Langmuir 1998, 14, 5757-5763
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Investigation of the Gel to Coagel Phase Transition in Monoglyceride-Water Systems G. Cassin,*,‡ C. de Costa,† J. P. M. van Duynhoven,† and W. G. M. Agterof*,† Unilever Research Vlaardingen, Olivier van Noortlaan 120, 3133AT Vlaardingen, The Netherlands, and Unilever Research Colworth, Colworth House, Sharnbrook, Bedfordshire, MK44 1LQ, U.K. Received March 26, 1998 The gel to coagel phase transition of monoglyceride-water systems has been studied by nuclear magnetic resonance and differential scanning calorimetry. It is shown that the molecular arrangements within the bulk β-crystal of monoglyceride and the coagel phase are identical. The mobility of the glycerol backbone is high in the gel phase. On a decrease of temperature the mobility drops and then crystallization of the gel into a coagel phase takes place where hydrogen bonds among the glycerol groups are formed. A prerequisite for this is that D and L isomers of monoglyceride rearrange within the bilayers through chiral discrimination. The gel to coagel transition can be discussed in the frame of the Avrami theory of crystallization kinetics. The values of the Avrami exponent indicate a transition from a 2-dimensional to a 3-dimensional crystal growth on increasing the monoglyceride concentration. In the case of a 2-dimensional crystal growth the phase transition is diffusion controlled. Moreover, on applying shear to the gel phase, the phase transition evolves from a random to a spontaneous nucleation mechanism.
I. Introduction A monoglyceride is a lipid molecule constituted of a fatty acid chain esterified to a glycerol backbone. It is an optically active molecule that presents two isomers: D and L. Due to their strong emulsifying property, monoglycerides are widely used in the food industry. The polymorphic behavior of anhydrous 1-monoglycerides is wellknown:1,2 the following sequence of phase on temperature increase has been reported: sub-R, R-crystals, melt. A time dependent phase transition has been observed from the metastable R-crystals to the more ordered β-crystals. Moreover, it has been shown that the β-crystalline structure consists of a regular stacking of layers of D and L isomers.3,4 This symmetrical stacking is stabilized by a network of hydrogen bonds between the polar headgroups.3 Hence, on crystallization from a racemic mixture (the R-crystalline phase) the two isomers form D and L layers. The influence of chirality on the structure of monolayers of saturated monoglyceride has been studied5,6 and revealed that the racemic mixture shows on compression a phase transition characterized by the change of the tilt azimuth, i.e., the angle of the fatty acid chain with respect to the methyl end group plane. The phase diagram of saturated 1-monoglyceride/water systems has been characterized, and various liquid and gel crystalline phases have been reported.1-2 Above the Krafft temperature of the aliphatic chains, a lamellar phase appears over a wide monoglyceride concentration regime. Below this temperature, the lamellar phase transforms into a gel phase, which is the hydrated form of the R-polymorph. This phase consists of monoglyceride * To whom correspondence should be addressed. † Unilever Research Vlaardingen. ‡ Unilever Research Colworth. (1) Krog, N.; Larsson, K. Chem. Phys. Lipids 1968, 2, 129. (2) Krog, N.; Borup, A. P. J. Sci. Food Agric. 1973, 24, 691. (3) Larsson, K. Acta Crystallogr. 1966, 21, 267. (4) Guo, W.; Hamilton, J. A. Biophys. J. 1995, 68, 1383. (5) Gehlert, U.; Vollhardt, D.; Brezesinski, G.; Mohwald, H. Langmuir 1996, 12, 4982. (6) Gelhert, U.; Weidemann, G.; Vollhardt, D. J. Colloid Interface Sci. 1995, 174, 392.
bilayers, whose alkyl chains are partially frozen, separated by water layers.1,2,7 A detailed study of the headgroup hydration in the lamellar and the gel phase of monoglyceride has been made by Tiddy et al.7 It was established that three water molecules were bound to one surfactant molecule in the lamellar phase. In the gel phase this number equals to 1. For uncharged surfactants such as monoglycerides, the gel phase stability is mainly governed by van der Waals attractive forces within the lipid layer and short-range repulsive forces between the bilayers. Israelachvili and Wennerstro¨m propose that the exponentially decaying short-range repulsion as observed in numerous colloidal systems is mainly due to the confinement of thermally mobile surface groups.8-10 Measurement of this repulsive force has been performed on a large number of systems.11-15 In the case of an adsorbed layer of saturated monoglycerides,12,13 the repulsive pressure in the lamellar phase is significantly larger than in the gel phase. The initial gel phase formed of monoglycerides is metastable. When stored below 45 °C, it shows a time dependent phase transition to a phase called the coagel.1,2 This phase is a dispersion of monoglyceride β-crystals in water. Transmission electron microscopic studies have shown that the coagel phase consists of a three-dimensional network of platelike crystals entrapping water domains.16 It can be visualized as a percolated network (7) Morley, W. G.; Tiddy, G. J. T. J. Chem. Soc., Faraday Trans. 1993, 89 (15), 2823. (8) Israelachvili, J. N.; Wennerstrom, H. Langmuir 1990, 6, 873. (9) Israelachvili, J. N.; Wennerstrom, H. J. Phys Chem. 1992, 96, 520. (10) Israelachvili, J. N.; Wennerstrom, H. Nature 1996, 379, 219. (11) McIntosh, T. J.; Magid, A. D.; Simon, S. A. Biochemistry 1987, 26, 7325. (12) Claesson, P. M.; Erikson, J. C.; Herder, C.; Bergenstahl, B. A.; Pezron, E.; Pezron, I.; Stenius, P. Faraday Discuss. Chem. Soc. 1990, 90, 129. (13) Pezron, E.; Pezron, I.; Bergenstahl, B. A.; Claesson, P. M. J. Phys. Chem. 1990, 94, 8255. (14) Rand, R. P.; Parsegian, V. A. Biochem. Biophys. Acta 1989, 988, 351. (15) Simon, S. A.; Advani, S.; McIntosh, T. J. Biophys. J. 1995, 69, 1473.
S0743-7463(98)00340-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/01/1998
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of crystals in water. Surprisingly, for a widely used molecule such as monoglyceride the mechanism of this phase transition has not been described previously. To improve the understanding of the process driving the gel to coagel phase transition, some questions should be raised: (i) Are there any structural differences between the crystalline structure of the coagel phase and the anhydrous β-crystal phase of monoglyceride? (ii) What are the temperature and concentration dependence of the phase transition rate? The purpose of this paper is to answer these questions and to give a phenomenological description of the gel to coagel phase transition. Differential scanning calorimetry is used to determine the transition enthalpies for the mesophases and to monitor the kinetics of the phase transition. Various NMR techniques have been used for the structural analysis of monoglyceride/water gels and coagels phase. One of the advantages of NMR is its chemical selectivity, yielding information on 13C nuclei at natural abundance. High resolution and enhancement of signal intensity have been achieved using cross-polarization and magic angle spinning. The molecular mobility in the gel was studied by T1F(H) relaxation experiments in combination with single pulse 13C NMR. The coagel phase and the bulk β-crystals of monoglyceride were studied with 13C CP-MAS. In addition, 2H NMR experiments have been utilized to investigate for the presence of structured water in the coagel phase. II. Materials and Methods II.A. Materials. Distilled saturated monoglycerides (Hymono 8806) were purchased from Quest International Limited. In the samples, more than 95% is saturated 1-monoglyceride, the rest being triglyceride and diglyceride. The fatty acid chain length composition is roughly 60% of C18 and 40% of C16. To ensure a proper swelling of the 5% monoglyceride sample, 0.2% anionic cosurfactant was added. The cosurfactant we used was diacetyl tartaric acid ester of monoglyceride, also purchased from Quest. All the samples have been prepared with normal water (ionic conductivity ) 600 mS/cm). The 2H2O was supplied by Campro Scientific (99.9% purity). II.B. Sample Preparation. Samples containing various weight percentages of surfactant were prepared. Two preparation procedures have been followed. Type I. All components are heated to 60 °C and premixed with a magnetic stirrer for 30 min. The pH was adjusted to 4.2. After mixing, the system was sheared at 60 °C for 10 min. To prevent any inhomogeneities induced by air bubbles, the samples were treated in an ultrasonic bath for 2 min. Then the samples were poured into a beaker and cooled to room temperature. Type II. This is similar to processing I, except that when cooled to room temperature, the gel was sheared for 1 min at 100 s-1. The microstructure of the gel phase was studied using a light microscope (cross polarized light). Micrographs were taken before shear and immediatly after shear. II.C. Methods. Differential Scanning Calorimetry. Heat exchange involved in a phase transition yields exothermic or endothermic peaks that were recorded in a DSC experiment. From these peaks, the onset of the transition temperature and the transition enthalpy can be estimated. These experiments have been performed on a Perkin-Elmer power compensated DSC-7 equipped with a Perkin-Elmer controlled cooling accessory. Each sample (10-15 mg) was measured in 20 mL sealed aluminum pans at a scanning rate of 10 °C/min. Prior to the temperature program, the temperature was held constant for 1 min for equilibration. Nuclear Magnetic Resonance. In the NMR experiments the information about relaxation processes is contained in the bandwidth of the resonances.17,18 Relaxation processes are (16) Heertje, I. Food Struct. 1993, 12, 343. (17) Abragam, A. The principles of Nuclear Magnetism; Clarendon: Oxford, U.K., 1961.
Cassin et al. Table 1. Transition Enthalpies and Onset Temperature of the Three Polymorphs of the Commercial Monoglyceride (Hymono 8806), Β, r, and Sub ra temp (°C) ∆H (J/g)
β f melt
R f melt
sub R f R
70.0 199.0
64.0 106.0
13.5 17.0
a For comparison, the melting enthalpies of the β polymorph of pure stearic acid and pure C18 monoglyceride are 180 and 217 J/g, respectively.
intimately linked to the mobility of the chemical group bearing magnetic spins. An increase in the bandwidth is related to a decrease of the transverse relaxation time, T2, and is related to a diminution of the molecular mobility. 13C, 1H, and 2H single pulse spectra were recorded on a MSL300 NMR spectrometer operating at resonance frequencies of 75.47 and 300.13 MHz for 13C and 1H, respectively. These spectra were obtained under static conditions by means of a conventional 10 mm broad-band liquid NMR saddle coil. To acquire high-resolution 13C NMR spectra of β-crystals and coagel phases, the CP-MAS technique was utilized. First, magic angle spinning (MAS) results in a resonance line narrowing.19 Additionally, cross polarization (CP) of the 13C spin system by synchronous RF irradiation of the 1H spins system was used to enhance the signal intensity of the 13C spins. For a given measurement time, the effect of the enhancement by CP is even greater since the excitation of the system on the 1H channel benefited from the faster longitudinal relaxation time of the protons. The CP-MAS experiments were recorded on a broad-band MAS solenoid coil. The samples were spun at 2.5 kHz in a variable-temperature double-resonance probe. The 13C and1H radio frequency field strengths during cross-polarization and decoupling were 60 kHz. The intensities in the crosspolarization build-up curves were analyzed by deconvolution of the respective CP-MAS spectra into Lorenztian contributions. Proton rotating-frame relaxation times, T1F(1H), were determined from the decay of the carbon signal as a function of 13C-1H contact time, tc.20 Prior to NMR experiment, each sample was characterized by DSC in order to determine whether they were in the gel or coagel state. For the 2H NMR experiments, the surfactant was deuterated following the method as described in ref 7.
III. Results III.A. Differential Scanning Calorimetry. III.A.1. Anhydrous Monoglyceride. DSC experiments have been performed on anhydrous samples of commercial monoglyceride. As found in the literature, two sequences of peaks were observed between 0 and 90 °C.7 The first heating curve presents only one peak corresponding to the melting of β-crystals. In a second heating curve two peaks appeared, the first one corresponds to the sub-R-phase melting, the second one is the R-phase melting. Transition enthalpies and temperatures are reported in Table 1. As a comparison, the melting enthalpies of the β-polymorph of pure stearic fatty acid and pure C18 monoglyceride have also been given. The enthalpy of melting of β-crystals is about twice the value obtained for the melting of R-crystals. This difference in melting enthalpies can be employed to monitor the kinetics of the gel to coagel phase transition. To do so, we defined a parameter called the coagel index (CI), which is given by:
CI )
∆H(first heat) ∆H(second heat)
(1)
CI ) 1 indicates that the sample is still in the gel state, (18) Bovey, F. A. High-Resolution NMR of Macromolecules; Academic Press: New York, 1972. (19) Fyfe, C. A. Solid State NMR for Chemist; CFC Press: Guelph, 1983. (20) Stejskal, E. O.; Schaefer, J.; Steger, T. R. Faraday Discuss. Chem. Soc. 1979, 13, 56.
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Figure 1. Evolution of the fraction of coagel phase (defined as φ ) CI -1) as a function of time for gels of various monoglyceride weight percentages. Samples were prepared following preparation type I and stored at 5 °C. The solid curve is a fitting to eq 2.
a value of 2 means that the sample is in the coagel state. The evolution of this unitless parameter as a function of time can be used to monitor the kinetics of the phase transition. The influence of the concentration, the storage temperature, and the sample preparation procedure has been assessed. III.A.2. Gels of Monoglyceride. Samples containing 5, 10, and 20% of surfactant in water were prepared following the preparation type I. These samples have been stored at 5 °C over 60 days in order to follow the coagel formation. On the other hand, to investigate the influence of the storage temperature and the procedure utilized to prepare the gels, samples containing 5% of surfactant in water were prepared following the preparation procedure typeII and stored at different temperatures. When the Coagel Index equals 2, the whole sample is in the coagel state. The fraction of the coagel phase φ is given by φ ) CI - 1. Hence, we analyzed these curves with the following equation:
φ ) CI - 1) 1 - exp[-(t/τ)n]
(2)
where 1/τ is a rate constant and n is a fitting parameter. Later in the discussion we shall see that the kinetics of the phase conversion is discussed in the frame of the wellknown Avrami theory of crystallization. Figures 1 and 2 report the evolution of the fraction of the coagel phase as a function of time for gels prepared following procedures I and II, respectively. The values obtained from a nonlinear least-squares regression of the experimental curves are reported in Tables 2 and 3. For the gels prepared following procedure I, the rate constant takes on the lowest value for the 20% monoglyceride sample and the parameter n is found to increase with the monoglyceride concentration. As shown in Table 3, the rate constant shows a steep increase with storage temperature for the gels prepared following procedure II. The fitting parameter, n, is found to be temperature dependent and take small values in comparison with the values reported in Table 2. A strong difference is observed in the evolution of the coagel fraction as a function of time for
Figure 2. Evolution of the fraction of coagel phase (defined as φ ) CI -1) as a function of time for gels prepared following preparation type II and stored at various temperatures. The monoglyceride content is 5% in weight. The solid curve is a fitting to eq 2. Table 2. Values for the Rate Constant, 1/τ, and the Avrami Exponent, n, Obtained for the Gel to Coagel Phase Transition as a Function of the Monoglyceride Weight Percentagea 1/τ × 107 (s-1) n
5%
10%
15%
20%
6.2 3
4.2 3.4
4.3 3.6
2.7 4
a Gels have been prepared by following preparation type I and stored at 5 °C.
Table 3. Values of the Rate Constant, 1/τ, and the Avrami Exponent, n, Obtained for the Gel to Coagel Phase Transition as a Function of Storage Temperaturea 1/τ × n
106
(s-1)
5 °C
10 °C
15 °C
20 °C
3.7 0.4
7.8 0.8
12.2 1.6
12.8 1.6
a Gels (5% monoglyceride) have been prepared by following preparation type II.
gels prepared according to the two procedures. In the case of a 5% monoglyceride sample prepared according to procedure II, the rate constant, 1/τ, increases from 0.62 × 10-6 s-1 to 3.7 × 10-6 s-1. However, an instantaneous phase conversion under shear is ruled out since the coagel index still equals 1 just after the shear step. Figure 3A,B shows cross-polarized light microscopy micrographs of the gel phase before and after shear. A change of microstructure on shearing is observed. The characteristic appearance of the lyotropic gel phase can be observed in Figure 3A, while in Figure 3B a display of close-packed small units, probably multilamellar vesicles, is shown. Such transition of the bilayer topology under shear has been observed before.21,22 An interpretation of these results will be given in the discussion. III.B. Static Single Pulse NMR Study of the Gel Phase. III.B.1. Static Single Pulse 1H Spectroscopy. Figure 4 represents the 1H spectrum of a 15% monoglyceride gel of type I at different temperatures. 1H spectroscopy yields information about the mobility of the (21) Gulik-Krzywicki, T.; Dedieu, J. C.; Roux, D.; Degert, C.; Laversanne, R. Langmuir 1996, 12 (20), 4668. (22) Sierro, P.; Roux, D. Phys. Rev. Lett. 1997, 78 (8), 1496.
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Figure 5. 13C NMR spectrum of a 15% monoglyceride gel as a function of temperature.
Figure 3. Cross-polarized light micrographs of a 5% monoglyceride gel: (A) after cooling from the lamellar phase; (B) after shearing at 100 s-1 for 1 min. Figure 6. Evolution of the transverse relaxation time T2 as a function of temperature for a 15% monoglyceride gel: (O) CH2OH glycerol; (9) CH2 alkyl chain.
Figure 4. 1H NMR spectrum of a 15% monoglyceride gel as a function of temperature.
protons of water and the alkyl chains. At room temperature, only one resonance was observed at 4.9 ppm, this peak corresponds to the water protons. When the temperature was raised to 65 °C, the system entered into the lamellar phase region of the phase diagram.1,2 Then a sharp resonance was also observed around 1.7 ppm, corresponding to protons of the monoglyceride alkyl chain. The appearance of the CH2 resonance is a consequence of the melting of the chains. Below 65 °C these chains present a low degree of mobility in the bilayer, leading to strong dipolar interactions between 1H nuclei. As a consequence, the resonance peak of CH2 groups is broadened beyond detection. Around 65 °C the chains are molten; then they show a fast trans-gauche isomer-
ism. As their mobility is drastically increased, the dipolar interactions are averaged out and the CH2 resonance can be observed. III.B.2. Static Single Pulse 13C Spectroscopy. To have insight into the thermal motions of the monoglyceride headgroup in the gel phase, we utilized 13C NMR spectroscopy. Figure 5 presents the static single pulse 13 C spectrum of a 15% gel of monoglyceride at different temperatures. At room temperature three main resonances were observed. One broad and structureless resonance around 35 ppm that corresponds to the alkyl chain (13C-H2) and two sharper lines around 70 and 65 ppm that can be attributed to the glycerol backbone resonances: 13C-HOH and 13C-H2OH, respectively.4 The evolution of the line shape of these resonances with temperature is peculiar. The alkyl chain resonance remains broad and structureless up to 60 °C although the headgroup resonances start to sharpen beyond 40 °C. This shows dynamical heterogeneity of the monoglyceride molecules in the gel phase. From the bandwidth of the peaks at half-height, an estimate of the effective transverse relaxation time, T2, can be obtained. It is related to a correlation time proportional to the molecular motions.17 Figure 6 depicts the evolution of T2 as a function of the temperature for the CH2OH and CH2 groups. The alkyl chain mobility does not depend on temperature until the temperature reaches 60 °C. Above this temperature range, the transverse relaxation time shows a drastic
Gel to Coagel Transition in Monoglyceride-Water
Figure 7. Solid state 13C CP-MAS spectrum of the monoglyceride β-crystals and the coagel phase (20% monoglyceride).
increase due to the melting of the chains, (not presented in Figure 5). On the other hand, the mobility of the glycerol backbone shows a constant increase as the temperature increases from 35 to 60 °C. The molecular mobility of the alkyl chains being low below 60 °C, the motions of the glycerol backbone cannot be related to a translation of the full molecule in the plane of the bilayers but rather to rotation or vibration. III.C. Solid State 13C CP-MAS and 2H NMR Study of the Coagel Phase. III.C.1. 13C CP-MAS Spectroscopy of Monoglyceride β-Crystals and Coagel. From these measurements a strong similarity between the coagel and anhydrous β-crystals has been observed. The solid state 13 C CPMAS spectrum of the anhydrous β-crystals and the coagel are reported in Figure 7. The two spectra are very similar. The 13C resonances of the polar headgroups (CHOH, CH2OH, CdO) appear as a split resonance. This phenomenon, called chemical shift degeneracy, is linked to the characteristic crystallographic structure of the β-polymorph of monoglyceride. The resonance splitting is caused by the difference in the chemical environment of D and L isomers of the monoglycerides in β-crystals.4 The difference in signal-to-noise ratio is due to the large amount of water contained in the coagel crystal network. The correspondence in the isotropic chemical shifts indicates that the magnetic environment of the 13C nuclei in the β-crystal and the coagel is identical. This means that in the coagel phase and in the bulk β phase the same symmetrical arrangement of isomers is observed. A closer insight in the molecular dynamics of the coagel and the bulk crystalline structure (β-crystal) is provided by the sideband patterns of the carbonyl resonances. The sideband patterns of β-crystals and coagel show much resemblance, indicating that the underlying CSA tensors must also be equal. Then the structure and dynamics of the carbonyl region are the same in the two systems. Unfortunately, the signal-to-noise ratio of these experiments hampers quantitative determination of the chemical shift’s tensor elements. In the β-crystalline structure proposed by Larsson, the monoglyceride glycerol backbones are stabilized by a hydrogen bond network3 where the carbonyl of the monoglyceride headgroup, a strong hydrogen bond donor-acceptor, is not involved. Any change in the crystalline structure of the coagel phase involving some hydrogen bonding of the carbonyl groups would have caused a shift in the resonances. This is not the case, consequently, the coagel and the β-crystal phases
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have the same hydrogen bond pattern among the headgroups. Note that coagels obtained from gels prepared following preparation types I and II yield the same CPMAS spectrum. This indicates that the processing only influences the phase conversion rate but not the structure of the final state. III.C.2. 13C CP-MAS Dynamic Experiments on Monoglyceride β-Crystals and Coagel. We studied the dynamics of molecules in the coagel and β-crystal phases. To do so, we determined the relaxation times of monoglyceride molecules in these two phases. In a typical 13C CP-MAS dynamic experiment the cross-polarization condition is applied for a certain time. During this contact time, the magnetization from 1H nuclei is transferred to 13C nuclei. The magnetization transfer from 1H to 13C is intimately linked to the number and the dynamics of 1H nuclei in the vicinity of 13C nuclei. Hence, the evolution of the resulting 13 C FID as a function of the contact time can be related to the dynamic behavior and to the molecular structure of the material. The cross-polarization dynamic of 13C nuclei is characterized by the relaxation times; TCH and T1F. 1H T1F relaxation times are a sensitive monitor of dynamic heterogeneities at the nanometer scale.23 On the other hand, TCH depends on the number of 1H nuclei in the direct vicinity and of the dynamics of the 13C-1H vectors. Hence, it gives information on the local structure and mobilities of the nuclei. These parameters can be determined from the evolution of the magnetization as a function of the contact time. The 13C CPMAS buildup curves obey the following relations:24
M(tc) )
M0 (1 - λ)
[ (
exp -
λ)
)
( )]
tc tc - exp T1FH TCH
TCH T1FH
(3)
(4)
where tc, TCH, and T1F are the contact time, the crosspolarization time, and the 13C relaxation time in the rotating frame, respectively. The CP buildup curves for the second carbon position on the glycerol backbone of both β-crystals and coagel are presented in Figure 8. G2A and G2B indicate that the magnetization is determined for the two deconvoluted peaks of the CHOH resonances. From these curves the 13 C relaxation parameters TCH and T1F have been determined. The values are calculated from a straight-line fit of the logarithm of the carbon signal intensity versus tc, where tc is varied from 5 to 200 ms. The TCH parameters for the two systems do not differ within experimental error, whereas the T1F relaxation values differ significantly (Table 4). The fact that the TCH values are similar means that on a local scale the chemical environment of the glycerol backbone in coagel and bulk β-crystals is identical. On the other hand, the decrease of the1H T1F value in a coagel may be indicative for the presence of some motional heterogeneities in the crystalline structure on a larger scale. For1H spins, the longitudinal relaxation time (T1) is often influenced by a spin-diffusion process, which can be understood as an efficient spin-spin “communication” (dipolar coupling mechanism) between pools of immobile protons and pools of mobile protons in the direct neighborhood. Thus, due to spin-diffusion, magnetization can be transferred efficiently over distances up to tens of (23) Rethwisch, D. G.; Jacintha, M. A.; Dybowski, C. R. Anal. Chim. Acta 1993, 283, 1033. (24) Mehring, M. Principles of High Resolution NMR in Solids; Springer Verlag: Berlin, 1983.
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of the water resonance as a function of the surfactant concentration will be affected by the presence of the bound water NMR signal.7 The 2H NMR spectra of coagel/D2O samples that differ in monoglyceride concentration (50, 70, and 80% in monoglyceride) have been recorded. All these samples show only one sharp 2H NMR resonance. Furthermore, the width of this resonance is independent of the monoglyceride weight percentage and remains equals to 100 Hz. Therefore, the presence of some hydrogen-bound water in the coagel crystalline structure is very unlikely. However, structured water at the crystals-water interface may be present but the concentration is too low to be detected by NMR. IV. Discussion
Figure 8. CP dynamic curves for the CHOH resonance of the monoglyceride β-crystals (A) and the coagel phase (B). (O) G2A and (9) G2B indicate that magnetization is determined from the two deconvoluted peaks of the second carbon position on the glycerol backbone. Table 4. Values of the Relaxation Parameters (13C) TCH and (1H) T1G Obtained from 13C CP-MAS Buildup Curves of the CHOH Resonances of β-crystals and Coagel Phase (20% Monoglyceride)a TCH (ms) T1F (ms)
β-crystal
coagel
0.10 ( 0.05 90 ( 9
0.11 ( 0.05 62 ( 5
a Magnetization is determined from the deconvoluted peak of the second carbon position on the glycerol backbone.
nanometers. For solid materials T1F relaxation times of rigid nuclei can be affected by spatially remote mobile spin pools, so-called relaxation sinks. Hence, the decrease of the T1F relaxation time in the coagel phase is probably related to effective spin diffusion from the monoglyceride crystalline phase to bulk water, III.C.3. 2H NMR Studies on Monoglyceride Coagel. In the previous section we examined the monoglyceride structure in the coagel phase. Now we focus on the other component: the water. Due to its hydrophilicity, the glycerol moiety is expected to trap some water molecules during the crystallization process leading to the coagel phase. This hypothesis was tested with the help of 2H NMR, the signal of which is sensitive to ordering phenomena.7,25 The free bulk D2O resonance was observed as a narrow line. When a D2O molecule is involved in a hydrogen bond, it is observed as a broad resonance due to nonaverage quadripolar interaction: the Pake-doublet line shape. In a system where bound and free water is in fast exchange, the observed 2H NMR spectrum is a combination of the two signals. To increase the signal related to bound water, it is useful to study systems where the surfactant concentration is high. Hence, if some immobilized water is present in the system, the evolution (25) Halle, B.; Wennerstrom, H. J. Chem. Phys. 1981, 75 (4), 1928.
Now, we concentrate the discussion around two main topics: (i) the influence of the molecular mobility on the phase behavior of the system; (ii) the processes driving the gel to coagel phase transition. Stored between 45 and 55 °C, gels of monoglyceride are stable.1,2 From the results reported in section III.B.2, the gel stability in this temperature range can be related to thermally induced motion of the glycerol backbone. This entropic phenomenon has two consequences. First, it yields a strong repulsive interaction between bilayers in the nanometer range.9-10 Although lipid protrusion is not supposed to occur in the gel state, this brings evidence of the entropic contribution to the short-range repulsive force in monoglyceride/water mesophases.13,26 Second, motion of the glycerol backbone could prevent the formation of the β polymorph of monoglyceride, which is formed of opposing layers of D and L isomers stabilized by hydrogen bonds.3 Stored below 45 °C a strong driving force leads the system to crystallization. We have shown that the structural arrangements of monoglyceride in bulk β-crystal and coagel are identical. Hence, on crystallization from the gel phase, the two isomers of monoglyceride have to separate into complete layers of D and L isomers in order to form the β-crystal structure. This is related to a favorable interaction between the same enantiomers that give rise to homochiral discrimination. The phase transition is from a disordered system (a racemic mixture of D and L isomers) to a highly ordered system. Thus, the enthalpy variation involved in the gel to coagel phase transition must be positive enough to counteract this unfavorable entropy change. This large enthalpy variation has two origins: (i) the establishment of an inter-, and intrabilayer hydrogen bond network between the glycerol backbones, which can easily offset the low hydration of the monoglyceride headgroups in the gel phase;7 (ii) the crystallization of the alkyl chains into a more dense packing that causes strong interchain van der Waals attraction. The difference between the melting enthalpies of the β-polymorph of pure stearic fatty acid and pure C18 monoglyceride gives a rough estimate of the energy gain involved in the H-bond network among the glycerol groups. A reasonable value of 13 kJ/mol (assuming a molar mass of 372 g for monoglyceride) is found. The equation we used to fit the kinetics of the phase transition is formulated in the frame of the KolmogorovAvrami theory.27-30 In eq 2, 1/τ is the rate constant of the (26) Gordeliy, V. I.; Cherezov, V. G.; Anikin, A. V.; Chupin, V. V.; Teixera, J. Prog. Colloid Polym. Sci. 1996, 100, 338. (27) Avrami, M. J.Chem. Phys. 1939, 7, 1103; 1940, 8, 212; 1941, 9, 177. (28) Takahashi, H.; Hatta, K.; Hatta, I. J. Phys. II Fr. 1996, 6, 1657. (29) Evans, U. R. Trans Faraday Soc. 1945, 41, 365. (30) Yang, C. P.; Nagle, J. F. Phys. Rev. A 1988, 37, 10, 3993.
Gel to Coagel Transition in Monoglyceride-Water
phase conversion, and the time power, n, is the Avrami exponent. The latter takes on values that depend on the mechanism of crystallization. In the case of a spontaneous crystallization, the Avrami exponent is equal to the growth dimensionality. It takes on a value of 2 for disk-shaped crystals. Under those conditions it is assumed unrealistically that all nuclei start to grow simultaneously at t ) 0. The model can be refined to allow for sporadic nucleation, where the number of nuclei in the system is a function of the time. The rate of formation of nuclei in the sample is given by N′2πr dr, where N′ is the random nucleation frequency. This has the effect of increasing the value of the Avrami exponent by 1,29-30 now having a value of 3 for disk-shaped crystals. Thus, the values reported in Table 2 for the 5% monoglyceride sample are in agreement with a phase transition taking place within bilayers. The variation of the parameters derived from the Avrami equation with the monoglyceride concentration is a complex phenomenon to explain. The best fit to the crystallization kinetics of the 20% monoglyceride sample is obtained when the Avrami exponent takes on a value of 4. Such a value of the Avrami exponent corresponds to a 3-dimensional crystal growth with a sporadic nucleation mode. Hence the increase of the Avrami exponent could indicate that on increasing the monoglyceride concentration the growth dimensionality of the new phase evolves from 2 to 3. As the bilayer spacing in the gel phase is inversely proportional to the surfactant concentration, at high concentration in monoglyceride (i.e., small bilayer spacing), coupling between bilayers could allow for the new phase to grow to adjacent bilayers instead of being confined within bilayers. This renders the crystallization mechanism concentration dependent. We assume that the gradual increase of the Avrami exponent is indirectly related to an increase in the efficiency of the interbilayer coupling allowing for 3-dimensional crystal growth. Although it is not possible to resolve the rate constant, which depends both on nucleation and on growth on increasing monoglyceride concentration, the decrease of the rate constant is probably due to the change in the crystallization process. In the case of a two-dimensional growth of the new phase, the necessity to form D and L isomer layers indicates that the phase transition should depend on molecular diffusion within bilayers. A mechanism where patches of isomers randomly formed attain a sufficient size to yield a nucleus that locally initiates the crystallization is highly probable. In this case the nucleation frequency and the radial growth rate depends on the diffusion coefficient of molecules within bilayers. This is illustrated by the increase in the rate constant with temperature reported in Table 3. On the other hand, the increase of the Avrami exponent with temperature may be related to an increase in the radial growth rate. Nevertheless above 45 °C, the monoglyceride’s headgroup mobility prevents homochiral interactions from occurring. The formation of domains of identical isomers that could act as nuclei is subsequently hampered. This, combined with the onset of steric repulsive force between bilayers account for the long-term stability of the gels above 45 °C.
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Strong differences in the phase transition rate and Avrami exponent are observed when the gel phase is sheared (Figures 1 and 2). This result could be explained by an increase of the nucleation frequency. The effect of the applied shear on the sample is to disrupt the lamellar structure of the gel in small bilayer units. Once the shear stops, these small bilayer units have to cluster in order to isolate the alkyl chains from water. In this case the lamellar structure of the gel is built at low temperature where attractive chiral interactions between isomers are dominant. This will increase the probability for identical isomers of monoglyceride to meet and thus form nuclei initiating the phase transition. This situation being similar to a spontaneous crystallization where all nuclei are formed at t ) 0, the value of the Avrami exponent is decreased by 1.29-30 Nevertheless, this cannot explain the fractional values of the Avrami exponent, which are still lower than the expected domain growth dimensionality of 2 for disk-shaped crystals. A similar result has been reported previously, the authors invoked the topology of the lipid bilayers to explain it.30 Indeed, the radial growth rate of the new phase within planar bilayers is a constant.31,32 Nevertheless, in the case of spherical or more complicated bilayer stacking, this is no longer the case. The radial growth rate of the new phase slows down for larger domains, and the value of n is then reduced. We observed that shearing the gel phase leads to the formation of small multilamellar units (Figure 3A,B). Hence, the small value of the Avrami exponent could be explained by the complex topology of the lipids bilayers induced by shearing the gels. V. Conclusion The gel to coagel transition can be discussed in the frame of the Avrami theory of crystallization kinetics. The high level of molecular motion of the polar headgroups that induces a steric repulsion at the bilayer surface and prohibits the formation of nuclei initiating the phase transition explains the long-term stability of the gel above 45 °C. The evolution of the Avrami exponent indicates a transition from a 2-dimensional to a 3-dimensional crystal growth on increasing the monoglyceride concentration. In the case of a 2-dimensional growth the crystallization is diffusion controlled. On applying shear to the gel phase, the phase transition evolves from a random to a spontaneous nucleation mechanism. Acknowledgment. The authors thankfully acknowledge Dr. A. S. Kulik for performing NMR experiments on the gels of monoglyceride. Pr. G. J. Tiddy is acknowledged for his fruitful comments on the manuscript. Dr. G. Cassin acknowledges the financial assistance from EEC, in the form of a postdoctoral fellowship (Human Mobility Capital Fund, Contract No. ERB-CHDG-CT-93-0426). LA980340M (31) Price, F. P.; Fritzsche, A. K. J. Phys. Chem. 1973, 77, 369. (32) Jabarin, S. A.; Stein, R. S. J. Phys. Chem. 1973, 77, 409.
