Mississauga, ON, Canada). Nitrogen was used as the purge gas with the flow of 18 mL/min. In this experiment, melting points (endothermic peak) of samples (2−5 mg) were determined by heating from 5 to 60 °C at a heating rate of 5 °C/min. Powder X-ray spectra of the POS polymorphs identified in this study are shown in Figure 1. In Figure 1A, a neverreported-before short-spacing (WAXS) fingerprint for a β polymorph of POS is shown, the β3 polymorphic form. Diffraction peaks in the WAXS region were almost identical to the form V crystal polymorph of cocoa butter. Cocoa butter TAGs display two β polymorphic forms, called form V and form VI, in increasing order of stability. Form V is considered the optimal crystal form for high-quality chocolate. This newly identified β form, here called β3, displayed peaks at 4.61 Å (vs), 3.98 Å (m), 3.87 Å (m), 3.75 Å (m), and 3.66 Å (m) (Figure 1A). Wille and Lutton8 and Chapman et al.13 showed very similar diffraction patterns for cocoa butter in form V at 4.58 Å (vs), 3.98 Å (m), 3.87 Å (m), 3.73 Å (m), and 3.65 Å (m). The second β POS polymorph (β2) had a very strong peak at 4.6 Å, two medium peaks at 3.97 and 3.85 Å, and three small peaks at 3.74, 3.69, and 3.65 Å (Figure 1B). After twelve weeks storage of POS crystals in oven at 20 °C, no change was observed in SAXS or WAXS patterns of the β3 and β2 crystal polymorphs. Willie and Lutton8 reported a diffraction pattern for POS (they called it “freshly prepared stable β-3 crystal form”) which displayed reflections at 5.41 Å (m), 5.09 Å (w), 4.57 Å (vs), 3.98 Å (m), 3.86 Å (w), 3.73 Å (vw), and 3.65 Å (w), that was similar to the diffraction pattern that we obtained for our β2 POS polymorph. The main difference was the relative intensity of the diffraction peak at 3.86 Å that they reported as a weak peak, while we observed a medium intensity peak at 3.85 Å. The third β crystal polymorph of POS (β1) was obtained here after storage for two years at room temperature (Figure 1C). In previous research, Arishima et al.1 referred to this form as the only β polymorph, while van Mechelen et al.14 called it a β1 polymorph of POS. In our study, β1 POS diffraction peaks were observed at 4.60 Å (vs), 4.04 Å (m), 3.93 Å (w), 3.86 Å (m), and 3.70 Å (s). Interestingly, these correspond closely to those of form VI of cocoa butter, reported by Wille and Lutton8 and Loisel et al.15 Although WAXS reflection positions, or short spacings, were not reported by van Mechelen et al.,12 based on inspection of the diffraction patterns reported in his paper, we would suggest that the β2

(m), 5.09 Å (w), 4.57 Å (vs), 3.98 Å (m), 3.86 Å (w), 3.73 Å (vw), and 3.65 Å (w). They showed that after six months storage at room temperature, the melting point of POS increased to 35.0 °C, while the WAXS reflections changed to 5.41 Å (w), 5.15 Å (vw), 4.59 Å (vs), 4.24 Å (vw), 4.05 Å (w), 3.89 Å (m), and 3.70 Å (s). About 25 years later, Arishima et al.1 reported four crystal polymorphs for POS and named them α, δ, pseudo-β′, and β. These were obtained by crystallization from the melt. They found β-3 POS had a melting point of 35.5 °C and an enthalpy of fusion of 147.8 kJ/mol. They reported WAXS reflections for this polymorph at 4.58 Å (vs), 4.02 Å (w), 3.92 Å (m), 3.85 Å (m), and 3.70 Å (s), and a long spacing of 64.0 Å. Arishima also commented on the fact that POS only displayed a single β polymorphic form, in contrast to SOS and POP, which display two β polymorphic forms, β2 and β1 in increasing order of stability. In the same year, Koyano et al.9 reported that the β1 polymorphs of POP and SOS formed only via melt-mediated polymorphic transformation from the β2 forms or from solvent crystallization, while β-3 POS was obtained from pseudo-β′ crystals only. Later in 1993, Yano et al.10 studied the molecular structures of different crystal forms of SOS, POP, and POS using Fourier transform infrared (FTIR) spectroscopy. They reported that the polymorphic behavior of POS was similar to that of SOS with the exception that no second β crystal form of POS was present. Later in 1996, the crystallization kinetics of POP, SOS, and POS were studied by Rousset and Rappaz.11 They found that POS had the lowest crystallization growth rate compared to the POP and SOS. More recently, in 2006, van Mechelen et al.12 isolated two β polymorphs of POS, and named them β2 and β1, where β1 was the most stable form. They obtained the β2 form by rapid heating above the melting point (about 40 °C), followed by cooling down to 23 °C. After several months storage at 22 °C, this crystal form transformed to the β1 crystal form. Unfortunately, WAXS reflection positions, or short-spacings, were not reported in this paper. The melting point of β2 and β1 POS was reported as 34.9 and 38.4 °C, respectively.12,14 In contrast to all the studies above, here we report the isolation and characterization of three β polymorphs of POS, β3, β2, and β1, in increasing order of stability, where β1 is the most stable polymorph. In our experiments, a racemic mixture POS of 98% purity (Lot: T 054:2, from Larodan AB, Karolinska Institutet Science Park, SOLNA, Sweden) was crystallized from acetone (Fisher Scientific, ACS reagent, >99.5%). To obtain β3 and β2 polymorphs, a 10% (w/w) POS solution in acetone was placed on an X-ray slide and left in an incubator to crystallize by evaporation of acetone at 18 and 5 °C, respectively. This procedure was repeated several times to obtain a proper layer of POS crystals on the X-ray slides. Crystallized POS in β3 and β2 crystal forms were stored in oven at 20 °C for further analysis. The most stable crystal form of POS (β1) was obtained by melt crystallization of POS, and incubation for two years at 22−23 °C. The occurrence of these B

DOI: 10.1021/acs.cgd.8b00598 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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Table 2. Melting Points and Enthalpies of Fusion for the Three β Polymorphic Forms of POS Obtained in This Studya polymorph

temperature (°C)

enthalpy (kJ/mol)

β3 β2 β1

32.91 ± 0.14a 33.77 ± 0.17b 38.69 ± 0.06c

141.51 ± 2.01a 147.93 ± 1.62b 158.46 ± 2.50c

a

Values represent means and standard deviations. Means in a column with a different letter are significantly different at the 0.05 level.

Figure 1. Small- and wide-angle powder X-ray diffraction spectra of the different polymorphs of POS, (A) β3, (B) β2, and (C) β1.

POS reported by them corresponds to an intermediate crystal form from form β2 to β3.12 No dramatic differences between the long spacings of the β3, β2, and β1 polymorphs of POS (64.1, 63.5, and 63.9 Å, respectively) were found in our study. Wille and Lutton8 and Arishima et al.1 both reported a long spacing of 64.0 Å for their β polymorphs of POS. The melting points and enthalpies of the three beta POS crystal forms found in this study (β3, β2, and β1) are shown in Table 2 and Figure 2. The melting points of these polymorphs were 32.9 °C for β3, 33.8 °C for β2, and 38.7 °C for the most stable polymorph, β1. The enthalpies of fusion for these crystal forms were 141.5, 147.9, and 158.7 kJ/mol, respectively. The melting point of 33 °C reported for the β′-3 of POS by Lutton4 was similar to the melting point of the β3 form obtained by us (32.9 °C). Lutton4 also reported the melting point of their β-3 form as 39 °C which was very similar to the melting point of our β1 form of POS (38.69 °C). It seems that the two-year storage of POS crystals at room temperature helped achieve

Figure 2. DSC heating thermograms of the different polymorphs of POS, (A) β3, (B) β2, and (C) β1.

the most stable crystal form of POS, namely, form β1. The enthalpy of fusion for this crystal form was the highest of the β forms (158.46 kJ/mol). Landmann et al.5 found similar melting points for POS crystal form β2 (33 °C) and form β1 (37.4 °C) compared to 33.77 and 38.69 °C for the β2 and β1 forms obtained in our study, while the melting points reported for β POS by Arishima et al.1 (35.5 °C) and β2 POS by van Mechelen et al.10 (34.9 °C) lie between the melting points obtained in our study for β2 and β1 of crystals of POS, 33.8 and 38.7 °C, respectively. The enthalpy of fusion for β POS reported by Arishima et al.1 was 147.8 kJ/mol compared to 147.93 kJ/mol obtained by us for β2 POS. Since the C

DOI: 10.1021/acs.cgd.8b00598 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Communication

crystallization of β POS is extremely slow,7 the crystal polymorphs reported in Arishima et al.1 and van Mechelen et al.10 most likely represented intermediate crystal forms and not distinct polymorphic states. Based on our understanding of the literature, there are no previous reports of the existence of β3 POS. In conclusion, the discovery of two new β polymorphs (β3 and β2) of POS could help explain the confusion in the literature regarding the number of polymorphs of POS, particularly the reported fact that POS did not possess a second β polymorphic form (Arishima et al.,1 Yano et al.,8 and Koyano et al.7). Moreover, we report a melting point for β1 POS of 38.68 °C, which lies in between β1 POP (36.7 °C) and β1 SOS (43 °C), which makes sense from a chemical composition perspective. Previously, the melting point of β POS was reported as 35.5 °C,16 which is below the melting point of β POP, which does make sense. Moreover, our newly discovered β3 crystal polymorph has identical long and short spacing patterns to cocoa butter in crystal form V. We believe that the present work will clarify the polymorphism of POS and the relationship of POS polymorphic transitions with bloom formation in chocolate.

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(12) Van Mechelen, J. B.; Peschar, R.; Schenk, H. Structure of Mono-unsaturated Triacylglycerols. II. The β2 Polymorph. Acta Crystallogr., Sect. B: Struct. Sci. 2006, 62, 1131−1138. (13) Chapman, D.; Akehurst, E. E.; Wright, W. B. Cocoa Butter and Confectionary Fats. Studies Using Programmed Temperature X-ray Diffraction and Differential Scanning Calorimetry. J. Am. Oil Chem. Soc. 1971, 48, 824−830. (14) Van Mechelen, J. B.; Peschar, R.; Schenk, H. Structures of Mono-unsaturated Triacylglycerols. I. The β1 Polymorph. Acta Crystallogr., Sect. B: Struct. Sci. 2006, 62, 1121−1130. (15) Loisel, C.; Keller, G.; Lecq, G.; Bourgaux, C.; Ollivon, M. Phase Transitions and Polymorphism of Cocoa Butter. J. Am. Oil Chem. Soc. 1998, 75, 425−439. (16) Arishima, T.; Sagi, N.; Mori, H.; Sato, K. Density Measurement of the Polymorphic Forms of POP, POS and SOS. J. Oleo Sci. 1995, 44, 431−437.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +1-519-824-4120, ext. 54340. Fax: +1-519-824-6631. ORCID

Alejandro G Marangoni: 0000-0002-3129-4473 Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Arishima, T.; Sagi, N.; Mori, H.; Sato, K. Polymorphism of POS. I. Occurrence and Polymorphic Transformation. J. Am. Oil Chem. Soc. 1991, 68, 710−715. (2) Abrahamsson, S.; Dahlén, B.; Löfgren, H.; Pascher, I. Lateral Packing of Hydrocarbon Chains. Prog. Chem. Fats Other Lipids 1978, 16, 125−143. (3) Chapman, D. The Polymorphism of Glycerides. Chem. Rev. 1962, 62, 433−456. (4) Lutton, E. S. The Polymorphism of the Disaturated Triglycerides-OSS, OPP, POS, OPS and OSP. J. Am. Chem. Soc. 1951, 73, 5595−5598. (5) Chapman, D.; Crossley, A.; Davies, A. C. The Structure of the Major Component Glyceride of Cocoa Butter, and of the Major Oleodisaturated Glyceride of Lard. J. Chem. Soc. 1957, 0, 1502−1509. (6) Lavery, H. Differential Thermal Analysis of Fats. II. Melting Behavior of Some Pure Glycerides. J. Am. Oil Chem. Soc. 1958, 35, 418−422. (7) Landmann, W.; Feuge, R. O.; Lovegren, N. V. Melting and Dilatometric Behavior of 2-Oleopalmitostearin and 2-Oleodistearin. J. Am. Oil Chem. Soc. 1960, 37, 638−643. (8) Wille, R. L.; Lutton, E. S. Polymorphism of Cocoa Butter. J. Am. Oil Chem. Soc. 1966, 43, 491−496. (9) Koyano, T.; Hachiya, I.; Arishima, T.; Sagi, N.; Sato, K. Polymorphism of POS. II. Kinetics of Melt Crystallization. J. Am. Oil Chem. Soc. 1991, 68, 716−718. (10) Yano, J.; Ueno, S.; Sato, K.; Arishima, T.; Sagi, N.; Kaneko, F.; Kobayashi, M. FT-IR Study of Polymorphic Transformation in SOS, POP, and POS. J. Phys. Chem. 1993, 97, 12967−12973. (11) Rousser, Ph.; Rappaz, M. Crystallization Kinetics of the Pure Triacylglycerols Glycerol-1,3-Dipalmitate-2-Oleate, Glycerol-1-Palmitate-2-Oleate-3-Stearate, and Glycerol-1,3-Distearate-2-Oleate. J. Am. Oil Chem. Soc. 1996, 73, 1051−1057. D

DOI: 10.1021/acs.cgd.8b00598 Cryst. Growth Des. XXXX, XXX, XXX−XXX