Dextran Mixture Studied by

Biomacromolecules 2003, 4, 928-936

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“Delayed” Phase Separation in a Gelatin/Dextran Mixture Studied by Small-Angle Light Scattering, Turbidity, Confocal Laser Scanning Microscopy, and Polarimetry Michael F. Butler* and Mary Heppenstall-Butler Unilever Research and Development, Colworth House, Sharnbrook, Bedfordshire, MK44 1LQ United Kingdom Received January 29, 2003; Revised Manuscript Received March 24, 2003

Small-angle light scattering, turbidity, and confocal laser scanning microscopy were used to study microstructure formation and evolution in a gelatin/dextran mixture. There was a time-delay of up to tens of minutes between reaching the quench temperature and the onset of phase separation, because demixing only occurred once a certain amount of ordering of the gelatin molecules, measured by polarimetry, was attained. The accompanying phenomenon of gelation retarded the development of the microstructure to different extents, depending on the quench temperature. At low temperatures, the structure was rapidly trapped in a nonequilibrium state with diffuse interfaces, characteristic of the early and intermediate stages of phase separation. At higher temperatures, coarsening continued for a certain amount of time before the structure was trapped. The duration of the coarsening period increased with increasing temperature and the interface between the phases became sharp, characteristic of the late stages of phase separation. Because the ordering process continued after the target quench temperature was reached, the effective quench depth continued to increase after the initial phase separation. At high quench temperatures, the system was able to respond to the thermodynamic requirements of the increasing effective quench depth by undergoing secondary phase separation to form a droplet morphology within the preexisting bicontinuous one. Introduction When different polymer solutions are mixed, phase separation often occurs. This phenomenon has been thoroughly studied, mainly because of its practical implications for the material properties of the resulting two-phase mixture and, in quiescently cooled liquid mixtures, is well understood. Two possible means of phase separation exist: nucleation and growth and spinodal decomposition.1 Nucleation and growth occurs when mixtures are taken to temperatures in the vicinity of the binodal, where the system is stable to small concentration fluctuations and phase separation only occurs after a nucleus in excess of the critical size has formed (the size of which will depend on whether nucleation is homogeneous or heterogeneous). Spinodal decomposition occurs for deeper quenches, when all concentration fluctuations below a certain size (that is determined by the competition between the free energy cost to create new interfaces and the transport of material) are stable and phase separation therefore occurs immediately. Nucleation and growth results in a random, polydisperse, array of droplets with sharp interfaces. Spinodal decomposition results in either a droplet or bicontinuous morphology that is characterized by one particular lengthscale (so that the droplets, for example, are roughly evenly spaced and of similar sizes when they are formed). Initially the interface between the phases is diffuse, although it later becomes sharp as the phases evolve toward their equilibrium compositions.

Spinodal decomposition is often studied using small-angle light scattering, because the scattering pattern is the spatial Fourier transform of the real-space concentration fluctuations, which is the most convenient way to quantify the concentration fluctuation distribution. The scattering intensity can be written as I(q) ) I(q,0)e2R(q)t

(1)

where the scattering vector, q (which is related to the scattering angle, θ, and the wavelength, λ, by the relation q ) (4π/λ) sin(θ/2)) is related to the real-space length, d, by q ) 2π/d, and R(q), known as the amplification factor, describes the growth in concentration fluctuations at scattering vectors q corresponding to real-space lengths, d. In the linear theory of spinodal decomposition, R(q) (and therefore the scattering intensity) has a maximum at a q value denoted by qm and is given by

(

R(q) ) Deff(q)q2 1 -

)

q2 2qm2

(2)

where Deff is the effective diffusion coefficient of the solutes. There is a maximum in the amplification factor and scattering intensity, the position of which yields the characteristic length-scale, ξ, that is observed in the structure, given by

* To whom correspondence should be addressed. Michael.Butler@ Unilever.com. Phone: +44 (0)1234 222958. Fax: +44 (0)1234 22 2757. 10.1021/bm0340319 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/07/2003

ξ)

2π qm

(3)

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Delayed Phase Separation in Gelatin/Dextran Mixtures

Additionally, information on the interface between the different phases can be obtained by measuring the asymptotic shape of the scattering intensity, Iasympt, at high q values. For the case of sharp interfaces, the following relations, known as Porod’s law,2 gives the intensity, Iasympt (provided that the length-scales in which there are inhomogeneities in refractive index are much larger than 1/q), which differs depending on whether droplet or a bicontinuous morphologies exist3 A Iasynpt(q) ∝ 〈δn2〉 4 q A Iasynpt(q) ∝ 〈δn2〉 6 q

droplet

(4)

bicontinuous

(5)

δn is the refractive index difference between the two phases and A is the amount of interfacial area between the two phases. A plot of ln(I) vs ln(q), known as a Porod plot, should yield a straight line with a gradient of -4 for droplets and -6 for a bicontinuous morphology if the interfaces are sharp compared to the wavelength of light and with an intercept with the ordinate whose value is proportional to the amount of surface area between the phases in the sample. Once demixing has occurred, and regardless of the mechanism by which it has formed, the microstructure coarsens. The power law relationships between characteristic size and time that are observed in the late stage can be separated into three distinct mechanisms, depending on the means of mass transport. The first two mechanisms, which both give an exponent of 1/3 in liquid systems but have been observed to give lower exponents in gelled systems,4-6 are the growth of larger droplets at the expense of smaller droplets (Ostwald ripening) with mass transport occurring by diffusion through the matrix7 and movement of the droplets toward each other leading to coalescence.2 The third mechanism, which gives an exponent of 1, occurs when hydrodynamic flow is the cause of mass transport.8 This mechanism occurs when there is sufficient material within the coarsening phase for flow fields to become established inside the coarsening phase which are coupled to flow fields in the matrix via the interface. Because it requires fluid flow, it is only observed in liquid systems and will not be observed if gelation occurs. Eventually, in liquid mixtures, and regardless of the phase separation mechanism, sedimentation and creaming occurs. When one or both of the components can form a gel, however, it becomes possible to slow the coarsening process and therefore “trap” microstructures.4,5,9,10 In the case where the gelation kinetics follow a first-order rate equation, it is possible to predict the time at which coarsening should cease.11,12 Biopolymers usually exhibit more complicated behavior, however, and therefore, most previous work on the phase separation of biopolymer mixtures has concentrated on the qualitative differences between phase separation in a system quenched to temperatures either above or below the gelation temperature, thereby allowing a comparison between the phase separation mechanism and kinetics of liquid and gelled systems, respectively.4-6,13 In these systems, by necessity, the gelation temperature lay below the binodal temperature. Another scenario, which is the subject of the present study, is when the gelation temperature lies above the binodal temperature.

Experiments on the gelatin/maltodextrin system have shown that the tendency for phase separation to occur is promoted by the coil-helix conformational change, which is associated with gelation, experienced by gelatin at temperatures in the vicinity of 30 °C.5,14-16 When the binodal temperature (for a particular composition) lies below the coil-helix transition temperature, phase separation does not occur immediately upon reaching the quench temperature but only begins once a certain degree of ordering of the gelatin molecules has been attained.16 Because the ordering kinetics are temperature dependent, it follows that in these systems there will be a temperature dependent time delay before phase separation occurs. Furthermore, because the extent of gelation determines how rapidly the microstructure becomes trapped, it is possible that phase separation might only begin once the gel has become strong enough to prevent any coarsening, thus trapping an early stage microstructure containing diffuse interfaces. This situation is less likely to occur in systems with a binodal temperature in excess of the gelation temperature, where the higher drive for phase separation ensures that the system is more likely to evolve from the early stage microstructure before gelation has occurred sufficiently to trap it.6 The aim of the present study was to study the microstructure development of a demixed gelatin/dextran system in which phase separation was triggered by the conformational change of the gelatin molecules, over a range of temperatures. Small-angle light scattering (SALS) was used to provide quantitative information on the phase separation mechanism and coarsening kinetics and to qualitatively study the interfacial width and phase composition changes. Confocal laser scanning microscopy (CLSM) was used to provide visual evidence assisting the interpretation of the SALS data and polarimetry was used to quantitatively study the ordering kinetics of the gelatin molecules in the mixtures. Experimental Section Materials and Sample Preparation. A mixture of gelatin and dextran was used in the following experiments. The gelatin was a lime-treated gelatin (LH1e) supplied by SKW, with values for Mn and Mw of 83 300 and 146 000 g mol-1 measured from size exclusion chromatography coupled with light scattering. The dextran was supplied by Sigma Chemical Co. and had a value of Mw of 413 000 g mol-1. A mixed solution was prepared as follows: gelatin granules were dispersed in a 0.5 M sodium chloride solution, heated to 60 °C, and stirred for 30 min. Similarly, dextran powder was dissolved in water, heated to 60 °C, and stirred for 30 min. After this time, the solutions were mixed together and held at 60 °C until used. The final concentrations of the components in the mix were: gelatin, 5%; dextran, 4%. No preservatives were added, and all solutions were used on the day that they were made. A pinch of rhodamine B powder was added to the mixtures used in the confocal microscopy experiments in order to fluorescently label the gelatin-rich phase and thus provide additional contrast between the phases in the images. Small-Angle Light Scattering. Samples for study by small-angle light scattering were made by placing a drop of

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the mixed solution onto a glass coverslip (thickness 0.17 mm, diameter 22 mm) in the center of a 50 µm stainless steel spacer which was heated to 60 °C on a Linkam THMS600 microscope stage. Another, identical, glass coverslip was placed on top of the sample to ensure that it was a constant thickness of 50 µm. The influence of the spacer thickness on possible spatial confinement of morphologies containing length-scales of the same order is discussed in the discussion section. Small-angle light scattering was performed using the apparatus described previously,5 with the exceptions that the light source was a 0.5 mW HeNe laser and the SALS patterns were recorded using a Peltier cooled CCD camera (Photonic Science Fast Digital Imager). Scattering patterns were recorded every 10 s. Data collection began at the start of the temperature profile, beginning with an isotherm at 60 °C for 3 min followed by a rapid quench, at 60 °C/min to the quench temperature. Quench temperatures were chosen in 2 °C steps from 12 to 28 °C (inclusive). The sample was maintained at the quench temperature for 95 min. The scattering patterns collected during the first 3 min at 60 °C were averaged, and this average was subtracted from the subsequently collected scattering patterns. The 2D scattering patterns were radially averaged to produce plots of intensity vs scattering vector q. Calibration of the scattering vector on these plots was performed using the scattering pattern from a 25 µm diameter pinhole. Turbidity. A Perkin-Elmer Lambda 40 UV/vis spectrophotometer connected to a programmable cooler was used to measure the phase separation temperature of a series of gelatin/dextran mixtures, from the turbidity at a wavelength of 365 nm. Gelatin and dextran concentrations between 4 and 7 wt % were studied, and the salt content varied from 0 to 1 M. A total of 3 mL aliquots of each gelatin/dextran mixture were placed in a pre-warmed 10 mm path length PMMA cuvette, held at 60 °C for 2 min and then cooled as rapidly as possible to 10 °C. The gelatin used was LH1e, as for the SALS experiments. Dextrans, supplied by Sigma Chemical Co., with Mw values of 200 000 and 413 000 g mol-1 were used, although the majority of the experiments were performed with the former material. Confocal Laser Scanning Microscopy. Samples for study by confocal microscopy were made in the same way as for SALS. The sample was placed on a temperature controlled stage (Linkam THMS600). The temperature profile used was the same as for the SALS experiments, and the sample was held at 60 °C prior to beginning the profile. Micrographs of the mixtures were acquired using a Biorad MRC 600 CLSM. A 488 nm Argon laser excited the rhodamine B, which provided the contrast between the gelatin-rich and dextranrich phases. Micrographs were captured using COMOS software. A ×20 lens was used with a digital zoom of ×1, ×5, or ×10. Polarimetry. Samples for the optical rotation experiments were made by placing the mixture into a jacketed quartz sample holder with a 5 mm path length. The cell was thermostated using water circulating through the jacket, and the sample temperature was measured using a thermocouple (K-type) inserted into the cell. Quench experiments were

Butler and Heppenstall-Butler

Figure 1. Evolution of the total scattered intensity with time for quenches to 20, 22, 24, 26, and 28 °C, from which the delay times before phase separation occurred were measured.

performed using two water baths by switching flow to the cell from one bath, maintained at 65 °C, to the other, maintained at the quench temperature. Optical rotation (OR) measurements were made using a Perkin-Elmer 241C polarimeter. A mercury lamp with a filter was used to provide a light source with a wavelength of 365 nm. The output signals from the polarimeter and the sample thermocouple readings were fed into a personal computer via a data-logging card (Strawberry Tree) controlled by signal logging and analysis software (Labware), allowing precise correlation of the OR signal with the sample temperature. The influence of sample turbidity on the ability of the instrument to reliably measure the optical rotation was assessed using a series of glucose solutions containing different concentrations of 500 nm diameter latex spheres. The instrument readings were reliable at all times for all samples over the time in which phase separation and coarsening was studied. In addition to the mixtures of gelatin and dextran studied in the SALS and CLSM studies, the change of rotation angle with time at selected quench temperatures was measured for individual gelatin and dextran solutions. Results Small-Angle Light Scattering. Quenches to temperatures below 28 °C were accompanied by an increase in the amount of scattered light as phase separation occurred. Figure 1 shows the variation in the integrated intensity for samples quenched to temperatures in the range from 14 to 28 °C. Quenches to temperatures below 20 °C resulted in an immediate increase in the amount of scattered light and turbidity. Above this temperature, however, an induction period was present after reaching the quench temperature, during which no scattered light was detected, and after which the amount of scattered light increased. The duration of the induction period decreased with increasing quench depth, with a delay of approximately 50 min occurring before phase separation occurred in the sample quenched to 28 °C but only 0.5 min at a quench temperature of 20 °C. At any time, the values of the total scattered intensity increased with increasing quench depth.



Figure 2. (a) Evolution of the scattering pattern with time and (b) Porod plot, for the quench to 22 °C. In the Porod plot, the gradients of -4 and -6 are superimposed for comparison with the experimentally measured high scattering vector region. The peak intensity increases with time.

Figure 3. (a) Evolution of the scattering pattern with time and (b) Porod plot, for the quench to 26 °C. In the Porod plot, the gradient of -6 is superimposed for comparison with the experimentally measured high scattering vector region. The peak intensity increases with time.

The evolution with time of the radially averaged intensity as a function of the scattering vector, q, is shown in Figures 2a, 3a, and 4a for quenches to 22, 26, and 28 °C, respectively. In all cases, there was a maximum in the scattered intensity at a scattering vector qm. For quenches to temperatures below 18 °C, the scattering pattern remained relatively unchanged with time. The initial peak position moved to lower scattering vectors, and the peak width became smaller, as the quench depth decreased. Figures 2b, 3b, and 4b are double logarithmic (Porod) plots of the structure function for quenches to 22, 26, and 28 °C, respectively. For quenches to 24 and 26 °C, the gradient of the curve in the limit of high q vectors was -6 (see Figure 3). The gradient of the curve in this limit deviated from -6 as the quench temperature decreased to 22 °C (see Figure 2). At 28 °C, however, the gradient was -4 (see Figure 4). In all cases above 22 °C, the intercept with the ordinate of the straight line in the high scattering vector limit increased with increasing time after phase separation. For the deeper quenches, the curves in the high scattering vector region of the scattering pattern overlapped. Figure 5 shows the evolution of the value of qm with time after the onset of phase separation for a series of quench temperatures, revealing that the position of the maximum actually moved to very slightly higher q vectors for the

quenches to 14 and 16 °C. For progressively shallower quenches, the scattering patterns changed to greater extents. For all quenches between 18 and 26 °C (inclusive), the peak initially moved toward lower q vectors with a power law coarsening exponent of about -1/6, reached a minimum value, and then asymptotically returned toward a slightly higher value. The time taken for the peak to reach the minimum value increased with decreasing quench depth. At the shallowest quench, to 28 °C, however, the peak initially moved very definitely to larger scattering vectors before moving back to lower ones but eventually became fixed at a certain value in the same way as for the deeper quenches. Figure 6 shows the variation in peak height for the quenches to 24, 26, and 28 °C. For quenches to temperatures lower than, and including, 26 °C the peak height rose rapidly toward an asymptotic limit as soon as it appeared. For the quench to 28 °C, however, the peak height rose much more slowly after it appeared. Turbidity Measurement of the Phase Separation Temperature. Phase separation was detected via an increase in turbidity either below the gelation temperature of the gelatin, which was 30 °C, or way above it (>60 °C). Phase separation was not observed at any temperature where the system

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Figure 6. Variation of the value of the peak height with time for quenches to 24, 26, and 28 °C.

Figure 7. Micrograph showing a trapped bicontinuous microstructure at 20 °C. Image width 718 µm.

Figure 4. (a) Evolution of the scattering pattern with time and (b) Porod plot, for the quench to 28 °C. In the Porod plot, the gradient of -4 is superimposed for comparison with the experimentally measured high scattering vector region. The peak intensity increases with time.

Figure 5. Variation of the value of the peak position with time for quenches to 14, 18, 20, 22, 26, and 28 °C.

remained liquid in the range between 30 and 60 °C for mixtures containing either molecular weight dextran studied. Confocal Laser Scanning Microscopy. The confocal microscope images revealed that the phase-separated microstructure was bicontinuous for quenches to 26 °C and below. At temperatures below 20 °C, shown in Figure 7 as an example, the bicontinuous microstructure was trapped before

it could coarsen substantially. At 26 °C, markedly different behavior was observed that is shown in Figure 8. At this temperature, the microstructure was able to coarsen considerably, albeit slowly, by Brownian coalescence, as shown by the mobility of the gelatin-rich inclusions (several of which can be seen merging) in Figure 8h-l. Secondary phase separation also occurred in both phases at some time after the initial phase separation event (in Figure 8f) and formed a droplet morphology within the original phases. Figure 9 shows a magnified image of a structure that underwent secondary phase separation, revealing the presence of substantial depletion layers in the original phases within which no secondary phase separation occurred. Polarimetry. Figure 10a shows the development of the rotation angle during quenching to the final temperature for a series of gelatin/dextran mixtures. The time to reach the quench temperature was about 3 min, during which the temperature dependence of the rotation angle for gelatin molecules caused it to decrease. However, upon reaching the quench temperature, the rotation angle continued to increase with time toward an asymptotic limit that became smaller as the quench depth decreased. Solutions of gelatin studied individually showed the same behavior as the gelatin/ dextran mixtures, and solutions of dextran studied individually showed that dextran had a negligible effect on the change in optical rotation during cooling to temperatures below the gelation temperature of gelatin. Figure 10b shows the change in rotation angle with time for the quench to 26 °C, as a typical example, fitted with a first order Avrami function. The ordering kinetics were not adequately explained as resulting from a first-order process. The values of the rotation angle at the time that phase separation began in the samples that experienced a delay



Figure 8. Sequence of micrographs showing the evolution of the microstructure in a sample quenched to 26 °C. Time after quench temperature was reached: (a) 24, (b) 26, (c) 28, (d) 29, (e) 32, (f) 34, (g) 39, (h) 41, (i) 49, (j) 52, (k) 57, and (l) 62 min. Image width 718 µm.

Discussion

Figure 9. Close up of secondary phase separation, showing droplets within the earlier formed phases and depletion regions within the gelatin and dextran rich phases. Image width 72 µm.

before phase separation are shown in the final column of Table 1. Within experimental uncertainty, the value of the rotation angle, and hence the amount of ordering of the gelatin molecules, was the same at the start of phase separation regardless of the quench temperature.

Phase Separation Mechanism. The quenches to temperatures below 26 °C occurred via spinodal decomposition. These samples were shown by CLSM to possess a bicontinuous morphology that can only be formed by the spinodal decomposition mechanism.1 Furthermore, the quenches to temperatures between 18 and 26 °C were characterized by a SALS peak that moved to lower scattering angles and rapidly increased in height from the outset,1 which is another behavior that is characteristic of spinodal decomposition. At temperatures below 18 °C, the gelation kinetics were sufficiently rapid to trap the structure as soon as the phase separated morphology formed and there was therefore relatively little movement of the SALS peak. The quench to 28 °C was believed to occur via nucleation and growth, however. In this case, the SALS peak initially moved to higher scattering angles, which may be explained in the nucleation and growth mechanism by the formation

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Figure 10. (a) Change in rotation angle with time, measured by polarimetry, for quenches to 14, 16, 18, 20, 22, 24, 26, and 28 °C. (b) A comparison of the measured change in rotation angle with a fitted first-order Avrami function for the quench to 26 °C. Table 1. Delay Times Measured by SALS and the Corresponding Rotation Angle Measured at that Time by Polarimetry quench temp (°C)

delay time (minutes)

rotation angle (°) ((10%)

20 22 24 26 28

0.5 2.0 5.1 7.9 46.4

-0.18 -0.19 -0.19 -0.19 -0.18

of new droplets between preexisting ones. If the volume fraction of droplets is sufficiently high, some degree of correlation will exist between their positions that will cause a peak, whose position is representative of the droplet spacing, to appear in the SALS pattern. The presence of a peak in the SALS pattern for the nucleation and growth mechanism has been predicted and observed in colloidal and polymer systems,17-23 including a liquid/liquid gelatin/ maltodextrin mixture near the binodal temperature.6 Once nucleation has stopped, coarsening leads to the increase in droplet size that causes the scattering peak to move to lower scattering angles, as observed. In addition, the transition of the behavior of the peak height, from a relatively rapid increase at the onset of phase separation in the spinodal decomposition samples to the slower increase in the sample quenched to 28 °C, is also indicative of a transition from spinodal decomposition to nucleation and growth.6,24 Finally, the Porod slope of -4 provided evidence that at 28 °C a

droplet morphology, as would be expected for the nucleation and growth mechanism, was present. If spinodal decomposition had occurred, a bicontinuous morphology would be expected because this was the morphology observed for the samples that definitely underwent spinodal decomposition. Delayed Phase Separation. Spinodal decomposition occurs when the system is quenched into the unstable region of the phase diagram. When a temperature change is the sole reason for incompatibility, phase separation occurs as soon as the quench temperature is reached in the unstable region. In the present case, spinodal decomposition was demonstrated by SALS and CLSM to be the phase separation mechanism for a number of samples where a delay was measured between reaching the quench temperature and the onset of phase separation. The temperature change was therefore not the primary reason for phase separation in these cases. Indeed, recent work has similarly reported a lack of sensitivity of the phase separation behavior of gelatin/dextran mixtures to temperature in the temperature range from 40 to 80 °C.25 The observation of the same degree of optical rotation at the onset of phase separation in the samples that experienced a delay suggests an explanation for the phase separation trigger, however. Optical rotation is caused by the coil to helix transition in gelatin26 that occurs at temperatures below about 30 °C. The optical rotation results suggest, therefore, that a certain degree of helix formation (i.e., ordering of the gelatin molecules) was required to trigger phase separation, presumably because the values of enthalpy and entropy of mixing of gelatin helices and dextran differed from those for gelatin coils and dextran. Previous results on a gelatin/ maltodextrin system without salt, in which phase separation was measured by an increase in turbidity, have also observed the requirement for a certain amount of helix formation prior to phase separation.16 Less directly, results on gelatin/ maltodextrin/salt systems that phase separate at temperatures above and below 30 °C have shown that below the gelation temperature there is an increased drive for phase separation that cannot be explained by the temperature quench alone.5,14,15 Because the gelatin ordering kinetics were slower for the shallower quenches, progressively longer delay times were observed before phase separation was initiated in these cases. Microstructure Development. Once phase separation had occurred, the resulting microstructure was determined mainly by the relative rates of three kinetic factors: those of gelation, coarsening, and continued conversion of gelatin coils to helices. Below about 18 °C, gelation rapidly trapped the microstructure and prevented coarsening, as shown by the CLSM images. The slight initial increase in peak position for these samples may be explained as a small effect of the continuing conversion of gelatin coils to helices. In these samples, a substantial amount of gelatin helices formed after phase separation began. The effective quench depth therefore increased with time, altering the thermodynamic conditions that determined the characteristic size in the spinodal structure. Deeper quenches are associated with smaller characteristic length scales1 and hence larger values of the scattering angle at which the peak in the scattering pattern is situated. It is therefore proposed that the system responded


to the continued increase in effective quench depth by diffusion of the components leading to a slight increase in the characteristic length scale. Gelation prevented coalescence, however, and rapidly trapped the microstructure in a nonequilibrium state leading to the pinning of the characteristic length scale at one particular value. The deviation of the Porod gradient from the value of -6 that is expected for a bicontinuous morphology3 suggested that rapid gelation kinetics trapped the interface in a relatively diffuse state characteristic of the early or intermediate stages of spinodal decomposition. Similar deviations have been observed during the intermediate stages of phase separation in synthetic polymer solutions.27 The increasing breadth of the peak in the SALS pattern at lower temperatures was also indicative of a less well-formed structure in these cases. The increasingly slower gelation kinetics for the samples quenched to progressively higher temperatures above 20 °C enabled the structure to coarsen via coalescence, as shown in Figure 8, for an increasing amount of time after the onset of phase separation. At 20 °C, only a small amount of coarsening occurred, but at 26 °C, coarsening of the microstructure occurred for up to about 1 h after phase separation began. In liquid systems that coarsen in a selfsimilar manner (i.e., systems that obey dynamic scaling), the coarsening exponent is predicted to be 1/3.2 Nevertheless, lower values, such as the value of 1/6 measured in the present study, have been predicted and observed in systems where coalescence and diffusion is hindered by the low mobility of the components.28 As for the deeper quenches, the eventual trapping of the microstructure was a consequence of increasingly hindered diffusion and coalescence caused by gelation. This behavior agreed qualitatively with predictions from a model that assumed first-order gelation kinetics,11,12 although a quantitative comparison of the time before coarsening stopped was precluded because the gelation kinetics in the present system, which were assumed to be related to the gelatin ordering kinetics, were not first order. Interestingly, the coarsening exponent measured in previous studies of gelatin/maltodextrin5,6,9 and gelatin/dextran mixtures4,13 quenched to temperatures below the gelation temperature was found to decrease steadily with increasing quench depth. In the present study, the coarsening exponent appeared to be generally constant regardless of quench temperature. The main difference between the present and previous systems is that the former system could only phase separate in the gelled state, triggered by the gelatin coil-helix transition, whereas the latter systems could phase separate in the liquid state due simply to temperature changes. It is not clear, however, why this difference should cause differences in the behavior of the coarsening exponent below the gelation temperature. It should be noted that, at the quench temperature of 26 °C, the domain size measured by SALS (approximately 30 µm) was similar to the thickness of the sample holder (50 µm). It is possible that this prevented true droplet structures from forming. Nevertheless, although this may have had some influence on the coarsening rate, it does not alter the main finding of microstructural trapping after a certain time that was a consequence of gelation. For deeper quenches, the domain size was significantly smaller than the


cell thickness, and the structure was therefore unlikely to have been affected by spatial confinement of the microstructure. For the quenches to 24 and 26 °C, the Porod gradient of -6 suggested that the gelation kinetics were sufficiently slow compared to the phase separation kinetics for the interface between the two phases to become sharp at times when the structure was definitely bicontinuous. The Porod gradient of -6 in the sample quenched to 26 °C at later times when CLSM showed an apparent droplet morphology may have been due either to some bicontinuity in the structure caused by spatial confinement of the larger structures whose size was similar to the cell thickness or because there was some bicontinuity in the much smaller secondary phase separated morphology. Figure 10 showed that the apparent droplets in the secondary phase separated structure were indeed very closely spaced in a particular plane, and it is not unreasonable to speculate that some connections may have existed at different levels in the structure. Deviations from -6 for the samples quenched to 20 and 22 °C suggested that in these cases the components were not sufficiently mobile for a completely sharp interface to develop. The increasing intercept with the ordinate made by the Porod slope for the samples quenched to temperatures above 22 °C indicated that the amount of surface area between the different phases increased with time.2 For the quench to 28 °C, where nucleation and growth was believed to occur, this behavior is expected because extra surface area is created by the continual nucleation of new droplets. For the samples quenched to lower temperatures, where spinodal decomposition occurred, this behavior is not expected from simple coarsening of the morphology. The occurrence of secondary phase separation, as shown in the CLSM images for the quench to 26 °C, does explain an increase in surface area for the spinodal samples, however, and demonstrates the influence of the continuing process of gelatin ordering after the initial phase separated morphology had formed. Secondary phase separation occurred as a result of the increase in effective quench depth because of continued gelatin ordering, when the components were sufficiently mobile enough for concentration fluctuations to develop in the existing phases with the compositions dictated by the effectively deeper quench. Similar results were observed in a rapidly quenched near-critical gelatin/maltodextrin mixture taken to temperatures between 1 and 25 °C.29 Conclusions The presence of a similar amount of gelatin helix formation at markedly different delay times between reaching the quench temperature and the onset of phase separation suggested that conformational ordering of the gelatin molecules was responsible for demixing when the binodal temperature lay below the gelation temperature of gelatin. Both spinodal decomposition and nucleation and growth mechanisms were observed, with the former being the predominant mechanism but both giving a peak in the smallangle light scattering pattern. Differences in the behaviour of the peak position combined with direct visual observation

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of the microstructure by optical microscopy could, however, be used to distinguish between the mechanisms. Bicontinuous morphologies were formed via spinodal decomposition whereas nucleation and growth yielded a droplet morphology. The use of conformational ordering combined with gelation allowed the formation of a range of microstructures that were determined by how soon phase separation was initiated and how rapidly microstructural development could be restricted by the reduction in mobility of the components. These ranged from structures characteristic of the early stages of spinodal decomposition, with diffuse interfaces and small domain sizes, to late-stage morphologies with sharp interfaces and significantly increased domain sizes due to coarsening via coalescence, to droplet morphologies with sharp interfaces characteristic of the nucleation and growth mechanism of phase separation. The continuing increase in effective quench depth caused by the continued ordering of the gelatin molecules following a temperature quench allowed secondary phase separation to occur for the shallower quenches, leading to the formation of complex embedded multiphase microstructures. In the case of the deeper quenches, the nonequilibrium conditions established by the temperature quench and the continuing ordering of the gelatin molecules were believed to be responsible for anomalous initial behavior of the spinodal decomposition peak in the SALS pattern. Acknowledgment. The authors thank Allan Clark, Ian Norton, and Bill Williams, of Unilever R&D Colworth, for their input and enthusiasm for the work presented in this paper. References and Notes (1) Strobl, G. The Phyics of Polymers; Springer-Verlag: Berlin, 1996. (2) Porod, G. In Small-Angle X-ray Scattering; Glatter, O., Kratky, O., Eds.; Academic Press: London, 1982.

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Dextran Mixture Studied by

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