Negative Pressure Effects during Pure Triacylglyerol Crystallization

Article pubs.acs.org/crystal

Negative Pressure Effects during Pure Triacylglyerol Crystallization Robert W. Lencki* and R. John Craven Department of Food Science, University of Guelph, Guelph, Ontario, Canada ABSTRACT: Under certain crystallization conditions, βtending triacylglyerol (TAG) can form cavities within the crystal network, leading to situations where the macroscopic solid density is less than the melted liquid phase. A similar phenomenon, termed negative pressure effect, has been observed during the crystallization of some synthetic polymers. Crystallization of pure β-tending trilaurin just below its melting point with seeding created a crystal structure with millimeter-sized star-like crystals and large air-filled pores, resulting in a macroscopic density (887.5 ± 2.9 kg m−3) that was significantly less than the liquid TAG (897.9 ± 0.6 kg m−3) just above its melting point (Tm). A moderate degree of supercooling (11.5 °C below Tm) led to a platy crystal habit and a higher macroscopic density (1030 ± 2.1 kg m−3). However, higher degrees of supercooling (>25 °C below Tm) created a very fine microporous crystal network with a lower density of 993.2 ± 2.5 kg m−3. It is evident from this work that negative pressure effects can have a significant influence on the macroscopic density of β-tending TAG.

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INTRODUCTION In theory, the dependence of material volume (V) on temperature at constant pressure is given by the expression: V = Vo + α(T − To)

(1) 3

where Vo is the solid or liquid volume (m ) at a specified reference temperature (To), T is the temperature (°C), and α is the coefficient of thermal expansion (°C−1). Values for α range from 10−7 °C−1 for hard solids to 10−3 °C−1 for organic liquids. Equation 1 can also be modified to provide an expression for density (ρ) as a function of temperature: m ρ= Vo + α(T − To) (2) where m is the sample mass (kg). The solid line in Figure 1 represents the volume change of a pure triacylglycerol (TAG) as it passes through the solid-toliquid phase transition. Since volume change tends to be less of a function of temperature for solids than for liquids, the slope of the line below the melting point (Tm) is relatively flat.1 When the TAG’s Tm is reached, a large volume step change is observed (ΔVC), a direct result of crystal melting. After the phase change is complete, liquid TAG volume increases with temperature at a rate approximately three times that of the solid.1 Equations 1 and 2 apply to the ideal case of a continuous pure crystal. However, single TAG crystals can only be produced under very controlled conditions. More often than not, a large quantity of crystals are generated, with their number and size determined by the relative rates of nucleation and growth. Generally, a low degree of supercooling favors growth (smaller quantities of larger crystals), whereas higher degrees of supercooling produce a larger number of small crystals.2 © 2012 American Chemical Society

Figure 1. Plot of volume versus temperature for a pure TAG.

Another phenomenon commonly observed with crystallizing TAG is the formation of a gel or fat crystal network.3 With strongly interacting crystals, network formation can occur at relatively low solids concentrations, with the remaining liquid phase occluded within the solid network (Figure 2). Even with very weak solid particle interactions, a maximum packing density will inevitably be reached, with liquid TAG filling the intercrystalline space. Once a continuous network or the maximum packing density is achieved, the crystal network volume should remain relatively constant. However, if more Received: July 3, 2012 Revised: August 16, 2012 Published: August 22, 2012 4981

dx.doi.org/10.1021/cg300901v | Cryst. Growth Des. 2012, 12, 4981−4986

Crystal Growth & Design

Article

change the total volume occupied by TAG molecules (Vmicro ), T but does affect the macroscopic volume defined by VTmacro = VS + VL + VG

(5)

where VG is the volume of gas-filled cavities. Macroscopic density (ρmacro) then simply is the total sample mass (m) divided by Vmacro . Evidently, if there is a gaseous phase present, T then Vmacro > Vmicro . T T Pulse NMR determines the SFC (i.e., the relative masses of the liquid and solid phases) by measuring the intensity of signals arising from hydrogen nuclei in the liquid and solid phases.8 If the mass of the sample is known, then its total volume can be calculated by the following expression: ⎛⎛ SFC ⎞ 1 ⎛ SFC ⎟⎞ 1 ⎞ ⎟ ⎟⎟ + ⎜1 − VTmicro = m⎜⎜⎜ 100 ⎠ ρL ⎠ ⎝⎝ 100 ⎠ ρS ⎝

Figure 2. Cross-section of a fat crystal network at high solids concentration: crystals - black; liquid oil - gray; gaseous/vapor phase white.

where ρS and ρL are the solid and liquid densities, respectively, at the measurement temperature. Substituting eq 2 into eq 6 gives ⎛⎛ SFC ⎞ ⎛ SFC ⎞⎟ ⎟(V ⎜ VTmicro = ⎜⎜ oS + αS(T − ToS)) + ⎝1 − ⎝ ⎠ ⎝ 100 100 ⎠ ⎞ (VoL + αL(T − ToL))⎟ ⎠ (7)

heat is withdrawn from the sample, the occluded liquid phase continues to contract due to further solidification. Gałeski and Piórkowska4 have examined a similar phenomenon with synthetic polymers that form large spherulitic crystals. Once the maximum packing density is reached, crystallization of the occluded liquid no longer takes place at constant pressure. Instead, further crystallization more closely resembles constant volume conditions. The resulting ΔVC of the crystallizing liquid creates localized areas of negative pressure, which can produce air-filled pockets within the polymer, either by cavitation or through the convective transport of air from the polymer surface. These cavities reduce the apparent density of the solid polymer and create structural weak spots.5 There is some indirect evidence in the literature that indicates that negative pressure effects can in fact occur within fat crystal systems. For example, Hvolby6 observed that many β-tending fats expand during crystallization, resulting in solids that are less dense than the original liquid. This is surprising since at low solids concentrations, fat crystals inevitably collect at the bottom of the liquid due to their higher relative density. Cebula et al.7 also observed that some solid triglycerides have abnormally high ultrasound attenuation. Their small angle neutron scattering (SANS) analysis showed a large quantity of what they termed “molecular defects” in solidified trilaurin, but which could be cavities formed due to negative pressure effects. If a completely liquefied pure TAG is cooled back to its melting point and only enough heat is removed at Tm so that a very small quantity of the solid phase is present, the total microscopic volume occupied by TAG molecules (Vmicro ) can T simply be expressed as VTmicro = VS + VL

(6)

Since pulse NMR is unaffected by the presence of a third phase because it only detects the TAG molecules in the sample, eq 7 is a measure of microscopic volume (i.e.,Vmicro ). Consequently, T the microscopic density (ρmicro) of trilaurin would simply be equal to m/VTmicro. In contrast, with methods such as dilatometery, volume is measured using a contacting medium such as a physical probe or liquid, so VT would depend on how well this medium penetrates into solid imperfections, pores, and cavities. If the water used in the dilatometer is not able to infiltrate gas-filled pores, the volume measured by this technique represents the combination of all three phases, and thus measures macroscopic volume (i.e., Vmacro ) as defined in T eq 5. In this case, the macroscopic density (ρmacro) would equal m/Vmacro . T By comparing the volume (or density) calculated from SFC measurements with those obtained by dilatometry, it is possible to quantify cavity formation resulting from negative pressure effects. Consequently, this work will compare the crystallization of a pure TAG (trilaurin) using these two techniques, and what is observed using polarized light and cryo scanning electron microscopy to determine if negative pressure effects do indeed occur during fat crystallization at high solids concentrations.

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EXPERIMENTAL SECTION

Glyceryl tridodecanoate (trilaurin) with purity greater than 98% was purchased from Spectrum Chemicals (New Brunswick, NJ). All other reagents were acquired from Fisher Scientific (Mississauga, ON). Solid Fat Content (SFC) Determination. Analysis was conducted using AOCS Official Method Cd 16b-93.9 A Minispec mq20 pulse NMR analyzer equipped with Biospin software (Bruker Optics, The Woodlands, TX) was used for the determination of SFC. Each data point represents the average and standard error of measurements obtained on three sample replicates. Density Measurement. Liquid density was determined by weighting 25 mL of trilaurin that was allowed to equilibrate at constant temperature for 1 h. The 25 mL volumetric flask used to measure volume was calibrated using deionized water as a standard.

(3)

where VS and VL are the solid and liquid volumes, respectively. By substituting eq 1, this expression can be rewritten as VTmicro = (VoS + αS(T − ToS)) + (VoL + αL(T − ToL)) (4)

If more heat is removed such that the maximum packing density is reached or a crystal network is created, gas-filled cavities may form. The presence of a gaseous phase does not 4982



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Solid bulk density was determined by first crystallizing approximately 4.0 g of liquid trilaurin that was placed in a vertical 6 mL syringe with the end cut off. The trilaurin was held isothermally for 24 h then held at 5 °C for another 24 h. Solid trilaurin was removed from the syringe by pushing on the plunger and weighed to the nearest μg. The sample was then placed in the reservoir of a dilatometer of known volume and degassed 5 °C water was added until a volume could be read on the capillary scale. Care was taken to first remove all air bubbles before sealing the reservoir, and then the weight of water added was determined. After letting the dilatometer reach thermal equilibrium for 1 h in a water bath set at 5.0 °C, the total volume was read on the capillary scale. Trilaurin volume could then be determined by subtracting the water volume (calculated using the measured mass and literature value for density) from the known total dilatometer volume at 5 °C. The measured mass was divided by the calculated volume to give the solid density. For both liquid and solid samples, each data point represents the average and standard error of measurements obtained on three sample replicates. Dilatometry. Analysis was conducted using AOCS Official Method Cd 10-57 (1994).10 Trilaurin samples were loaded into the dilatometer as described in the methodology for density measurement. Polarized Light Microscopy. Liquid trilaurin was placed on a microscope slide and incubated for 24 h at the prescribed crystallization conditions. After holding the samples for another 24 h at 5 °C, images were taken using an Olympus BX 60F5 (Olympus Optical, Tokyo, JP) light microscope fitted with a Sensys HRD 060NIK 0.60x camera (Photometrics, Tucson, AZ). Image Pro Plus Version 6.0 (Media Cybernetics, Bethesda, MD) was used for the light microscope image analysis. Cryo Scanning Electron Microscopy. A technique for cold-stage SEM developed by Schmidt et al.11 and applied in dairy research by Kaláb12 for the cold-stage SEM observation of cheese was used to study trilaurin crystal structure. Images were taken with a Hitachi Type S-570 scanning electron microscope (Hitachi, Tokyo, JP). An Emscope SP2000A cooling unit (Canberra Packard, Meriden, CT) and Emitech K550X turbo sputter coater (Emitech, Ashford Kent, UK) were used for sample preparation.

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RESULTS AND DISCUSSION The SFC versus temperature curve of 100% pure trilaurin would simply be a step function (Figure 3a), with a sharp transition occurring at the literature value melting point (Tm) of 46.5 °C.1 Corresponding V/VoS and density plots for 100% pure trilaurin were derived using the following literature values: ρS of the β-form at −38.6 °C = 1057 kg m−3; and αS = 2.3 × 10−4 °C−1.13 If 0 °C is chosen as the solid reference temperature (ToS), using a 1.0 kg basis, VoS = 9.530 × 10−4 m3. Consequently, VS/ VoS = 1.011 and ρS = 1038 kg m−3 at Tm. Liquid trilaurin density was experimentally determined at 55, 65, and 75 °C (Table 1). From a least-squares fit of the data, an αL value of 6.97 × 10−4 °C−1 was derived, which is comparable to the literature value of 7.0 × 10−4 °C−1.1 If Tm is chosen as the trilaurin liquid reference temperature (i.e., ToL) then, on a 1.0 kg basis, VoL = 1.105 × 10−3 m3. Thus, for liquid trilaurin, VL/VoS = 1.159 and ρL = 904.8 kg m−3 at Tm. The derived ΔVC of 1.420 × 10−4 m3 kg−1 is the same as the literature value,1 adding independent verification to the analysis. The experimental trilaurin SFC versus temperature curve was close to ideal but was slightly sigmoidal (Figure 3a) because the TAG used was approximately 98% pure. Equation 7 was used /VoS and ρmicro versus T to convert this data to predicted Vmicro T curves (Figure 3b,c, respectively). The ρmacro of solid TAG at 5 °C as determined by dilatometry was found to be a strong function of crystallization

Figure 3. Trilaurin (a) SFC, (b) Vmicro/VoS, and (c) microscopic density as a function of temperature: --- pure compound;  experimental.

conditions (Table 1 and Figure 4). Trilaurin crystallized from solvent consisted of small columnar crystals with lengths up to 150 μm (Figure 5). The density of this crystal form (subsequently referred to as Solid 1) at 5 °C (1044 ± 1.1 kg m−3) was comparable to the value calculated from the literature 4983



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Table 1. Macroscopic Density of Various Forms of Trilaurin temperature (°C)

density (kg m−3)

75 65 55

liquid liquid liquid

886.8 ± 0.4a 892.1 ± 0.9a 897.9 ± 0.6a

5 5

Solid 1 - crystallized from solvent at 20 °C Solid 2 - crystallized in dilatometer at 45 °C after 1 h at 38 °C Solid 3 - crystallized from melt at 45 °C with seeding Solid 4 - crystallized from melt at 35 °C Solid 5 - crystallized from melt at 20 °C Solid 6 - crystallized from melt at 5 °C

1044 ± 1.1 961.1 ± 0.1

5 5 5 5 a

form

887.5 ± 2.9 1030 ± 2.1 993.2 ± 2.5 982.9 ± 3.2

α = 0.000697 °C−1.

Figure 5. Polarized light micrograph of Solid 1 (trilaurin crystallized from solvent) (white bar = 200 μm).

medium), either in the liquid or vapor phase, being siphoned into some of the cavities. Because this influences density measurement accuracy, subsequent sample crystallizations were first performed externally before being inserted inside the dilatometer. When trilaurin was crystallized at 45 °C under very low supercooling conditions (1.5 °C below Tm) with seed addition (Solid 3), large star-like crystals formed with diameters as large as several millimeters (Figure 6a). As is often the case with crystals formed via the aggregation of nanosized platelets, the resulting size distribution was relatively monodispersed.13 This resulted in a crystal mass with a macroscopic density (887.5 ± 2.9 kg m−3) significantly less (p < 0.05) than that of the trilaurin liquid phase just above Tm (897.9 ± 0.6 kg m−3). This expansion effect was so powerful that the walls of the syringe in which the TAG was incubated were significantly dilated after crystallization. Hvolby7 also observed similar expansion upon solidification of bulk quantities of several β-tending fats. Figure 7 illustrates a possible mechanism by which macroscopic density can actually decrease during crystallization. Because of the crystal habit that forms under such a low degree of supersaturation (large rough-surfaced star-like crystals), large continuous intercrystalline channels with an approximate diameter of 10−3 m are present. Initially these channels are liquid-filled (Figure 7a). However, when the solids concentration increases to the point where the crystals reach their maximum packing density, further crystal formation (and the resulting ΔVC) results in the depletion of liquid within the channels, and the creation of cavities. Furthermore, since the crystals adopt spherical star-like structures with high surface roughness that favors weak intercrystalline interactions, their growth would disrupt the surrounding structure, pushing neighboring crystals outward (Figure 7b). The result is a porous, low macroscopic density crystalline structure. Solid 4, which was crystallized at 35 °C under a moderate degree of supersaturation (11.5 °C below Tm), had a more continuous platy crystal habit (Figure 6b). These conditions

Figure 4. Trilaurin macroscopic density as a function of temperature: ○ - crystallized in dilatometer; ● - crystallized from solvent; ■ crystallized at 35 °C; ▲ - crystallized at 20 °C; ▼ - crystallized at 5 °C; - - - theoretical.

(1048 kg m−3).1 The density versus temperature profile of these finely dispersed crystals also corresponded well to theoretical values (Figure 4). Since these crystals were well dispersed and did not significantly interact to form a crystal network (Figure 4), the dilatometer contacting medium (i.e., water) easily penetrated the intercrystalline volume, so ρmacro and ρmicro were approximately equal. A large hysteresis effect was observed when the melted trilaurin powder was crystallized within the dilatometer (Solid 2). In order to produce a significant quantity of nuclei, the liquid had to be first supercooled to 38 °C for 1 h before it could be warmed back up to 45 °C and allowed to solidify for 24 h. The resulting densities as the sample was subsequently cooled incrementally to 5 °C were 80−100 kg m−3 lower than were observed during the heating cycle. The final density at 5 °C was 961.1 ± 0.1 kg m−3, 87 kg m−3 less than the calculated literature value. If trilaurin is crystallized within the dilatometer, negative pressure phenomena can result in water (the contacting 4984



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Figure 7. (a) Four spherulitic crystals (black) surrounded by liquid oil (gray) with a seed crystal (black) in the center; (b) seed crystal grows into a spherulite, consuming the surrounding liquid oil and expanding the crystal network.

reduced the macroscopic density by 18 kg m−3 at 5 °C. Furthermore, in contrast to what was observed with Solid 3, the cavities were much smaller (on the order of 5 × 10−6 m) and had a more closed-cell structure. At high degrees of supersaturation (i.e., 26.5 °C (Solid 5) and 41.5 °C (Solid 6) below Tm), small crystals are formed that associate into a fine network consisting of primary particles that were more spherical than plate-like (Figure 6c). Once the critical gel concentration is reached, the Vmacro of the solid T lattice remains relatively constant (Figure 2). However, further crystal formation siphons the surrounding occluded liquid, leading to cavity formation. In this case, the crystal diameter was much smaller than those of the liquid channels, so no disruption of the lattice is observed. Nevertheless, the final structure consisted of continuous 10−6 m diameter pores, with a macroscopic density of 982.9 ± 3.2 kg m−3 at 5 °C that was 65 kg m−3 below the calculated literature value. Because trilaurin, the contacting water and apparatus were all equilibrated at 5 °C before the sample was inserted into the dilatometer, and the total mass and volume were measured within a few minutes, the air inside the cavities did not have time to significantly dissolve in the surrounding water. The small amount of water head that was present at the time the volume was measured could have forced some of the liquid into air-filled cavities, resulting in a slightly higher ρmacro value. When solid 3 was incrementally heated within the dilatometer, at 25 °C, air bubbles started to form on the solid’s surface, likely a result of the expansion of air inside the crystal cavities. When this solidified mass was completely melted inside the dilatometer, a large air pocket was observed inside the bulb, indicating that a significant gaseous phase was present inside the crystalline structure. Needless to say, the presence of such a large gaseous volume invalidated these density measurements (data not reported). Similar gas bubble production was not observed with solids 4 to 6. These samples contained significantly less gas than solid 3. Furthermore, cavities were much smaller, and at least with solid 4, had a more closed cell structure (Figure 6b), making it more difficult for the air to escape. Once the crystal structure melted, the surrounding degassed water appeared to have enough capacity to dissolve the liberated gas. As a result, the ρmacro versus T curves for these solids (Figure 5) were less affected by the phenomenon discussed above. The literature values for trilaurin density and α where determined at −38.6 °C with mercury as the contacting liquid.14 Using mercury at such a low temperature greatly reduces errors resulting from gas dissolution in the contacting phase or liquid vapor entering solid cavities. Interestingly,

Figure 6. SEM images of trilaurin crystallized under different conditions: (a) Solid 3 (45 °C with seeding); (b) Solid 4 (35 °C); (c) Solid 5 (20 °C) (black bar = 500 μm; white bar = 20 μm).

create a better balance between the rates of nucleation and growth, resulting in a network with a relatively high bulk density (1030 ± 2.1 kg m−3). Nevertheless, it is evident from Figure 6b that cavitation did occur to some degree during solidification, producing voids within the crystal network that 4985



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Bailey and Singleton14 suggested that the TAG samples should be solidified in very thin layers before being added to the dilatometer in order “to avoid errors presumably caused by vacuole formation in samples solidified in large masses.” Apparently, they were aware of the potential problems created by cavity formation but did not speculate on their cause.

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CONCLUSIONS When crystallized from the melt, trilaurin can have a wide range of macroscopic densities, the direct result of negative pressure effects within the fat crystal network. At very low levels of supersaturation, seeding produced spherical crystals with diameters of several millimeters, creating a very porous network with ρmacro that were less than those of the corresponding liquid immediately above Tm. On a macroscopic scale, moderate supercooling produced a relatively dense solid, whereas high degrees of supercooling led to the formation of a fine microporous crystal network with moderate density. These results have important implications for confectionery products because it is not only the microscopic density, but also changes in the macroscopic density during solidification that strongly affect the efficacy of the demolding process. In addition, chocolate porosity governs liquid TAG transport to the confection’s surface, thus influencing the rate of chocolate bloom defect formation. Therefore, an in-depth understanding of negative pressure effects is essential for obtaining an optimal confectionery product.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 519-824-4120 x54327; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Formo, M. W. Physical Properties of Fats and Fatty Acids. In Bailey’s Industrial Oil and Fat Products, 4th ed.; Swern, D., Ed.; Wiley: New York, NY, 1979; Vol. 1, pp 177−232. (2) Mullin, J. W. Crystallization, 4th ed.; Oxford: Boston, MA, 2001; p 594. (3) Walstra, P.; Kloek, W.; van Vliet, T. Fat Crystal Networks. In Crystallization Processes in Fats and Lipid Systems; Garti, N.; Sato, K., Eds.; CRC Press: Boca Raton, FL, 2001; pp 289−328. (4) Gałeski, A.; Piórkowska, E. J. Polym. Sci. Polym. Phys. Ed. 1983, 21, 1299−1312. (5) Gałeski, A.; Piórkowska, E. J. Polym. Sci. Polym. Phys. Ed. 1983, 21, 1313−1322. (6) Hvolby, A. J. Am. Oil Chem. Soc. 1974, 51 (3), 50−54. (7) Cebula, D. J.; McClements, D. J.; Povey, M. J. W. J. Am. Oil Chem. Soc. 1990, 67 (2), 76−78. (8) Van den Enden, J. C.; Rossell, J. B.; Vermaas, L. F.; Waddington, D. J. Am. Oil Chem. Soc. 1982, 59 (10), 433−439. (9) Anonymous. AOCS Official Method Cd 16b-93; Firestone, D., Ed.; AOCS Press: Urbana, IL, 2005. (10) Anonymous. AOCS Official Method Cd 10−57; Firestone, D., Ed.; AOCS Press: Urbana, IL, 1994. (11) Schmidt, D. G.; Henstra, S.; Theil, F. Mikroscopie (Wien) 1979, 35, 50−55. (12) Kaláb, M. Scan. Electron Microsc. 1981, 3, 453−472. (13) Privman, V.; Goia, D. V.; Park, J.; Matijević, E. J. Colloid Interface Sci. 1999, 213, 36−45. (14) Bailey, A. E.; Singleton, W. S. Oil Soap 1945, 22, 265−271.

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Negative Pressure Effects during Pure Triacylglyerol Crystallization

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