Characterization of Phase Separation in Film Forming Biopolymer

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Characterization of Phase Separation in Film Forming Biopolymer Mixtures Maria Petersson, Niklas Lore´ n, and Mats Stading* SIK - The Swedish Institute for Food and Biotechnology, P.O. Box 5401, SE-402 29 Go¨teborg, Sweden Received October 8, 2004; Revised Manuscript Received November 19, 2004

Enhanced, tailor-made films can be achieved by combining the good gas barrier of the hydrophilic high amylose maize starch (hylon) with the water resistance of the hydrophobic protein zein. Two polymers are not always miscible in solution, and the phase separation behavior of the mixture is therefore important for the final film structure and its properties. Phase separation of a mixture of these two biopolymers was induced either by cooling, which was observed as growing droplets of the hylon phase which in some cases also formed small aggregates, or by solvent evaporation and studied in real-time in a confocal laser scanning microscope. Solvent evaporation had a much stronger effect on phase separation. During the early stage of phase separation, hylon formed large aggregates and subsequently smaller droplets coalesced with other droplets or large hylon aggregates. The later part of the separation seemed to take place through spinodal decomposition. 1. Introduction Phase separation is important in many polymeric materials and in many different applications. For example, phase separation can be an important factor in applications such as separation and purification methods, fiber production, membranes, foams, and films.1 Phase separation, in biopolymer mixtures for film preparation, governs the final film structure and thereby its properties, such as mechanical and barrier properties. Phase separation can be induced in a miscible solution, for example, by cooling the mixture or by changing the solvent composition (evaporation). Achieving a deeper knowledge of the phase separation process during film formation and understanding the structure evolution will make it possible to create tailor-made biopolymer materials with the desired mechanical, permeability, and release properties. The morphology of the final microstructure will depend on the kinetics of phase separation and, in the presence of gelling components, on gelation.2 Edible films and coatings made of biopolymer mixtures can be used for example as barriers or release carriers for food products. Applications of this kind have high demands on, for example, the gas and moisture permeability properties. Many edible hydrophilic biopolymers are quite moisture sensitive, but provide good barriers to gases such as oxygen, carbon dioxide, etc. However, the gas permeability of these materials might be too low for respiring food products. A combination of the good gas barrier of the hydrophilic hylon (high amylose maize starch) and the moisture resistance of the hydrophobic protein zein can be used to create tailormade films with the desired permeability properties. By controlling how the biopolymers phase separate during film formation, the permeability, mechanical, and release proper* To whom correspondence should be addressed. Phone: +46 31 335 5600. Fax: +46 31 83 37 82. E-mail address: [email protected].

ties can be tailored to match the requirements of specific applications. The morphology and microstructure of the film that is formed are highly dependent on the phase separation kinetics in connection with the evaporation process of the solvent. It is often desirable to produce a bilayer film directly from solution, consisting of one hydrophobic, moisture resistant layer and one hydrophilic, gas resistant layer. In some applications, it can also be desirable to create a film or coating where one of the phases is evenly distributed in a continuous matrix of the other phase to achieve appropriate permeability or release properties. There is thus a great desire in many applications for more detailed knowledge of the phase separation in film forming solutions during film formation. Although studies have been done on phase separation in edible films related to the permeability and mechanical properties,3,4 the phase separation process and structure evolution in real-time during film formation have not yet been elucidated to any great extent. Three different events can take place in a binary polymer solution: (1) the solution remains in a homogeneous phase; (2) segregation of the two polymers occurs and the mixture separates into two distinct phases; (3) the polymers associate, which results in precipitation or gelation.5 In a phase diagram of a binary system, the binodal separates the one-phase (compatible) region from the two-phase region (incompatible). The two-phase region can be divided into the metastable region, where the mixture separates through nucleation and growth, and the unstable region, where the mixture separates through spinodal decomposition.1,6 The spinodal separates the unstable region from the metastable region. Spinodal decomposition is characterized through simultaneous spontaneous formation of single-phase domains, which grow and coalesce and become more pure with time in order to reach an equilibrium concentration. Phase separation through nucleation and growth can be described as formed

10.1021/bm049365s CCC: $30.25 © 2005 American Chemical Society Published on Web 01/22/2005

Phase Separation in Film Forming Systems

droplets that will be irregularly spaced, appear at different times, have a broad size distribution, will have equilibrium concentration, and grow with time.7,8,9 An activation energy is required to initiate separation by nucleation, unlike the case of spinodal decomposition, which occurs spontaneously.7,10 The behavior of polymers is different at the external surface as compared to the bulk, due to surface interactions. Surface enrichment can occur when one of the polymers is attracted to a surface.11 Puri12 simulated phase separation in a homogeneous mixture when one of the components is attracted to the surface. It was seen that there was an enrichment of one phase (A) at the surface, which grew with time. Beneath this wetting layer, there was a depletion layer that was rich in the other phase (B). The depletion layer consisted of two zones: one that was rich in the B phase and another less rich in B. The isotropic bulk followed beneath these domains with the morphology of bicontinuous spinodal decomposition. It is not always easy to find two biopolymers that are soluble in the same solute and which are miscible at the same temperatures. However, the mixture of the hydrophilic hylon and the hydrophobic zein seems to possess these properties, and these biopolymers were thus used in this study. The aim here was to evaluate the effect on phase separation of changes in temperature and solvent composition during evaporation in the biopolymer system. The cooling rate was chosen in order to mimic the cooling rate during film formation, and the concentration of hylon was varied. The phase separation process was studied in real-time using a CLSM (confocal scanning laser microscope). 2. Materials and Methods 2.1. Materials. Phase separation was studied in a mixture of zein and acetylated high amylose maize starch (hylon). Zein, the prolamin protein of maize, was purchased from Sigma-Aldrich Chemie (Deisenhofen, Germany). Hylon had an approximate degree of substitution of 1.3 and was kindly supplied by Lyckeby Sta¨rkelsen (Kristianstad, Sweden). The high amylose maize starch was modified with acetyl groups to achieve greater hydrophobicity than ordinary high amylose maize starch. 2.1.1. Defatting of Zein. The zein powder contained some fat (7-8%) and thus had to be defatted before use. Approximately 45 g zein was defatted with approximately 200 mL of n-hexane using 4-5 sequential extractions for periods of 60 min during stirring. The zein was allowed to settle for approximately 10 min after each step of extraction, and then the hexane was removed. After the final extraction step, the zein powder was left to dry at room temperature. After this procedure, the zein powder had a moisture content of ∼5.9%, and at least half of the initial fat content had been removed. The moisture content was not taken into account when making the mixtures for the CLSM experiments. 2.2. Dissolution of Hylon. A smear of 6% (w/V) hylon/ ethanol (80% V/V) solution heated to 70 °C for 10 min was made and studied in a light microscope (LM) (Nikon Microphot FXA) at 10× magnification, to investigate

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whether granules remained. The smear was also compared with a smear of a hylon solution heated to approximately 140 °C for 10 min in a Reacti-Therm heating/stirring module (Pierce Reacti-Therm, Rockford, Illinois, USA). The smear of hylon was stained with Lugols solution (iodide). 2.3. Samples for CLSM. The two biopolymers were dissolved and stained separately in 80% (V/V) aqueous ethanol before mixing. 10% (w/V) zein was dissolved in aqueous ethanol, in which the Texas Red had already been dissolved (1 mg of Texas Red in 10 mL of aqueous ethanol). The zein solution was heated and stirred gently. When it had reached 69 °C, it was held at that temperature for 10 min, before mixing with the hylon solution. Three different concentrations of hylon were used, 3, 6, and 10% (w/V). Hylon was dissolved in aqueous ethanol, in which the Acriflavine had already been dissolved (∼0.00125 g of Acriflavine in 100 g of aqueous ethanol). The solution was heated to approximately 140 °C in a glass vial (Reacti-Vial) in the Reacti-Therm and held there for 10 min. The ReactiVial was then removed from the Reacti-Therm and placed on a magnetic stirrer to allow it to cool to approximately 70 °C in order for the zein and hylon solutions to be of the same temperature at the time of mixing. The hylon and zein solutions were then mixed at a 1:1 ratio and stirred for 5 min in a hot water bath set to 72 °C. This results in a solution with half of the original concentrations of hylon and zein. In the final mixed solutions studied in the CLSM, the concentrations of hylon were 1.5, 3, and 5% and of zein 5% (w/V). These are the concentrations referred to in the results. All experiments were done at least in duplicate. 2.4. CLSM Experiments and Image Analysis. The phase separation processes were studied with a CLSM Leica TCS SP2 (Leica Microsystems Heidelberg GmbH, Germany). The light sources used were Ar/ArKr lasers using λex ) 488 nm (Acriflavine) and HeNe lasers using λex ) 594 nm (Texas Red). 2.4.1. Temperature-Induced Phase Separation (TIPS). An up-right Leica RXA2 microscope connected to the Leica TCS SP2 was used to study solutions while cooling. The microscope was equipped with a Linkam TMS 94 heatingand cooling table (Linkam Scientific Instruments, Surrey, United Kingdom). A metal test cup with good heat conductivity and an inner diameter of ∼15 mm and a depth of ∼420 µm was used. The microscope objective used was an oil objective with a magnification of 100× (N.A. ) 1.4) and a 2× digital zoom. The immersion medium was oil with a refractive index of 1.518. The test setup is shown in Figure 1a. The scanning speed was 400 Hz and eight scans were averaged during creation of each image, which were 1024 × 1024 pixels. The same spot in the horizontal plane in the solutions was studied during cooling. However, the confocal plane had to be shifted downward during cooling to be able to follow droplet aggregation and to achieve the appropriate intensity. A temperature ramp was used when studying temperatureinduced phase separation in the CLSM. The solutions were cooled from 70 to 50 °C at 10 °C/min and then to 20 °C at 5 °C/min in order to mimic the temperature profile during film formation in room temperature.13 The mixed solutions

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Figure 1. Test setup for the different experiments; (a) Temperatureinduced phase separation, (b) Solvent-induced phase separation.

were transferred from the mixing vessel with a preheated glass pipet to the preheated test cup in the CLSM. Preheated cover glasses were used to prevent evaporation and coolinginduced phase separation near the surface. The solution was studied at different depths in the test cup when a temperature of 20 °C had been reached, to investigate whether there were differences in the concentration of the solution. As a result of differences in refractive indices between the cover glass and the solution in the test cup, the nominal focus point (NFP) differs from the actual focus point (AFP).14 Hence, in this case, the AFP is less than the NFP. The NFP values do however give a hint of where in the test cup the pictures are taken, even though it is not the exact depth. 2.4.2. SolVent-Induced Phase Separation (SIPS). An inverted Leica DMIRE2 microscope connected to the Leica TCS SP2 was used to study phase separation induced by evaporation (changes in the composition of the solution). To reduce the objective’s working distance as much as possible, another thermal microscope stage, TS-4 (Sensortek Inc., Clifton, New Jersey), was used. Another test cup with a transparent bottom also had to be used. A quartz cup (THMS/ Q) with an inner diameter of 15 mm and a depth of approximately 2 mm was purchased from Linkam. An objective with a magnification of 10× (N.A. ) 0.3) and a 4× digital zoom was used in these experiments. It was not possible to use objectives with higher magnifications here, since this test setup (inverted microscope equipped with a heating stage) required objectives with long free working distance. The scanning speed was increased to 800 Hz in these experiments in order to minimize the time between pictures. Two scans were averaged during creation of each image, which were either 1024 × 1024 or 512 × 512 pixels. One or more experiments on each resolution were done for all solutions. Time series were created during the evaporation process and the time between taken images was minimized to 3 s and 12 ms for 1024 × 1024 images and 1 s and 681ms for 512 × 512 images, which resulted in series with 200 and 400 images. The temperature controller was set to give a constant temperature of approximately 50 °C in the bottom of the

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quartz cup. 200 µL of the mixed solution was transferred to the quartz cup from the mixing vessel with an Eppendorf pipet. The focus point was set as close to the center of the test cup as possible and the confocal plane was situated ∼15-20 µm from the bottom. The test setup is shown in Figure 1b. The process of taking pictures was started as soon as possible after the solution was added to the test cup. Following the evaporation experiments, images from the CLSM were transferred to and stored on a SUN Sparc Classic work station for phase volume measurements by image analysis. A Contextvisions microGOP 2000/S (Linko¨ping, Sweden) contextual image analysis system was used to create binary images and measure the phase volume of hylon. Before measurements were made of the phase volume, the CLSM images had to be transferred into binary images on the SUN Sparc Classic work station. In this case, the images from the Texas Red channel were used since they produced the best binary images. The phase volume of hylon was measured on the last image taken in the evaporation experiments because the structures were more or less locked at this stage. The basic steps in the program for measuring phase volume were noise reduction and thresholding. According to the Delesse principle, the areal density is equal to the volume density.15 Hence, the phase volume can be measured from the micrographs. After the evaporation experiments some of the films formed were scanned in the microscope to detect any sedimentation behavior. 3. Results and Discussion 3.1. Dissolution of Hylon. The LM micrographs showed many undissolved hylon granules left in the hylon solution when heated to only 70 °C in 80% (V/V) aqueous ethanol. However, a smear of hylon heated to 140 °C showed few remaining granule fragments. Thus, all hylon solutions were heated to ∼140 °C before mixing with zein and studying them with CLSM. Due to poor stirring in the Reacti-Vials, the dispersion of 10% hylon was a bit difficult to dissolve, while 6 and 3% were easier (original hylon concentrations, before mixing with zein). It was concluded from CLSM, however, that hylon was sufficiently dissolved. 3.2. TIPS. A number of processes can take place during cooling. When the temperature is reduced, other parts of the phase diagram are reached. The binodal is passed at some temperature, and the region of incompatibility is reached. The concentrations of the components can have an effect on when this region is reached. As cooling proceeds, droplets are formed and aggregation of droplets can take place. Cooling had a clear effect on all of the zein/hylon solutions, see Figure 2. Overall, the mixtures appeared to be well mixed and looked to be a one-phase system at 70 °C. A few hylon droplets could be detected at 70 °C, however, probably due to cooling of the hylon solution from 140 to 70 °C and mixing with zein. Some hylon granules or granule residues could also be seen. These two findings were most consistent in the samples with 3 and 5% hylon. As Figure 2 shows, more hylon droplets appeared in the solution during cooling. Small, spherical droplets were first formed.



Figure 2. Variation of hylon growth, depending on temperature and concentration. Hylon appears in red.

In the solutions with higher concentrations of hylon, these droplets aggregated. The largest aggregates were found in the solution with the highest concentration of hylon, 5%. Thus, a greater amount of hylon implied more aggregation. The hylon granules seen at the beginning of the cooling process starting at 70 °C seemed to sink in the solution during cooling and are hence not visible later on. It seems from Figure 2 that the region of incompatibility where the phase separation began was reached at different temperatures. The solutions that had more hylon reached this region before the solutions containing less hylon. At 45°, almost no small droplets had appeared in the solution with only 1.5% hylon, whereas more droplets had formed and aggregation had begun in the solution with 5% hylon. At 20 °C, the hylon droplets in the solution with 3% hylon had also begun to aggregate. At 20 °C in the solution with 5% hylon, the aggregates that had formed earlier during cooling had grown larger and some had probably also sunk in the solution. Thus, there was less hylon visible here. Information on how hylon was distributed in the sample was obtained by moving to different depths in the test cup after the cooling process. Small and almost unaggregated hylon droplets had ascended to the upper part of the solution close to the cover glass, at a depth of ∼0-10 µm, probably due to surface effects. It also seemed that aggregated and thereby heavier hylon aggregates had descended in the solution, thus leaving an area with almost no hylon between, see Figure 3. This was most clear for the solution with 3% hylon. The depths given in the legend to Figure 3 are the

NFP values. It was difficult to focus deeply in the cup for the solution with 5% hylon, probably because of scattering effects and thus a loss of intensity. The present findings, an enrichment of hylon close to the surface of the cover glass, beneath which is a hylon-depleted layer followed by aggregated hylon droplets at a greater distance from the surface, are comparable to parts of the model presented by Puri.12 In conclusion, there existed a sedimentation and surface enrichment behavior that may influence the film formation and the final film properties during cooling. 3.3. SIPS. Several processes can take place during evaporation of the solvent. The most important factors affecting phase separation are the changes in the composition of the solution caused by evaporation of the solvent. As the solvent evaporates the total composition of the mixture changes and other parts of the phase diagram are reached. Furthermore, the movement in composition space is not linear since ethanol evaporates at a higher rate than water. At some specific composition of the mixture, the binodal is passed and the region of incompatibility is reached. Similar behaviour has been observed in other types of systems in which the phase separation was induced by addition of a modifier instead of solvent evaporation.16 Another important factor during evaporation is that the viscosity of the mixture increases as the solvent evaporates. This implies kinetic trapping of structures as a result of reduced mobility. Finally, the phases are solidified and a film is formed. Solvent evaporation was found to have a much greater effect on phase separation than cooling with respect to the

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Figure 3. Solution with 3% hylon at 20 °C taken at different depths in the test cup. Depth (NFP) measured from the cover glass; (a) ∼5, (b) ∼9, (c) ∼60, and (d) ∼100 µm. Hylon appears in red.

structures that are formed, see Figure 4. In the solvent evaporation experiments the temperature was kept constant at 50 °C; hence the only thing that affected the phase separation was the changes in solution composition. Two main steps could be noted during the evaporation process. 3.3.1. Structure EVolution during the First Step of Phase Separation. During the first step, one hylon fraction separated from solution. The first step of the phase separation lasted from 0 to approximately 2 min. This fraction gave rise to a thready, coarse, irregular network with unspherical elements. In all solutions, some of these structures were visible as diffuse patterns almost from the beginning of the evaporation process (see Figure 4, ∼2 min). The influence of the surface tension was not very strong in the first step of phase separation (see Figure 4, ∼2 min), since spherical structures were not formed which would have minimized the surface area. Hence, this first step of the phase separation did not seem to fit into a model involving nucleation and growth or spinodal decomposition. Instead, this first step was probably induced by high molecular weight hylon or hylon aggregates consisting of slightly undissolved granules. Two other things that can have affected the phase separation are the cooling from 70 to 50 °C when the solution was transferred to the test cup and the immediate evaporation of the solvent when the solution was transferred to the test cup. This could also be a reason some structures were visible at t ∼ 0 min in Figure 4. More hylon structures were visible in the solution with 5% hylon than 1.5% at t ∼ 0 min in Figure 4. However

the situation had changed by ∼2 min. The solution with 1.5% hylon then showed a greater phase volume of hylon than the solution containing 5% hylon. According to the apparent location of the binodal found during cooling (Figure 2) and that the entropy of the system is reduced when more polymer is added and hence facilitating phase separation, the hylon structures in the solution containing 5% hylon should have developed more than the ones in the solutions containing less hylon, which could be an effect of slower transport of material in the solution with 5% owing to the presence of more material in solution and thereby less free space. In addition, since the CLSM is sensitive to differences in fluorochrome concentration, i.e., hylon concentration, this could be a sign of the differences in concentration between the phases. Thus meaning that, if there existed small hylon droplets around the diffuse structures, the differences in fluorochrome concentration between them were not great enough for all of the hylon droplets to be visible. 3.3.2. Structure EVolution during the Second Step of Phase Separation. As more solvent evaporated, the state of the mixture moved in the phase diagram, and the second step of the phase separation subsequently took place after approximately 2 min. As the solvent evaporated, hylon and zein became more concentrated. In the mixtures with 1.5 and 3% hylon, many small hylon droplets of similar size appeared simultaneously during the second step of the phase separation (Figure 4, ∼3 min). This was most clear for the mixture with 3% hylon. Hence, during this step, the phase



Figure 4. Selection of images from the evaporation process (1024 × 1024). Images taken at the same location in the solution. Hylon appears in red.

separation seemed to take place through spinodal decomposition. These small droplets of hylon subsequently coalesced with either other small hylon droplets or with the larger hylon domains formed during the first step. The appearance of small hylon droplets was not very clear in the mixture with 5%, probably because of reduced mobility because there was

less free space or because they were taken up directly in the thready hylon phase. In this mixture, however, the second step of the phase separation took place very rapidly and the first structures formed were suddenly enriched with more hylon. The final hylon structure became almost fully developed in the mixture with 5% hylon somewhere between

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Figure 5. Images showing coalescence during the evaporation process. Hylon appears in red.

3 and 4 min (Figure 4). This process took a longer time in the mixtures with less hylon, between 2 and 4 min (Figure 4, 1.5 and 3% hylon). The reason the larger structures in step one appeared earlier than the smaller droplets in step two during the evaporation process could have to do with differences in the molecular weights of polymer chains in the polydisperse hylon. According to simulation experiments carried out by Termonia,17 low molecular weight chains in a mixed solvent behave as if they were dissolved in a pure “good” solvent, whereas the solubility of the longer chains is controlled by the average composition of the mixture. The longer chains precipitate first and tend to populate the polymer rich phase while shorter chains remain in solution because of their greater solubility. In the present study, the composition of the solvent changed during evaporation. At first there was 80% (V/V) ethanol in the solution. The ethanol evaporated faster than the water, however, giving a solvent with higher water content in the later stages of the evaporation process. Water is a poorer solvent for both hylon and zein than ethanol. When enough ethanol has evaporated from the solution even the low molecular weight hylon chains separated from the solution. 3.3.3. Coalescence and Depletion. The small hylon droplets that appeared at ∼2-3 min in the mixtures containing 1.5 and 3% hylon (Figure 4) either aggregated with other small droplets or with the large hylon aggregates that had formed during the first step of phase separation through coalescence,18 see Figure 5. This implied that the large hylon aggregates became more compact, distinct, and enriched with hylon. Some of the small droplets that appeared during the

Figure 6. Small droplets of hylon appearing during evaporation; (a) 1.5% hylon (512 × 512), (b) 3% hylon (1024 × 1024), and (c) small remaining droplets of hylon in a film with 1.5% hylon (1024 × 1024). Hylon appears in red.

evaporation process even remained in the formed films, especially in the case of 1.5% hylon, see Figure 6. A hylon-depleted layer consisting of zein was formed in the area between small droplets and the large aggregates (see Figure 6c). This hylon-depleted layer was most clear for the


mixtures with only 1.5% hylon. Some small droplets could be seen even in the films with a greater amount of hylon. In the solutions with more hylon, smaller droplets that appeared during evaporation coalesced with the larger aggregates to a greater extent than in the solutions with only 1.5% hylon. There is always a driving force to reduce the free energy in a system. In this case, there was a struggle between creating new surfaces, which increases the free energy of the system, and transportation of material. Free energy could be saved by reducing the total surface area by transportation of small amounts of material to larger areas that already exist. In the mixtures with 3 and 5% hylon the distances over which material was transported from the small droplets to the larger aggregates were shorter than for many of the droplets in the mixture with only 1.5% hylon. Hence, in the mixtures with 3 and 5% hylon, the free energy was reduced by a reduction of total surface area since material was transported from the droplets to the large aggregates. In the mixture with 1.5% hylon however, it was more energetically favorable for some of the droplets situated far from the large aggregates to remain as droplets instead of there occurring a transport of material to the larger aggregates. Also, a greater amount of hylon would result in higher surface tensions and thereby a stronger driving force for coalescence. This could explain the loss of droplets in the mixtures with more hylon and also why there were droplets and a hylon-depleted layer in the mixtures with only 1.5% hylon. 3.3.4. ObserVations during the Later Stages of the EVaporation Process. After ∼5 min (Figure 4), the structures formed were almost fully developed and the phases were solidified due to formation of a film. Before that, however, the hylon areas were reduced in size, resulting in larger zein areas (see Figure 7). This was most clear in the mixture containing 5% hylon. Basically, the structural changes shown in Figure 7 are mainly an effect of the distribution of the solvents between the hylon-rich and the zein-rich phase. The distribution of solvents may depend on the fact that the composition of the solvent changed during evaporation and on the differences of rates of syneresis. Since ethanol evaporated faster than water, hylon, which is more hydrophilic than zein, was able to stay in solution longer than zein. Figure 7a shows that the hylon aggregates contained the remaining water-rich solvent. When the water-rich solvent evaporated, the hylon aggregates shrank and became more compact, which resulted in larger zein areas (Figure 7b). At ∼4 min, it was clear (Figure 4) that the mixture with 5% hylon had a much greater phase volume of hylon than the mixture with 1.5% hylon, in contrast to the situation at ∼2 min. Even though the reduction of the hylon phase was most clear in the mixture with 5% hylon, the results of the phase volume measurements and the images in Figure 4 at ∼10 min clearly showed that more hylon present implied a greater phase volume in the films, see Table 1. The samples with 3% hylon had almost twice the phase volume than the samples with only 1.5%. The phase volume may differ depending on where in the sample the image was taken. However, there was a trend that the samples with more hylon resulted in a greater phase volume, which was expected. The


Figure 7. Shrinkage of the hylon phase during evaporation. Images taken at the same location in a mixture of 5% hylon (1024 × 1024); (a) and (b) taken at an interval of approximately 1 min. Hylon appears in red. Table 1. Results of Measurements of Phase Volume of the Hylon Phase concentration of hylon [%]

phase volume of hylon

1.5 1.5 3.0 3.0 5.0 5.0

0.20 0.18 0.39 0.28 0.43 0.42

results of the measurements of phase volume also showed that there was no clear expelling of either hylon or zein due to surface effects in the quartz cup. In conclusion, the differences in the concentration of hylon clearly affected the phase separation process and the final structure of the film. A bicontinuous system was formed in all of the mixtures during the evaporation process. In the films with only 1.5% hylon, there were both large hylon aggregates as well as large areas of zein with small droplets of embedded hylon. A dense hylon network was formed in the films with a greater amount of hylon. This was most clear in the case of 5% hylon, and almost no small droplets

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were visible in these films. In the films with 3% hylon, however, both types of structures could be detected. The final forms and structures of the large hylon aggregates were of the same shape and form as the hylon structures first formed and were only more dense or compact. There were no clear differences in concentration through the films, for example as a result of sedimentation, as was the case in the TIPS experiments. This could be attributed to a formation of a space-filling network with little free volume which hindered sedimentation of smaller particles. 4. Conclusions It was found that CLSM was an appropriate technique for following phase separation in a mixture of hylon and zein during cooling and evaporation. Segregative phase separation of zein and hylon occurred during cooling. Small hylon droplets appeared in the solutions and aggregated. Aggregation was more pronounced when more hylon was present in the solution. The region of incompatibility was reached at different temperatures depending on the hylon concentration. More hylon droplets were visible at 45 °C in the solution with 5% than in the one with 1.5% hylon. Furthermore, during the temperatureinduced phase separation experiments, larger hylon aggregates descended in the test cup, small droplets were enriched at the surface close to the cover glass and a zone depleted of hylon had formed in between. This sedimentation behavior may influence the film formation and the final film structure. Overall, the temperature effect was small. Evaporation of the solvent, however, had a greater impact on phase separation than cooling for this system. More hylon could be seen in the films that were formed than in the solutions in the cooling experiments. It was found during the solvent evaporation process that the phase separation took place in two steps. One hylon fraction separated from solution during the first step. This fraction formed a coarse, irregular network with unspherical elements. The second step of the phase separation took place as more solvent evaporated. During this step many small hylon droplets of the same size appeared almost simultaneously. This was most clear in the mixtures with 1.5 and 3% hylon. Hence, this second step seemed to take place through spinodal decomposition. These small droplets subsequently coalesced with either other small droplets or with the large aggregates that were formed during the first step, probably depending on what was energetically most favorable. Some of these droplets even remained in the films made of the mixtures with 1.5% hylon. As the solvent evaporated, the structures that had formed were kinetically trapped because of the increase in viscosity. The large aggregates that were formed during the first step of the phase separation were probably high molecular weight hylon, whereas the small droplets that appeared during the second step were low molecular weight hylon. Overall, the differences in hylon concentration affected both the phase separation and the final film structure. The large aggregates that were formed during the first step were enriched with hylon and became denser during the second step through coalescence. However, the aggregates retained their original

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shapes and forms during the evaporation process. A bicontinuous system was formed in all mixtures during the evaporation experiments, unlike during the process of cooling, where a bicontinuous system did not form. Further study is needed to achieve an understanding of phase separation in mixed films. Objectives with a higher magnification and longer free working distance would be helpful, and other concentrations and temperatures could also be investigated. It would also be interesting to investigate the influence of zein on phase separation by varying the zein concentration as well. Acknowledgment. Many thanks to Annika Altska¨r for her professional help with the CLSM experiments and for answering my questions about CLSM, labeling techniques, and much more. Thanks also to Paula Olofsson for a great deal of practical help with the CLSM, the light microscope, and phase volume measurements. Karin Svegmark is also acknowledged for her help with the model system. This work was financed by Vinnova and the EU project Enviropak (INCO-DEV ICA4-CT-2001-10062). References and Notes (1) Loreń, N. Structure Evolution during Phase Separation and Gelation of Biopolymer Mixtures. Ph.D. Thesis, Chalmers University of Technology, Gothenburg, Sweden, 2001. (2) Loreń, N.; Hermansson, A.-M. Phase separation and gel formation in kinetically trapped gelatin/maltodextrin gels. Int. J. Biol. Macromol. 2000, 27, 249-262. (3) Petersson, M.; Stading, M. Water vapour permeability and mechanical properties of mixed starch-monoglyceride films and effect of film forming conditions. Food Hydrocolloids 2005, 19, 123-132. (4) Anker, M.; Berntsen, J.; Hermansson, A.-M.; Stading, M. Improved water vapor barrier of whey protein films by addition of an acetylated monoglyceride. InnoVatiVe Food Sci. Emerging Technol. 2002, 3, 81-92. (5) Hoskins, A. R.; Robb, I. D.; Williams, P. A. Selective separation of proteins from mixtures using polysaccharides. Biopolymers 1998, 45, 97-104. (6) Tanaka, H.; Yokokawa, T.; Abe, H.; Hayashi, T.; Nishi, T. Transition from metastability to instability in a binary-liquid mixture. Phys. ReV. Lett. 1990, 65, 3136-3139. (7) Jones, R. A. L.; Richards, R. W. Polymers at Surfaces and Interfaces; Cambridge University Press: Cambridge, U.K., 1999. (8) Loreń, N.; Altska¨r, A.; Hermansson, A.-M. Structure evolution during gelation at later stages of spinodal decomposition in gelatin/ maltodextrin mixtures. Macromolecules 2001, 34, 8117-8128. (9) Norton, I. T.; Frith, W. J. Microstructure design in mixed biopolymer composites. Food Hydrocolloids 2001, 15, 543-553. (10) Califano, F.; Mauri, R. Drop size evolution during the phase separation of liquid mixtures. Ind. Eng. Chem. Res. 2004, 43, 349353. (11) Liu, B.; Zhang, H.; Yang, Y. Surface enrichment effect on the morphological transitions induced by directional quenching for binary mixtures. J. Chem. Phys. 2000, 113, 719-727. (12) Puri, S. Kinetics of phase separation near surfaces. Comput. Phys. Commun. 1999, 121-122, 312-316. (13) Anker, M.; Stading, M.; Hermansson, A.-M. Effects of pH and the gel state on the mechanical properties, moisture contents, and glass transition temperatures of whey protein films. J. Agric. Food Chem. 1999, 47, 1878-1886. (14) Hell, S.; Reiner, G.; Cremer, C.; Stelzer, E. H. K. Aberrations in Confocal fluorescence microscopy induced by mismatches in refractive index. J. Microsc. 1993, 169, 391-405.

Phase Separation in Film Forming Systems (15) Weibel, E. R. Stereological Methods. Vol 1, Practical methods for biological morphometry; Academic Press: London, 1979. (16) Gupta, R.; Mauri, R.; Shinnar, R. Liquid-liquid extraction using the composition-induced phase separation process. Ind. Eng. Chem. Res. 1996, 35, 2360-2368. (17) Termonia, Y. Polymer solubility in mixed solvents. J. Polym. Sci., Part B: Polym. Phys. 1999, 37, 2782-2787.

Biomacromolecules, Vol. 6, No. 2, 2005 941 (18) McGuire, K. S.; Laxminarayan, A.; Martula, D. S.; Lloyd, D. R. Kinetics of droplet growth in liquid-liquid phase separation of polymer-diluent systems: Model development. J. Colloid Interface Sci. 1996, 182, 46-58.

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Characterization of Phase Separation in Film Forming Biopolymer

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