Formulación y caracterización de emulsiones de de limoneno estabilizadas por un tensioactivo polimérico

FORMULACIÓN Y CARACTERIZACIÓN DE EMULSIONE DE LIMONENO ESTABILIZADAS POR UN TENSIOACTIVO POLIMÉRICO Luís María Pérez Mosqueda Universidad de Sevilla Sevilla, 2014 UNIVERSIDAD DE SEVILLA FACULTAD DE QUÍMICA Departamento de Ingeniería Química Luis Mª Pérez Mosqueda Tesis Doctoral FORMULACIÓN Y CARACTERIZACIÓN DE EMULSIONES MODELO DE POTENCIAL USO AGROQUÍMICO BASADAS EN UN DISOLVENTE VERDE: D-LIMONENO por Luis Mª Pérez Mosqueda Directores del trabajo: Dr. José Muñoz García Dr. Pablo Ramírez del Amo Profesor Titular Profesor Ayudante Doctor Trabajo presentado para aspirar al título de Doctor por la Universidad de Sevilla Sevilla, marzo de 2014 Dr. D. Felipe Cordobés Carmona, Director del Departamento de Ingeniería Química de la Universidad de Sevilla: CERTIFICA: Que la Tesis Doctoral que presenta D. Luis Mª Pérez Mosqueda ha sido realizada dentro de la línea “Reología aplicada y tenología de fluidos complejos”. Fdo: Dr. Felipe Cordobés Carmona AGRADECIMIENTOS En primer lugar creo que tengo que decir que voy a ser un poco injusto en estos agradecimientos porque seguro que no voy a ser capaz de transmitir el enorme sentimiento de gratitud que tengo hacia tanta gente que me ha ayudado, apoyado, soportado, sufrido, aguantado, etc…durante este tiempo. En primer lugar me gustaría acordarme de mis directores de tesis, José Muñoz y Pablo Ramírez por su inestimable ayuda en la realización de esta tesis doctoral. Me gustaría agradecer a Pepe la oportunidad que me dio de incorporarme en el grupo de investigación, hace ya algunos años, que al final me ha traído hasta aquí y por su ayuda durante este tiempo. A Pablo debería agradecerle su paciencia durante este tiempo y sobre todo el que sea un gran amigo. A los compañeros del departamento (Cecilio, Carlos, Cristina, Manolo, Juan Carlos, José Antonio, Nieves, Paco Toscano, Montse, Mª Carmen, Nuria, Lucía etc…) debería agradecerles el hacer el día a día más sencillo y generar ese buen ambiente que hace ir a trabajar con alegría y que negocios como “La bodeguita” y “El Aljibe” sean prósperos. En particular agradecer a Paco sus consejos, el tener la puerta del despacho siempre abierta y principalmente por ser un gran amigo. A mis hermanitos Luis y Jeni por seguir aguantándome día tras día, especialmente últimamente con los agobios de final de tesis Quisiera agradecer a Julia Maldonado la oportunidad que me dio de realizar una estancia de investigación en el grupo de Física de Fluidos y Biocoloides en la Universidad de Granada y a la gente de “la sala” (Azahara, César, Juan Pablo, Jose, Miguel, Felipe, Carmen Lucía, Germán, Miguel Ángel, Leonor) por hacer que ese experiencia fuera tan reconfortante tanto a nivel personal como profesional. De la misma manera debo agradecer al doctor Reinhard Miller el concederme la posibilidad de pasar seis meses trabajando en su grupo de investigación en el Max Planck for Collids and Interface en Berlín. También debo acordarme de toda esa gente que me ha facilitado y ha hecho tan llevadero mi tiempo en Berlin (Vamsee, Antonio, Georgi, Nenad, Sabine, Jooyoung Inga, Ali, Jurgen, Narges, Marzy, Altynay, Bota, Samee). A mi familia y amigos quisiera agradecerle toda la ayuda prestada durante todo este tiempo. Solo deciros que no sé cómo transmitiros el sentimiento de gratitud tan grande que produce el sentirse tan apoyado y acompañado como me he sentido yo en los momentos más feos. Al principio dije que los agradecimientos iban a ser injustos porque era imposible nombraros a todos, pero no os preocupéis porque sabéis que esto lo celebraremos convenientemente. Y por último, a las personas que más les debo, a mis padres. Gracias por tener siempre tanta paciencia y tantísima confianza en mí. A los dos os tengo que agradecer el haber sido siempre un ejemplo y el tener tantísimo coraje y fuerza de voluntad y sobre todo por enseñarme a vivir la vida como la vivís vosotros, siempre mirando hacia adelante. Gracias. A mis padres Summary ......................................................................................................................... 1 Resumen .......................................................................................................................... 5 Introduction .................................................................................................................... 9 1.1 Emulsions ......................................................................................................... 10 1.1.1 Definition .................................................................................................. 10 1.1.2 Destabilization mechanism ....................................................................... 11 1.1.2.1 Gravitational separation ........................................................................... 12 1.1.2.2 Flocculation ............................................................................................. 14 1.1.2.3 Coalescence ............................................................................................. 15 1.1.2.4 Ostwald ripening...................................................................................... 16 1.1.3 Droplet size ............................................................................................... 17 1.2. Emulsification techniques ................................................................................ 17 1.2.1 Rotor/stator systems ................................................................................. 18 1.2.2 Highpressure valve homogenizers ......................................................... 19 1.2.3 Ultrasonic homogenizers .......................................................................... 20 1.2.4 Microfluidization ...................................................................................... 20 1.2.5 Membrane homogenizers ......................................................................... 20 1.3 Adsorption of surfactants ................................................................................. 21 1.3.1 Interfacial tension ..................................................................................... 22 1.3.2 Interfacial rheology................................................................................... 25 1.4. D-limonene ...................................................................................................... 26 1.5. Pluronics .......................................................................................................... 30 1.6. Rosin gum ........................................................................................................ 32 References ................................................................................................................... 34 Interfacial characterization of Pluronic PE9400 at biocompatible (air-water and limonene-water) interfaces. ......................................................................................... 46 Abstract ......................................................................................................................... 47 2.1 Introduction ........................................................................................................... 48 2.2 Materials, methods and models ............................................................................ 50 2.2.1. Material ......................................................................................................... 50 2.2.2 Drop profile tensiometry ................................................................................ 51 2.2.3 Theoretical models ......................................................................................... 52 2.2.3.1 Equilibrium isotherm ............................................................................... 52 2.2.3.2 Dilatational rheology ............................................................................... 55 2.2.3.3 Adsorption kinetics .................................................................................. 55 2.3 Results and Discussion ......................................................................................... 57 2.3.1 Equilibrium surface pressure isotherms ......................................................... 57 2.3.2 Interfacial dilatational rheology ..................................................................... 61 2.3.4 Adsorption kinetics ........................................................................................ 64 2.3.5 Desorption process ......................................................................................... 68 2.4. Conclusions .......................................................................................................... 70 References ................................................................................................................... 71 Optimum formulation of D limonene emulsions by response surface methodology ........................................................................................................................................ 81 Abstract ......................................................................................................................... 82 3.1 Introduction ........................................................................................................... 83 3.2 Materials and methods .......................................................................................... 85 3.2.1. Materials ........................................................................................................ 85 3.2.2 Emulsion preparation ..................................................................................... 86 3.2.3 Droplet size measurements ............................................................................ 86 3.2.4 Creaming stability .......................................................................................... 86 3.2.5 Ostwald ripening ............................................................................................ 87 3.2.6. Overall destabilization index (Turbiscan Stability Index) ............................ 87 3.2.7. Rheology of emulsions .................................................................................. 87 3.2.8. Design of experiments ................................................................................... 88 3.3 Results and discussions ......................................................................................... 90 3.3.1. Surface response analysis .............................................................................. 90 3.3.2 Emulsion droplet size ..................................................................................... 92 Resumen 6 Esta tesis está encuadrada dentro de la ejecución del proyecto “Caracterización Reológica y Estabilidad Física de Emulsiones Formuladas con Disolventes Verdes” (CTQ2011-27371) financiado por el Ministerio de Economía y Competitividad (MINECO) y con fondos de la Comisión Europea (Programa FEDER). La realización de esta tesis fue posible gracias a la concesión de la beca “IV Plan Propio Universidad de Sevilla” a Luis Mª Pérez Mosqueda. El papel de los disolventes en la industria es cada vez más significativo. Muchos disolventes orgánicos han sido retirados, o se espera que sean prohibidos, en los próximos años debido a su toxicidad y a su contribución a las emisiones atmosféricas de compuestos orgánicos volátiles (VOC). De esta manera, los disolventes más dañinos para el medio ambiente están siendo sustituidos gradualmente por compuestos conocidos como “disolventes verdes” (Höfer, 2007). El D-limoneno, es un hidrocarbono natural, un monoterpeno cíclico, que se encuentra comúnmente en la cáscara de cítricos como pomelo, limón, lima y sobre todo en las naranjas. El D-limoneno presenta una buena biodegradabilidad y por este motivo puede ser presentado como una interesante alternativa a los disolventes orgánicos tradicionales. Además, presenta un peso molecular y estructura similar a la del ciclohexano y el tolueno, por lo que sus propiedades como disolvente son parecidas, haciéndole ser un prometedor candidato para sustituir a estos VOCs (Kerton, 2009). El principal objetivo de esta tesis es la formulación de una emulsión submicrónica estable que puede ser empleada en formulaciones con potencial uso industrial sustituyendo los disolventes orgánicos tradicionales. El copolímero tribloque, PE 9400, se ha empleado como emulsificante debido a su baja toxicidad y a la esperada adsorción irreversible en la interfaz D-limoneno-agua. Además, la adición de un Resumen 7 biopolímero lipofílico (goma rosín) inhibe el crecimiento del tamaño de gota por Ostwald ripening obteniéndose un sistema coloidal altamente estable. Esta tesis está formada por cinco capítulos. El primer capítulo introduce los principales concepto de la ciencia y tecnología de emulsiones así como el estado del arte de los principales compuestos empleados: D-limoneno, pluronics y goma rosín en relación con su aplicación en sistemas coloidales. El segundo capítulo está dedicado a la caracterización interfacial del surfactante polimérico no-iónico, PE 9400. Se ha estudiado la isoterma de adsorción, reología dilatacional, y los procesos de desorción obteniendo información interesante respecto al proceso de adsorción y las conformaciones del surfactante en la interfaz D-limonenoagua. El tercer capítulo muestra detalladamente un estudio de la influencia de dos de las principales variables en emulsificación; la fracción de fase dispersa, ф, y la relación tensioactivo/aceite, R, en la distribución del tamaño de gota (DTG) y en la estabilidad física de emulsiones de D-limoneno en agua estabilizadas por el pluronic PE 9400. Usando metodología de superficie de respuesta se ha obtenido un óptimo de las dos variables llegando a tener una emulsión submicrónica en un proceso de homogenización de una etapa en un homogenizador rotor/stator. Sin embargo, esta emulsión sufre un rápido crecimiento del tamaño de partícula debido al proceso de Ostwald ripening. En el cuarto capítulo de la tesis se muestra como el óptimo obtenido en la formulación en el capítulo previo puede ser mejorado por la adición de una goma lipofílica (goma rosín). Se muestra como una concentración de goma de en torno al 10% m/m inhibe casi completamente el proceso de Ostwald ripening. Además, la combinación de medidas reológicas, difacción láser y dispersión múltiple de luz Resumen 8 muestran la influencia de la concentración de surfactante en la estabilidad física de las emulsiones. Finalmente, el quinto capítulo resume las principales conclusiones de la tesis. 1 Introduction Chapter 1 10 1.1 Emulsions 1.1.1 Definition An emulsion is a type of disperse system which is formed by two immiscible liquids (usually a kind of oil and water). One of the liquids forms small spherical droplets in the other. The liquid which forms the droplets is known as the disperse phase and the liquid containing these droplets is the continuous phase. Several classes of emulsion could be distinguished depending on the relative spatial distribution of the oil and aqueous phase. A system which is formed by oil droplets dispersed in an aqueous phase is called an oil-in-water emulsion or O/W emulsion, such as milk, cream, dressings, mayonnaise, some beverages, different pharmaceuticals and cosmetics (Aulton, 2007; Troy, 2006). A system consisting of water droplets dispersed into an oil phase is called a water-in-oil or W/O emulsion, like margarine or butter (McClements, 2005; Tadros, 2009). In addition to the conventional O/W or W/O emulsions it is also possible to prepare various types of multiple emulsion as oil-in-water-in-oil (O/W/O) or water-in-oil-in-water (W/O/W) emulsions (Garti, 1997; Garti, 2004). If we try to mix a pure organic phase with pure water a fast destabilization will be seen, since the attainment of a homogeneous solution is thermodynamically unfavorable (Israelachvili, 1992). Eventually, the phase with the lowest density (oil) will form a cream layer at the top, whereas at the bottom a water layer will appear. The system evolves to the state of minimal contact area between the two phases. This is because droplets tend to merge together, eventually leading to a complete phase separation (McClements, 2005). Nevertheless, it is feasible to create emulsions that are kinetically stable (metastable) with a time window useful for many industrial applications (ranging from a few days to months). To that end, the addition of an emulsifier is required. An emulsifier is a surface-active chemical that adsorbs at the Chapter 1 11 interface of freshly formed droplets and also plays an important role in long-term stability (Binks, 1998; Tadros, 2005; Tadros, 2009). A wide variety of molecules can be used as emulsifiers, such as: surfactants, proteins, polysaccharides, etc. In this Ph.D. Thesis a non-ionic triblock copolymer surfactant has been used as emulsifier. Droplet concentration is usually defined as the disperse phase volume fraction (ф), which is the ratio between the volume occupied by all the droplets and the total volume of the emulsion (ф=V D /V E ) or as the disperse phase mass fraction (ф m ), which is the ratio between the mass of all emulsion droplets and the total mass of the emulsion (ф m =m D /m E ). ф m and ф are related according to (McClements, 2007): ( ) 1 1 2 1 m ρ φ φ φ φ ρ −   = + −     (1) ( ) 1 1 2 1 m m m ρ φ φ φ φ ρ −   = + +     (2) where, ρ 1 and ρ 2 are the densities of the continuous and the disperse phase, respectively. The process whereby two immiscible liquids form an emulsion is called homogenization. Normally, this process occurs in a device called a homogenizer, where this device supplies the necessary energy for the homogenization process. 1.1.2 Destabilization mechanism The term “emulsion stability” usually refers to the ability of an emulsion to resist changes in its physicochemical properties over time (McClements, 2005). Several physicochemical mechanisms could take place to generate an unstable emulsion such as: gravitational separation (creaming/sedimentation), flocculation, coalescence, partial Chapter 1 12 coalescence, Ostwald ripening and phase inversion (Dickinson, 1992; Friberg, 2004; McClements, 2005). Figure 1. Destabilization processes for emulsions. 1.1.2.1 Gravitational separation The density of the droplets in an emulsion is usually different from that of the liquid that surrounds them, and therefore a net gravitational force acts upon them (McClements, 2005; McClements, 2007). Chapter 1 13 On the one hand, if the liquid in the droplets has a lower density than the surrounding liquid, the droplets will tend to move upwards, a process known as creaming. On the other hand, if the liquid inside the droplets has a higher density than the surrounding liquid, the droplets will tend to move downwards, which is called sedimentation. The rate of creaming or sedimentation depends on the balance force. The most famous mathematical model which describes the gravitational separation rate is Stoke’s law. This approximation is only valid for an isolated rigid spherical particle drop in an ideal (Newtonian) liquid (McClements, 2007): ( ) 2 2 1 1 2 9 stokes gr v ρ ρ η − = − (3) where v stokes is the creaming velocity, r is the radius of the drop, g is the acceleration due to gravity, ρ, is the density, η, is the shear viscosity, and the numbers 1 and 2 refer to the continuous and the disperse phase, respectively. Although, this equation is valid only for ideal systems (a system is usually considered ideal when the volume disperse fraction is lower than 1%), the main factors influencing creaming/sedimentation rate, which are particle size, the viscosity of the disperse phase and differences between densities, also have to be taken into account for more concentrated emulsions. Furthermore, the polidispersity of the emulsion droplets as well as the likely occurrence of droplet flocculation have to be considered for more concentrated emulsions. Hence, in order to minimize the gravitational separation rate some strategies can be adopted such as minimizing the difference in densities between the phases, reducing the particle size, changing the rheology of the disperse phase, increasing the droplet concentration and/or avoiding flocculation (Bengoechea, 2006). Chapter 1 14 Chanamai (Chanamai, 2000a; Chanamai, 2000b) proposes dividing the creamed emulsion into three layers in order to quantify the extension of the creaming process: (a) a lower layer, where the concentration of droplets is very low or non-existent; (b) a middle layer, where the concentration of droplets is similar to the one in the original emulsion; and (c) an upper layer, where the concentration of droplets is higher than the initial one and they are closely packed. The trend of the droplets in most O/W emulsions, such as the ones studied in this Ph.D. Thesis, is to move upwards. It is observed that whereas the thickness of the lowest (H L ) and uppermost (H U ) layers increases over time, the thickness of the middle layer (H M ) decreases. In some cases, the middle layer disappears completely after a certain time (Figure 2), (Chanamai, 2000a; 2000b). Figure 2. Creaming zones 1.1.2.2 Flocculation This destabilization process occurs when several droplets suffer aggregation, forming larger entities but maintaining the integrity of the individual droplets. The interaction between droplets arises when the repulsion between them is not enough to avoid the van der Waals attraction (Tadros, 2009). Although this is a reversible destabilization process, it could drive the development of other destabilization processes such as coalescence. Chapter 1 15 One factor enhancing emulsion flocculation is the presence of non-adsorbing colloidal particles such as biopolymers or surfactant micelles. This destabilization mechanism is usually explained on the basis of the exclusion volume theory developed by Asakura and Osawa (1958). As shown in Figure 3, the surfactant micelles are excluded from a narrow region surrounding each droplet. This region extends from the droplet surface to a distance equal to that of the micelle radius. Hence an osmotic potential difference between the depletion zone and the bulk liquid is generated, whereby droplets tend to aggregate in order to reduce this difference (McClements, 2005; Pal, 2011; Quemada, 2002). Figure 3. Depletion Flocculation scheme 1.1.2.3 Coalescence This is an irreversible emulsion destabilization whereby two or more liquid droplets merge together to form a single, larger droplet (McClements, 2005). Interfacial elasticity of the adsorbed surfactant layer plays a key role in coalescence (Husband, 1997; Carrera-Sánchez, 2005). Emulsions which suffer coalescence evolve to a more Chapter 1 22 HLB Application Example 0-4 Antifoam Oleic Acid (1,0) Etilen glycol mono-sterate (2,9) 4-6 Emulsificer W/O Glycerol mono-sterate (3,8) Sorbed mono-stearate (4,7) 6-9 Emulsifier O/W Humectantes PEG – 4 di-laurate (6,0) sucrose di-palmitate (7,4) sorbed monolaurate (8,6) 9-13 Emulsifier O/W PEG – 4 monolaurate (9,8) polysorbate 61 (9,6) polysorbate 85 (11,0) PEG – 8 mono-estearate (11,6) octoxynol – 9 (12,8) 13-40 Emulsifier O/W, detergent PEG – 8 mono-laurate (13,1) sucrose mono-laurate (15,0) Sodium oleate (18,0) Potasium oleate (20,0) Sodium lauril-sulfate (40) Table 3. HLB Classification. 1.3.1 Interfacial tension “The change in free energy of a system that occurs when a surface-active solute is present manifests itself as a change in the interfacial tension, that is, in the amount of free energy required to increase the interfacial area between the water and the oil phases by a unit amount” (McClements, 2005). The presence of an amphiphilic molecule decreases the interfacial tension because the hydrophobic portion is located in the oil (air) and the hydrophilic portion is located in the water phase and, as a result, the thermodynamically unfavorable contacts between the oil and water phase are decreased (McClements, 2005). The interfacial tension is represented by γ , whereas the reduction of the interfacial tension by the presence of amphiphilic molecules could be referred to as surface pressure: Chapter 1 23 Π = γ o/w – γ (6) where γ o/w is the interfacial tension of the pure oil-water (air-water) interface and γ is the interfacial tension in the presence of surfactants (Hiemenz, 1997). The adsorption of surfactant at the interface is a dynamic process in which kinetics is very important, as, for example, in industrial processes (Torcello-Gómez, 2012). This process occurs in two steps. First, a diffusive process takes place, whereby surfactant molecules are transferred from the bulk phase to the interface. The concentration gradient is the driving force; this phenomenon is controlled by mass transfer. The second step is an adsorption/desorption process of the surfactants located at the interface. (Torcello-Gomez,2012). It has been shown that there is an induction time for the adsorption of proteins and macromolecules. Hence, at the beginning of the adsorption process the interfacial tension does not decrease due to the high molar area of proteins and macromolecules (Miller, 2000; Maldonado-Valderrama, 2006). Adsorption of surfactants is usually studied by equilibrium surface tension isotherms, i.e., plots of the equilibrium interfacial tension vs. surfactant bulk concentration γ(c). For conventional surfactants three different regions are usually observed: first, at low concentrations the surfactant adsorption provokes a decrease in the interfacial tension. Once the interface has been saturated, a constant surface concentration value is attained and a linear decrease of interfacial tension versus the log of concentration is observed according to the Gibbs adsorption isotherm. Eventually, at high surfactant bulk concentration monomers start to aggregate producing micelles. Increasing the surfactant bulk concentration does not increase the number of monomers in solution and, therefore, a constant interfacial tension value is attained. The surfactant Chapter 1 24 bulk concentration at which micelles start to form is called critical micelle concentration, cmc. However, for some polymeric surfactants, such as Pluronics or proteins, the interfacial tension isotherm shows a more complicated shape with intermediate regions of constant interfacial tension values, which are reported to be due to conformational changes of the adsorbed macromolecules (Ramírez, 2011; Ramírez, 2012; PérezMosqueda, 2013) Figure 4. Structural region CMC . Chapter 1 25 1.3.2 Interfacial rheology “Interfacial rheology is the science of the response of mobile two dimensional phases to deformation. Interfacial rheological properties are the main characteristics of the dynamic properties of a film” (Sun 2011). In many technological applications equilibrium values of the interfacial tension or cmc values are not enough to fully understand the interfacial properties of surfactants For example, the aim of this Ph.D. Thesis is the production of stable D-limonene emulsions, in these systems, the knowledge about how the interface will respond against any kind of interfacial perturbation is very important. This response will be conditioned by the composition of the molecules which are at the interface, and depends specially on the chemical structure of the molecules adsorbed, their conformation, concentration and interaction (Torcello-Gómez, 2012). The interfacial rheological properties could be characterized by the interfacial shear and dilational viscoelasticity. However, it seems that dilational viscoelastic parameters are much larger than the shear ones and show a more direct correlation to emulsion stability (Wasan, 1982; Clint, 1981; Neustadter, 1981). Gibbs defined the interfacial elasticity as the variation in the interfacial tension when a variation in the area is produced (Sun, 2011; Maldonado-Valderrama, 2006): ( ) ln / d d d A dA A γ γ ε = = (7) where ε is the dilational modulus, γ is the interfacial tension and A is the interfacial area. This expression gives a measure of the resistance of the interface to a change in the area. Usually the interfaces are not completely elastic because they could present some viscous behavior. This viscous process is related to relaxation processes, such as Chapter 1 26 the interchange of molecules between the bulk and the interface or molecular rearrangement processes in macromolecules (Williams, 1996; Maldonado-Valderrama, 2006). The dilatational modulus is formed in this case by an elastic component, called the storage modulus, and a viscous component, called the loss modulus: d d i ε ε ωη = + (8) where ε d is the dilational elasticity or storage modulus and η d is the dilational viscosity component or loss modulus that represents a combination of internal relaxation processes and relaxation due to transport of matter between the surface and the bulk. Usually, the storage modulus and the loss modulus can be determined by dilatational rheology using oscillatory perturbations of the area. The oscillations have to be done in the linear viscoelastic range which implies less than 10% variation in the area. The response is a sinusoidal signal. The storage modulus is obtained from the ratio of amplitudes of the sinusoidal input signal and the sinusoidal output signal. The loss modulus can be determined by the lag phase between the two signals. This is determined by the phase angle parameter (φ) and is defined by the following expression: tan d η ε ϕ ε = (9) If the φ= 0º the interfacial layer is purely elastic, otherwise if the φ= 90º the interfacial layer is completely viscous and if the phase angle is 0º < φ < 90º the interfacial layer is viscoelastic. 1.4. D-limonene D-limonene (4-isopropenyl-1-methylciclohexane) is a natural occurring terpene with the following chemical structure: Chapter 1 27 Figure 5. D-Limonene chemical formula. This interesting bio-derived solvent can be obtained from citrus peels, especially from orange peel, by steam distillation, both at lab scale (Saleh, 2008) and an industrial scale. “The vast majority of essential oils are produced from plant material in which they occur by different kinds of distillation or by cold pressing in the case of the peel oils from citrus fruits” (Kubeczka, 2010). D-limonene gives citrus fruits their typical odor (Fahlbusch, 2003) The option to obtain D-limonene from citrus peels could be an extra added value because it is possible to transform a by-product from the juice industry into an essential oil with interesting applications (Kerton, 2009). Chapter 1 28 Physical/chemical properties of Dlimonene Molecular formula C 10 H 16 Molecular weight 136.2 g/mol Physical state Colorless liquid, with characteristic mild citrus odor Melting point -75 ºC Boiling point 176 ºC Solubility 13.8 mg/l at 25ºC Density 0.84 g/ml Table 2. Physical/Chemical properties of D-limonene Limonene has a chiral center, i.e., an asymmetric carbon. There are two optical isomers. The IUPAC nomenclature for them is R-limonene and S-limonene. However, in this Ph.D. Thesis the most common nomenclature, D-limonene and L-limonene, is used. The two isomers present different properties; for example, the odor of Dlimonene is lemon flavor and the odor of L-limonene is pine flavor (WHO, 1998). The D-limonene isomeric form is more abundantly present in plants than the racemic mixture and L-limonene isomeric form (Wichtel, 2002). Usually, the purity of commercial D-limonene varies from 90% to 98%, the impurities usually being monoterpenes. D-limonene is likely to suffer a quick destabilization by oxidation. Hence, D-limonene should be stored in a cool, dry place without direct sunlight. Chapter 1 29 Figure 6. Product of D-limonene oxidation (Sonchick, 2003) D-limonene is one of the most commonly used terpenes and the estimated worldwide limonene production is of 70·10 6 kg (Kerton, 2009). Due to its molecular weight and low miscibility in water, D-limonene has similar characteristic to classical volatile organic compounds (VOCs). Therefore, D-limonene is an interesting environmentally-friendly chemical which can replace these typical volatile organic compounds (VOCs), such as hexane, ciclohexane, acetone, toluene or xylene (Kerton, 2009). D-limonene is widely used in household and personal cleaning products, fragrance additives and the food and cosmetic industries due to its characteristic lemon flavor. It is possible to use this solvent in other kinds of applications such as resins, paints, cooling fluids, pigments and, thanks to its low solubility in water, it is also employed as degreasing agent. Moreover, it has also been used as an insect repellent (Kerton, 2009). Chapter 1 30 Some studies have shown that D-limonene can act as an anticancer agent in some types of cancer, such as bladder (Xu, 2010) or colon cancer (Chidambara, 2012). Different researchers have proved that monoterpenes from dietary sources such as citrus are capable of inducing cytotoxicity in different human cancer cells (Crowell, 1999, Gould, 1995; 1997, Patil, 2010). In this Ph.D. Thesis, we have used D-limonene as an ecofriendly oil phase in the O/W formulation with the goal of producing more environmentally-friendly emulsions with potential agrochemical use. D-limonene has a low solubility in water due to its hydrophobic character and in many applications the emulsified form is the best way to apply D-limonene. We have seen that D-limonene could be used as substitute for classical oils. There are numerous references from several authors concerning oil-inwater emulsions with D-limonene as the disperse phase: D-limonene is widely used in spray-drying encapsulation due to its flavor properties (Fisk, 2013; Marcuzzo, 2012; Jafari, 2007a; 2007b), and it is also used in the production of drug delivery systems because terpenes are not toxic to the skin (Abil, 2000; Sapra 2008). Another interesting application could be its use in the food industry as a preservative (Hai-Yan, 2012; Aissat, 2012; Chikhoune; 2013) due to its antimicrobial properties and because it is considered as safe (GRAS) for use as a flavoring agent and food preservative (Sun, 2007) 1.5. Pluronics Pluronics are A-B-A triblock copolymer surfactants where the central chain is the hydrophobic part and is formed by PPO (poly propylene oxide) and the side chains are the hydrophilic parts and are formed by PEO (poly etoxi oxide). They are often denoted as (PEOx-PPOy-PEOx). Pluronics are also known as poloxamers, and kolliphor or Synperonic as commercial names. Due to the possibility of combining blocks, Chapter 1 31 surfactants with different molecular weights and HLB can be obtained, (from 2000 to 20000 Da and the range in PEO contents between 20-80% (Alexandridis, 1997), and the properties of the resulting polymers vary in a wide range. This surfactant will present a different physical state at different temperatures, depending on the proportion between the PEO and PPO blocks. They are classified by a capital letter: F for flakes, P for Paste and L for Liquids. When a pluronic is named, the number which follows the capital letter provides information about the size and proportion of the different blocks: the first number (or the first two in a three-digit number) multiplied by 300 shows the approximate molecular weight of the hydrophobic part, and the last digit of the number multiplied by 10 gives the percentage of polyoxyethylene content (Pasquali, 2005). For example, we will focus on Pluronic PE9400, which is formed by the follow structure PPO 21 -PEO 50 -PPO 21 and will be a paste at room temperature. The molecular weight of PPO will be around 9x300=2700 g/mol and each molecule will possess 60% of PEO. . Normally, Pluronics adsorb at a liquid/liquid interface with the central chain (the hydrophobic PPO chain) at the hydrophobic interface while the two side chains (hydrophilic PEO chains) protrude into the water phase (Torcello-Gómez, 2012). 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Chapter 2 47 Abstract In this work, we provide an accurate characterization of non-ionic triblock copolymer Pluronic PE9400 at the air-water and limonene-water interfaces, comprising a systematic analysis of surface tension isotherms, dynamic curves, dilatational rheology and desorption profiles. The surface pressure isotherms display two different slopes of the Π -c plot suggesting the existence of two adsorption regimes for PE9400 at both interfaces. Application of a theoretical model, which assumes the coexistence of different adsorbed states characterized by their molar areas, allows quantification of the conformational changes occurring at the adsorbed layer, indentifying differences between the conformations adopted at the air-water and the limonene-water interface. The presence of two maxima in the dilatational modulus vs. interfacial pressure importantly corroborates this conformational change from a 2D flat conformation to 3D brush one. Moreover, the dilatational response provides mechanical diferences between the interfacial layers formed at the two interfaces analysed. Dynamic surface pressure data were transformed into a dimensionless form and fitted to another model which considers the influence of the reorganization process on the adsorption dynamics. Finally, the desorption profiles reveal that Pluronic PE9400 is irreversibly adsorbed at both interfaces regardless of the interfacial conformation and nature of the interface. The systematic characterization presented in this work provides important new findings on the interfacial properties of pluronics which can be applied in the rational development of new products, such as biocompatible limonene-based emulsions and/or microemulsions. Chapter 2 54 where c is the surfactant bulk concentration and b i is the adsorption equilibrium constant of the polymer in the state i. It is assumed that the adsorption equilibrium values are constant for all the states, hence the total adsorption constant is: . Post-critical regime In this regime three additional parameters are used: the compressibility coefficient, ε , the number of multilayers, m, and the adsorption coefficient for the adsorption of the second and subsequent layers, b 2 . ε is introduced to account for the decrease in the area occupied per molecule due to condensation. From this parameter a new auxiliary variable is defined: (6) where, Π * and Γ * are the values of the critical surface pressure and critical surface concentration, respectively. Therefore, the adsorption isotherm and the equation of state become: (7) ( ) ( ) ( ) 2 0 0 ln 1 RT a Π Γω Γ ω ω Γω Ψω   = − − + − +   (8) It is assumed that surface pressure values are not affected by the adsorption of the subsequent layers and the coverage of the multilayers is proportional to an adsorption coefficient b 2 and the preceding surface concentration. So, as a rough approximation the surface concentration of the second and subsequent layers, Γ Σ , is given by: 1 n i i b nb = = ∑ * exp *RT Γ Π Π Ψ ε ω Γ −   =     ( ) ( ) exp 2 1 i i i i a bc ωωΨ ω Γ ω Γω ωΨ Γω   −     =− Chapter 2 55 (9) The software package Protein M, (Aksenenko, 2012) has been used to fit surface pressure isotherm data to the model explained above. 2.2.3.2 Dilatational rheology According to the diffusional model (Zholob, 2009; Lucassen, 1972) the interfacial elasticity, which is directly linked to the dilatational modulus, E can be calculated by the following equation: 0 2 1 2 2 E E ζ ζ =+ + (10) where E0 is the limiting elasticity which is defined as: (11) the parameter ζ is defined as: (12) where ω is the angular frequency of the measurement and ω 0 is the characteristic frequency of the diffusion process and can be written as: (13) Cs being the bulk concentration and D the diffusion coefficient. 2.2.3.3 Adsorption kinetics The experimental data (surface tension vs. time) have been transformed into the dimensionless variables, θ and t’, according to the scaling argument for diffusion controlled adsorption proposed by Ferri et al. (Ferri, 2000): 1 2 12 1 i m i b c b c Σ Γ Γ − =   =   +   ∑ 0ln eq d Π Ed Γ   =     0 ω ζ ω = 2 0 2 S dC D d ω   =   Γ   Chapter 2 56 (14) ' D t t τ = (15) where γ (t) , γ 0 and γ eq are the surface tension, the surface tension of the pure interface and the equilibrium surface tension, respectively. τ D is the characteristic time scale for the diffusion process and is a function of the diffusion coefficient, D , and the adsorption depth, h : (16) The adsorption depth can be calculated from mass balance on a differential area, dA : 0 0 eq eq dA C hdA h C Γ Γ = → = (17) where Γ eq stands for the equilibrium surface concentration and C 0 is the surfactant bulk concentration. In order to fit the experimental dynamic surface tension values a model which combines the diffusion of the polymer to the interface with the subsequent reorientation has been used (Serrien, 1992). The proposed equation for the dimensionless dynamic surface tension reads as follows: (18) where and . β and τ R are the amplitude and characteristic time of the reorganization process, respectively. When β = 0 and τ R >> τ D equation (18) reduces to a pure diffusional process: 0 ( ) eq eq t γ γ θ γ γ − =− 2 D h D τ = ( ) ( ) 12 4 ( ') 1 exp ' exp ' t B t B At θπ           = − − + −             0 eq B β γ γ =− D R A τ τ = Chapter 2 57 (19) This model has been previously used to explain the adsorption kinetics of other Pluronics (Muñoz, 2000b). 2.3 Results and Discussion 2.3.1 Equilibrium surface pressure isotherms Figures 1A and 1B show the interfacial pressure isotherms ( Π -c) obtained for Pluronic PE9400 adsorbed at the air-water interface and the limonene-water interface respectively. The interfacial pressure values plotted in Figures 1A and 1B were obtained after 3 hours of adsorption at constant interfacial area. After this period, the interfacial layer has settled and the changes of interfacial tension remain below 5%. Both figures show a non-monotonous increase in the interfacial pressure as a function of bulk concentration. Namely, Figures 1A and 1B show two separate regions: first an increase in the interfacial pressure as the bulk concentration increases and finally a constant value once the interfacial layer is saturated, accounting for a critical aggregation concentration, cac. Within the first region we can also distinguish two regimes (precritical and post-critical) which differ in the slope of the increasing tendency of Π . This type of behaviour has previously been reported for pluronics adsorbed at the air-water interface and at the hexane–water interfaces but not at the limonene-water interface (Muñoz, 2000a; Ramírez, 2011). The occurrence of this kink before the cmc is likely to be due to a conformational change at the interface in which the polymer changes its orientation from 2D flat conformation to a 3D brush or mushroom one as reported in the literature for other Pluronics (Muñoz, 2000a; Muñoz, 2000b; Ramírez, 2011; Ramírez, 2012; Torcello-Gómez, 2012; Llamas, 2013). Figures 1A and 1B reveal that adsorption 1 2 4 ( ') exp 't t θπ       = −         Chapter 2 58 in a flat conformation leads to a higher slope in the Π -c isotherm due to the high interfacial area occupied by each molecule leading to larger changes in the interfacial coverage, and hence, in the interfacial pressure. Once the interface is saturated with flat molecules the increase of Π is less pronounced. Further adsorption at the post-critical regime could lead to the formation of multilayers, condensation of the adsorbed polymer layer, aggregation within the interfacial layer and/or penetration into the oil phase. Interestingly, these regimes appear at both the air-water and the limonene-water interface. The main difference is the length of the first adsorption regime (2D-flat conformation) which is larger at the limonene-water interface (Figure 1B). This already suggests structural differences in the adsorbed layer of PE9400 at air-water and limonene-water interfaces. Furthermore, the interfacial pressure of a saturated interface appears higher for PE9400 at the limonene-water interface, hence demonstrating a higher adsorption recorded at the oil-water interface as compared to the adsorption at the air-water interface (Maldonado-Valderrama, 2005). The cac of PE9400 is slightly higher at the limonene-water interface (cac limonene-water = 0,05 molm -3 > cac air-water = 0,01 molm -3 ) also accounting for this feature. Chapter 2 59 Figure 1. A) Surface pressure isotherm for Pluronic PE9400 at the air-water interface at T = 25ºC. B) Surface pressure isotherm for Pluronic PE9400 at the air-water interface at T = 25ºC. The blue line is the best fit to the protein adsorption model with the values of the parameters given in Table 1. The crossing of the blue line with the red line indicates the value of the cac. The standard deviation of each data point is ± 0.5 mN/m. In order to gain further insight into the different conformation and condensation of the interfacial layer of PE9400 at the air-water and the limonene-water interface we applied the theoretical model explained above to the experimental data Chapter 2 60 displayed in Figures 1A and 1B. The lines in Figures 1A and 1B are the best fit of the experimental data obtained with parameters given in Table 1. Since there are many adjusting parameters the experimental data can be fitted with a high number of different combinations of them. In order to obtain a meaningful physical result, two of the parameters, ( ω 0 = 2,4·10 5 , ω m = 3,0·10 7 ), have been fixed according to the values given in a previous work for the adsorption of the same polymer at the hexanewater interface (Ramírez, 2011). Hence, we consider that the molar area occupied by a segment of the polymer, ω 0 , and the maximum molar area, ω m , are intrinsic properties of the polymer and do not depend on the nature of the non-polar phase. Accordingly, the only fitting parameters are: ω 1 , the minimum molar area (this parameter can be different since it is likely that the hydrophobic segments of the polymer can dissolve or penetrate into the oil phase while this is not possible at the air-water interface), a, the interaction parameter, ε , the condensation parameter and when multilayer formation is considered, b 2 and m in the post-critical regime. Table 1 shows a lower value of ω 1 of PE9400 at the limonene-water interface than that obtained at the air-water. This agrees with the experimental findings discussed above suggesting the formation of a more compact layer at the limonene-water interface at high interfacial coverage. This could be the result of PPO segments penetrating into the limonene in agreement with recent work which demonstrates the penetration of PPO segments into the hexane phase by ellipsometric measurements (Ramírez, 2012). The same value of a at both interfaces indicates that the attractive interaction between adsorbed polymer molecules is similar at the air-water and the limonene-water interface. Conversely, the value of ε appears much higher at the limonene-water interface. This accounts for an increased compressibility of the PE9400 at the oil-water interface that 2 m mol 2 m mol Chapter 2 61 would possibly promote higher interfacial coverage and lead to higher condensation of the interfacial layer. In general, the higher condensation of the adsorbed layer of PE9400 at the limonene-water interface, which was already inferred from the experimental Π -c isotherms, is corroborated and rationally quantified by the outcome of the fitting procedure. Moreover, the numerical values displayed in Table 1 importantly quantify this increased condensation by means of the lower molar area ( ω 1 ), the higher saturation Π and the higher compressibility (ε) of the molecule at the limonene-water interface. The values of the interfacial pressure isotherm are not affected by introducing the parameters of multilayer formation. However, they are given in Table 1 since they affect the fitting of the dilatational rheology, as will be explained in next section (Maldonado-Valderrama, 2005). 2.3.2 Interfacial dilatational rheology In order to explore in more detail the interfacial behaviour of the copolymer at the air-water and the limonene-water interfaces the dilatational rheology of these systems was measured (Figures 2A and 2B). The dilatational modulus was obtained by imposing harmonic perturbations to the interface at a frequency of 0,1 Hz after equilibration of the interfacial layer. Figures 2A and 2B show the dilatational modulus measured at the end of the adsorption process recorded for each of the bulk concentrations shown in Figures 1A and 1B. Also, since it has been reported previously that the interfacial elasticity of these copolymers is a unique function of the interfacial pressure (i.e. interfacial coverage) (Blomqvist, 2005; Torcello-Gómez, 2012), harmonic oscillations were performed throughout the whole adsorption process for a given concentration of PE9400 in the bulk. These values are also plotted in Figures 2A and 2B, hence confirming that E is directly linked to the interfacial pressure rather than the polymer bulk concentration. Chapter 2 62 Figure 2. A) Surface dilatational elasticity vs. surface pressure for Pluronic PE9400 at the air-water interface. B) Interfacial dilatational elasticity vs. interfacial pressure for Pluronic PE9400 at the limonene-water interface. Black circles are the surface elasticity calculated once the final surface pressure was reached for each bulk concentration. Red squares and blue triangles are two independent measurements of the surface elasticity for a given bulk concentration (2,2·10 -3 molm -3 ) at different adsorption times. Solid and dashed lines correspond to a theoretical dilatational modulus calculated via equation (10) for a harmonic perturbation of 0,1 Hz and a diffusion coefficient of 1·10 -10 m 2 s -1 (air-water) and 2·10 -10 m 2 s -1 (limonene-water) with the equilibrium model parameters of Table 1 and m = 2 (bilayer, solid line) and m = 1 (monolayer, dashed line), respectively. The standard deviation of each data point is ± 0.5 mN/m. Chapter 2 63 The existence of two maxima in the elasticity of the interfacial layer was expected in the literature owing to the conformational transition taking place (TorcelloGómez, 2012; Muñoz, 2003) but very few works specifically report two maxima (Blomqvist, 2005; Noskov, 2006; Ramírez, 2012; Muñoz, 2003). Recently, the occurrence of three maxima (two of them at low surface pressure) was reported for a more hydrophilic Pluronic F68 at the air/water interface by means of electrocapillary wave and quasi-elastic light scattering, a technique capable of measuring at very high frequencies (up to 1·10 6 Hz) (Llamas, 2013). The first maximum, located at low interfacial pressures, i.e. low interfacial coverage, corresponds to the 2D conformation of the molecules which lie fully extended at the interface (Blomqvist, 2005). This conformation provides similar elasticity values (20 mN/m) regardless of the nature of the interface. This is consistent with the theoretical analysis which provides the same maximum molar area ( ω max ) and interaction parameter (a) at both interfaces (Table 1) and only shows differences in the molecular conformation at high interfacial pressures, i.e. when the interaction is maximized. The second maximum would correspond to a 3D brush conformation of the polymer in which the PPO segment protrudes into the oil phase. Interestingly, this maximum appears displaced to higher interfacial pressures at the limonene-water interface, importantly corroborating the higher interfacial coverage attained at the oil-water interface. Figures 2A and 2B also show theoretical predictions of E as a function of Π . The theoretical values were obtained via equation (10). The limiting elasticity, E 0 and characteristic frequency are related to the equilibrium properties of the adsorbed film by equations, (11-13). Therefore, with the equilibrium model parameters (Table 1) the only adjusting parameter is the diffusion coefficient. The solid lines in Figures 2A and 2B show the Chapter 2 70 2.4. Conclusions A systematic surface characterization has been carried out on the equilibrium, dynamics, interfacial rheology and desorption profile of Pluronic PE9400 at the airwater and limonene-water interface in order to fully characterize the properties of the adsorbed layers. The equilibrium surface pressure isotherm displays two adsorption regimes in the pre-critical region which are characterized by two slopes in the Π -c plot. These regimes are related to a conformational transition of the adsorbed polymer layer from a 2D flat conformation to a 3D brush one which takes place at both the air-water and the limonene-water interface. The theoretical model provides quantitative information on this transition showing that Pluronic PE9400 has a lower minimum molar area and higher internal compressibility at the limonene-water interface than at the air-water interface, suggesting a more compressible layer formed at the former. Dilatational rheology offers further evidence of the two conformations at the interface by showing two maxima as a function of the surface pressure and the application of the theoretical model to these data provides good agreement, importantly confirming the reliability of the proposed model. Moreover, the dilatational response confirms the structural differences between adsorbed molecules at high interfacial coverage. Also, the model indicates that the adsorption proceeds in the form of a bilayer at higher interfacial coverage at the limonene-water interface. In general, the higher condensation of the adsorbed layer of PE9400 at the limonene-water interface is corroborated and rationally quantified by the outcome of the fitting procedure. Figure 6 displays a sketch of the transition from 2D flat conformation to a 3D brush structure when augmenting the surface pressure in both air/water and limonene/water interfaces showing the enhanced compressibility and the likely bilayer formation of the adsorbed PE9400 at the limonene/water interface. Analysis of the adsorption kinetics by transforming the Chapter 2 71 original data into a dimensionless form yields a unique curve for the dynamic surface tension at different concentrations, which is fitted to a model involving the diffusion time scale and the amplitude and the time scale of the reorganization process. Realistic diffusion coefficients were obtained at both interfaces and a more favourable interaction between PE9400 and limonene again inferred from the adjusting parameters. Finally, the desorption profile of PE9400 was studied at the air-water and the oil-water interface. These experimental results importantly demonstrate that Pluronic PE9400 is irreversibly adsorbed at the interface regardless of the nature of the interface and the conformation of the polymer, providing important functional information concerning the adsorbed layer. References Aksenenko, E.V., http://www.thomascat.info/Scientific/AdSo/AdSo.htm (accessed December 2012). Alahverdjieva, V. S., Fainerman, V. B., Aksenenko, E. V., Leser, M. E., & Miller, R. (2008). Adsorption of hen egg-white lysozyme at the air–water interface in presence of sodium dodecyl sulphate. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 317(1), 610-617. Alexandridis, P., & Lindman, B. (2000). Amphiphilic block copolymers: self-assembly and applications. Elsevier. Bai, Z., & Lodge, T. P. (2010). Pluronic micelle shuttle between water and an ionic liquid. Langmuir, 26(11), 8887-8892. Chapter 2 72 Berthier, D. L., Schmidt, I., Fieber, W., Schatz, C., Furrer, A., Wong, K., & Lecommandoux, S. (2010). Controlled Release of Volatile Fragrance Molecules from PEO-b-PPO-b-PEO Block Copolymer Micelles in Ethanol− Water Mixtures. Langmuir, 26(11), 7953-7961. Blomqvist, B. R., Wärnheim, T., & Claesson, P. M. (2005). Surface rheology of PEOPPO-PEO triblock copolymers at the air-water interface: Comparison of spread and adsorbed layers. Langmuir, 21(14), 6373-6384. Fainerman, V. B., Lucassen-Reynders, E. H., & Miller, R. (2003). Description of the adsorption behaviour of proteins at water/fluid interfaces in the framework of a twodimensional solution model. Advances in Colloid and Interface Science, 106(1), 237259. Ferri, J. K., & Stebe, K. J. (2000). Which surfactants reduce surface tension faster? A scaling argument for diffusion-controlled adsorption. Advances in colloid and interface science, 85(1), 61-97. Guzmán, E., Ortega, F., Baghdadli, N., Luengo, G. S., & Rubio, R. G. (2011). Effect of the molecular structure on the adsorption of conditioning polyelectrolytes on solid substrates. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 375(1), 209-218. Hambardzumyan, A., Aguié-Béghin, V., Daoud, M., & Douillard, R. (2004). β-Casein and symmetrical triblock copolymer (PEO-PPO-PEO and PPO-PEO-PPO) surface properties at the air-water interface. Langmuir, 20(3), 756-763. Chapter 2 73 Jafari, S. M., Beheshti, P., & Assadpoor, E. (2012). Rheological behavior and stability of d-limonene emulsions made by a novel hydrocolloid (Angum gum) compared with Arabic gum. Journal of Food Engineering, 109(1), 1-8. Kabanov, A. V., Lemieux, P., Vinogradov, S., & Alakhov, V. (2002). Pluronic® block copolymers: novel functional molecules for gene therapy. Advanced drug delivery reviews, 54(2), 223-233. Kerton, F. M. (2009) Alternative solvents for green chemistry, RSC Publishing, Kim, C., & Yu, H. (2003). 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Marcuzzo, E., Debeaufort, F., Sensidoni, A., Tat, L., Beney, L., Hambleton, A., ... & Voilley, A. (2012). Release Behavior and Stability of Encapsulated d-Limonene from Emulsion-Based Edible Films. Journal of agricultural and food chemistry, 60(49), 12177-12185. F. Monroy, F. Ortega, R.G. Rubio, B.A. Noskov, in R. Miller and L. Liggieri (2009) (Eds.), Interfacial Rheology, Brill, Leiden, , Chapter 6. Muñoz, M. G., Monroy, F., Ortega, F., Rubio, R. G., & Langevin, D. (2000). Monolayers of symmetric triblock copolymers at the air-water interface. 1. Equilibrium properties. Langmuir, 16(3), 1083-1093. Chapter 2 75 Munoz, M. G., Monroy, F., Ortega, F., Rubio, R. G., & Langevin, D. (2000). Monolayers of symmetric triblock copolymers at the air-water interface. 2. Adsorption kinetics. Langmuir, 16(3), 1094-1101. Muñoz, M. G., Monroy, F., Hernández, P., Ortega, F., Rubio, R. G., & Langevin, D. (2003). Anomalous damping of the capillary waves at the air-water interface of a soluble triblock copolymer. Langmuir, 19(6), 2147-2154. Murali, R., Karthikeyan, A., & Saravanan, R. (2013). Protective Effects of d‐Limonene on Lipid Peroxidation and Antioxidant Enzymes in Streptozotocin‐Induced Diabetic Rats. Basic & clinical pharmacology & toxicology, 112(3), 175-181. Myrvold, R., & Hansen, F. K. (1998). Surface elasticity and viscosity from oscillating bubbles measured by automatic axisymmetric drop shape analysis. Journal of colloid and interface science, 207(1), 97-105. Noskov, B. A., Akentiev, A. V., Loglio, G., & Miller, R. (2000). Dynamic surface properties of solutions of poly (ethylene oxide) and polyethylene glycols. The Journal of Physical Chemistry B, 104(33), 7923-7931. Noskov, B. A., Lin, S. Y., Loglio, G., Rubio, R. G., & Miller, R. (2006). Dilational viscoelasticity of PEO-PPO-PEO triblock copolymer films at the air-water interface in the range of high surface pressures. Langmuir, 22(6), 2647-2652. Noskov, B. A. (2010). Dilational surface rheology of polymer and polymer/surfactant solutions. Current Opinion in Colloid & Interface Science, 15(4), 229-236. Chapter 2 76 Papadimitriou, V., Pispas, S., Syriou, S., Pournara, A., Zoumpanioti, M., Sotiroudis, T. G., & Xenakis, A. (2008). Biocompatible microemulsions based on limonene: formulation, structure, and applications. Langmuir, 24(7), 3380-3386. Ramírez, P., Muñoz, J., Fainerman, V. B., Aksenenko, E. V., Mucic, N., & Miller, R. (2011). Dynamic interfacial tension of triblock copolymers solutions at the water– hexane interface. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 391(1), 119-124. Ramírez, P., Stocco, A., Muñoz, J., & Miller, R. (2012). Interfacial rheology and conformations of triblock copolymers adsorbed onto the water–oil interface. Journal of colloid and interface science, 378(1), 135-143. Schramm, L. L. (2006). Emulsions, foams, and suspensions: fundamentals and applications. John Wiley & Sons. Sedev, R., Steitz, R., & Findenegg, G. H. (2002). The structure of PEO–PPO–PEO triblock copolymers at the water/air interface. Physica B: Condensed Matter, 315(4), 267-272. Serrien, G., Geeraerts, G., Ghosh, L., & Joos, P. (1992). Dynamic surface properties of adsorbed protein solutions: BSA, casein and buttermilk. Colloids and surfaces, 68(4), 219-233. Svitova, T. F., & Radke, C. J. (2005). AOT and Pluronic F68 coadsorption at fluid/fluid interfaces: a continuous-flow tensiometry study. Industrial & engineering chemistry research, 44(5), 1129-1138. Chapter 2 77 Tadros, T.; in Holmberg, K. (Ed.). (2003). Novel Surfactants: Preparation Applications And Biodegradability, Revised And Expanded. Crc Press. Chapter 16 Tadros, T. F. (2005). Applied surfactants: principles and applications. John Wiley & Sons. Tavano, L., Muzzalupo, R., Trombino, S., Cassano, R., Pingitore, A., & Picci, N. (2010). Effect of formulations variables on the in vitro percutaneous permeation of Sodium Diclofenac from new vesicular systems obtained from Pluronic triblock copolymers. Colloids and Surfaces B: Biointerfaces, 79(1), 227-234. Torcello-Gómez, A., Santander-Ortega, M. J., Peula-García, J. M., MaldonadoValderrama, J., Gálvez-Ruiz, M. J., Ortega-Vinuesa, J. L., & Martín-Rodríguez, A. (2011). Adsorption of antibody onto Pluronic F68-covered nanoparticles: link with surface properties. Soft Matter, 7(18), 8450-8461. Torcello-Gómez, A., Maldonado-Valderrama, J., Martín-Rodríguez, A., & McClements, D. J. (2011). Physicochemical properties and digestibility of emulsified lipids in simulated intestinal fluids: influence of interfacial characteristics. Soft Matter, 7(13), 6167-6177. Torcello-Gómez, A., Jódar-Reyes, A. B., Maldonado-Valderrama, J., & MartínRodríguez, A. (2012). Effect of emulsifier type against the action of bile salts at oil– water interfaces. Food Research International, 48(1), 140-147. Vieira, J. B., Li, Z. X., & Thomas, R. K. (2002). Adsorption of triblock copolymers of ethylene oxide and propylene oxide at the air/water interface: The surface excess. The Journal of Physical Chemistry B, 106(21), 5400-5407. Chapter 2 78 Wulff-Pérez, M., Gálvez-Ruíz, M. J., De Vicente, J., & Martín-Rodríguez, A. (2010). Delaying lipid digestion through steric surfactant Pluronic F68: A novel in vitro approach. Food research international, 43(6), 1629-1633. Wulff-Pérez, M., de Vicente, J., Martín-Rodríguez, A., & Gálvez-Ruiz, M. J. (2012). Controlling lipolysis through steric surfactants: New insights on the controlled degradation of submicron emulsions after oral and intravenous administration. International journal of pharmaceutics, 423(2), 161-166. Zholob,S.A, Kovalchuk, V.I., Makievski,A.V., Krägel,J., Fainerman,V.B., Miller,R., in Miller, R., and L. Liggieri (2009) (Eds.), Interfacial Rheology, Brill, Leiden, , Chapter 3. Zoller, U. (Ed.). (2009). Handbook of detergents, part E: applications. CRC Press. 81 3 Optimum formulation of D limonene emulsions by response surface methodology Chapter 3 88 at 25ºC ± 0.1ºC, using a C5P Phoenix circulator (Thermo-Scientific) for sample temperature control. The results represent the mean of three measurements. Equilibration time prior to rheological tests was 180 s. 3.2.8. Design of experiments In order to rationally develop an optimal formulation of D-limonene-in-water emulsions prepared with a rotor/stator homogenizer, a second-order experimental design and mathematical model was required. In the present work, the two process variables selected were: surfactant/oil ratio, R, and disperse mass fraction, φ . With only two factors, a full factorial design 3 2 with three replications of the center point was selected. The quadratic model is given below:  =  +    +   ∅ +     +   ∅  +   ∅ (4) Each factor is measured at three levels which were coded to take the value -1 when the factor is at its low level, 0 when at its medium level and +1 when at its high level. The lowest value for R (0.02) was chosen such that the minimum size of stable droplets that can be theoretically produced was 1 µm, according to the following equation (McClements, 2005): (5) where C s is the surfactant concentration in the emulsion and Γ sat is the excess surface concentration of the surfactant at saturation. A value of Γ sat = 3 mg·m -2 was used 16 . The highest limit was set as 5 times the lowest one, so R varied in the range 0.02 – 0.1. φ was studied in the range 10 – 50 wt%, from dilute to slightly concentrated emulsion. min 6 sat s dC Γ φ = Chapter 3 89 It has been reported that emulsion droplet size is a function of the processing conditions; homogenization rate and emulsification time (Franco, 1998) in addition to the formulation used. The optimal processing conditions were found to be 17500 rpm and 60 seconds of homogenization rate and emulsification time respectively, as shown in Figure 1 for the center point (R= 0.06, φ = 30 wt%). r.p.m 5000 10000 15000 20000 25000 30000 d 3,2 [µm] 0.5 1.0 1.5 2.0 2.5 3.0 3.5 t [s] 0 50 100 150 200 d 3,2 [µm] 0.5 1.0 1.5 2.0 Figure 1. Sauter mean diameter as a function of homogenization rate for D-limonenein-water emulsion with R = 0.06 and φ = 30%. Emulsions were produced by Ultraturrax T-25 at room temperature. The inset shows the influence of emulsification time on droplet size for the optimal homogenization rate (17500 rpm). Symbols are the mean of three replicates and the error bars show the standard deviation of the measurements. The effect of the two independent variables, surfactant/oil ratio (R) and disperse mass fraction ( φ ) on emulsion mean droplet size, d 3,2 , and on the destabilization processes: creaming index, CI, Ostwald ripening rate k OR and the overall destabilization index TSI (Turbiscan Stability Index) of D-limonene-in-water emulsion was studied using a 3 2 experimental factorial design. Experiments were randomly carried out in Chapter 3 90 order to minimize the effects of random error in the observed responses. The center point in the design was determined in triplicate to calculate the repeatability of the method 27 and to check the fitting quality of the mathematical model (Khuri, 1996). The quadratic terms of the response surface models were obtained by the least squared method. For model construction, terms with p > 0.05 were removed and the analysis was recalculated without these terms. The suitability of the models was determined by using the coefficient of determination (R 2 ) and the lack of fit test (F lof ). Joklegar and May proposed that R 2 should be higher than 0.80 to obtain a good fitting (Jokeglar, 1987). F lof is the ratio between mean squares due to lack of fit and mean squares for pure error. This F lof value must be compared with a table value of F crit for α level of significance and the degrees of freedom of the mean squares employed. It was assumed that the proposed model did not exhibit lack of fit at α level of significance when F crit exceeded F lof . 3.3 Results and discussions 3.3.1. Surface response analysis The variation of each response variable was initially assessed as a quadratic function of surfactant/oil ratio and disperse mass fraction (Table 1). The lack of fit shown by the fittings of creaming index and turbiscan stability index was solved by adding a new cubic interaction term in these models: β 122 R*ϕ 2 . The results indicated that the models employed were adequate, showing no significant lack of fit (F crit > F lof with α = 0.05) and very satisfactory values of R 2 for all responses. The R 2 values for droplet size, creaming index, Ostwald ripening rate and turbiscan stability index were found to be 0.959, 0.998, 0.967 and 0.991 respectively. Comparison between predicted and actual values for the response variables also indicated that the polynomial Chapter 3 91 regression models were suitable to determine optimum formulation for preparing Dlimonene-in-water emulsions with maximum stability (Fig. 2). Regression coefficients d 3,2 (µ µµ µm) C.I. (%) k OR (h - 1 ) TSI β ββ β 0 0.936667 2.49224 0.236833 5.78167 β ββ β 1 -0.348333 0.50695 0.262155 4.025 β ββ β 2 -0.1216667 -2.3496 0.230378 -4.63667 β ββ β 11 0.248333 1.48822 0.0923217 3.085 β ββ β 22 - (p > 0.05) 1.77868 -(p > 0.05) -(p > 0.05) β ββ β 12 - (p > 0.05) 2.6975 0.209317 2.5125 β ββ β 122 --- -1.47425 --- -3.6275 F lof 1.45 2.81 1.51 3.05 F crit 9.01 9.55 9.12 9.28 R 2 0.959 0.998 0.967 0.991 Adj-R 2 0.943 0.997 0.948 0.983 Table 1. Regression coefficients of the fitting models Chapter 3 92 Figure 2 A) Sauter mean diameter at t = 0 vs. surfactant/oil ratio, R. B) Creaming index (CI) as a function of R. C) Normalized Ostwald ripening rate (k OR ) vs. R and D) Turbiscan stability index (TSI) vs. R. Symbols correspond to experimental measurements: triangles ( φ = 10%), squares ( φ = 30%) and circles ( φ = 50%). Lines are the theoretical values obtained by the surface response models by using the parameters in Table 1. 3.3.2 Emulsion droplet size Droplet size is a key variable to study emulsion stability (McClements, 2005). Destabilization processes, such as creaming or Ostwald ripening growth, depend greatly on droplet size. It was therefore particularly important to determine the droplet size in order to characterize our system. Chapter 3 93 From full factorial design we deduced that the most significant factors affecting droplet size in our system were the linear and quadratic terms of surfactant/oil ratio and the linear term of disperse mass fraction (Table 1). The equation of the fitted model was: d 3,2 = 0.94 - 0.35*R -0.12* φ +0.25*R 2 (6) 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 0.02 0.04 0.06 0.08 10 20 30 40 50 d 3,2 [µm] R φ 0.8 1.0 1.2 1.4 1.6 1.8 Figure 3 Response surface 3D plot of the Sauter mean diameter as a function of the process variables R and φ . The color scale indicates the Sauter mean diameter values ranging from 0.8 µ m to 1.8 µ m. Figure 3 shows the surface response of the effect of variables R and φ on the d 3,2 . For a given disperse mass fraction, increasing R yielded a decrease in the droplet size until it reached a minimum value. This d 3,2 variation was to be expected since more emulsifier was available to fully cover the O/W interface with smaller droplets as R was Chapter 3 94 increased. However, once a certain ratio was achieved a further increase of surfactant did not result in smaller droplets. The limiting factor may be a) the actual emulsification process, which was not capable of generating enough energy to further reduce the droplet size, and/or b) the fact that the emulsifier did not adsorb fast enough to form a protective layer to prevent recoalescence phenomena (McClements, 2005). On the other hand, the influence of φ on the droplet size was certainly more striking. The lack of major differences in d 3,2 with φ was to be expected. However, a clear tendency to decrease d 3,2 with increasing φ was observed. For a given surfactant/oil ratio (R) if the amount of oil (disperse phase) is increased, the surfactant concentration in the continuous phase is also increased, enhancing the viscosity of the continuous phase, η c . It has been reported that the viscosity of the continuous phase influences the droplet size diameter obtained in rotor/stator homogenizers. It is stated that an increase in η c will lead to higher turbulent eddies which can be larger than the smaller droplets and therefore emulsification will proceed via a turbulent viscous regime producing smaller droplets (Vankova, 2007). 3.3.3 Destabilization processes It was seen that all emulsions were affected by both gravitational separation and increase in droplet size in a very short time (within 12 hours). Since the disperse phase is less dense than the continuous one the oil droplets tend to move towards the top, which is known as creaming. As an example, Figure 4 shows the droplet size distribution (A-C) and the backscattering profiles (D-F) for emulsions with R = 0.06. Depending on φ , two different behaviors were detected: for φ = 10 and 30 wt% both destabilization processes, creaming and increase of the droplet size were readily noticeable. Nevertheless, for emulsions with φ = 50 wt% creaming was not easily Chapter 3 95 perceived whereas the increase in the droplet size was very marked. Eventually, this emulsion was also affected by creaming, probably due to the increase in droplet size. Figure 4. A-C: Droplet size distribution for emulsions with R = 0.06 and φ = 10 wt% (A), φ = 30 wt% (B) and φ = 50 wt% (C). t = 0 (Squares and solid lines) and t = 12 h (Circles and dashed lines). D-F: Backscattering profiles as a function of the measuring cell height for emulsions with R = 0.06 and φ = 10 wt% (A), φ = 30 wt% (B) and φ = 50 wt% (C). t = 0 (solid lines) and t =12 h (dashed lines). In order to gain a deeper knowledge of the destabilization processes a 3 2 full factorial design was used, R and φ being the process variables. The dependent variables studied were creaming index (CI) to analyze gravitational separation, normalized Ostwald ripening rate (k OR ) for droplet size growth and turbiscan stability index (TSI) as an overall destabilization parameter. Chapter 3 96 3.3.4 Creaming Index (CI) Due to the lack of fit of the quadratic model the addition of a cubic interaction was considered, R* φ 2 . Hence, the model equation was: CI = 2.5 + 0.51*R - 2.3* φ + 1.5*R 2 + 1.8* φ 2 + 2.7*R* φ − 1.5∗R* φ 2 (7) The high value of the coefficient of determination, R 2 (0.998) indicates that experimental measurements agree well with the model. Figure 5 shows the surface response of the effect of process variables R and φ on the CI. The CI variation as a function of R was quite dependent on disperse mass fraction, φ . Hence, at φ = 10 wt% CI decreased with R, whereas for the highest φ = 50 wt% the opposite behavior was observed. Figure 5. Response surface 3D plot of the creaming index (CI) as a function of the process variables R and φ . The color scale indicates the creaming index values ranging from 0 to 16. 0 4 8 12 16 0.02 0.04 0.06 0.08 0.10 10 20 30 40 50 C.I. R φ 0 4 8 12 16 Chapter 3 97 From CI values, the creaming velocity (V c ) can be calculated as follow: (8) where H e is the total height of the emulsions in mm and 12 is the total time of measurement in hours, therefore V c is in mm·h -1 . Figure 6 shows the creaming velocity as a function of the squared radius of the droplets r 2 . A linear variation was observed for emulsions containing 10 wt% D-limonene. For non-dilute emulsions ( φ > 2 wt%) hydrodynamics interactions between particles are expected to slow down the velocity of the gravitational separation as expressed by Stokes’ law. If droplet flocculation is negligible a kind of Stokes’ law equation can be used to predict the creaming velocity (McClements, 2005). (9) where g is the acceleration due to gravity, r is the radius of the droplet, ρ 1 and ρ 2 are the densities of the continuous and disperse phases respectively and η e is the viscosity of the emulsion. As shown in Figure 7A the emulsions with φ = 10 wt% are Newtonian and the viscosity for all the emulsions was around 2 mPa·s. By using equation (9) with η e = 2 mPa·s, g = 9.81 ms -2 , ρ 1 = 1000 kg·m -3 and ρ 2 = 841 kg·m -3 creaming velocities very close to experimental values were obtained as seen in Figure 6. For φ values above 10 wt% the systems became more complex and it was likely that droplet-droplet interactions lead to droplet flocculation. Therefore, creaming velocity was not a linear function of r 2 . Vc=(CI ⋅He) 1 0 0 ⋅ 1 2 ( ) 2 2 1 2 9 c e gr V ρ ρ η − = − Chapter 3 104 intersection of both curves gave an optimal value with minimum destabilization and droplet size for R = 0.062. R 0.00 0.02 0.04 0.06 0.08 0.10 0.12 d3,2 [µm] 0.0 0.2 0.4 0.6 0.8 1.0 TSI 0.0 0.2 0.4 0.6 0.8 1.0 Figure 11. Normalized Sauter mean diameter vs R (dashed line) and normalized Turbiscan Stability Index vs. R (solid line) for emulsions with φ = 50 %wt. The intersection of both curves indicates the optimum formulation. 3.4 Conclusions Monomodal, submicron D-limonene-in-water emulsions were obtained by rotor/stator homogenizer by using pluronic PE9400 as emulsifier. In order to gain a deeper knowledge of the droplet size distribution and stability of the emulsions as a function of the variables, surfactant/oil ratio (R) and disperse mass fraction ( φ) a surface response methodology has been implemented. The mathematical models obtained from the full factorial design gave an adequate fit between the experimental and actual values. Chapter 3 105 D-limonene-in-water emulsions were destabilized by both, creaming and Ostwald ripening growth. The former was more marked for dilute emulsions, whereas the latter was faster at higher φ values. Furthermore, creaming destabilization of dilute emulsions ( φ = 10 wt%) can be quantified using a kind of Stokes’ law by substituting the viscosity of the continuous phase for that of the emulsion. From rheological measurements the transition from Newtonian to shear-thinning behavior of the emulsions pointed to the occurrence of flocculation induced by surfactant micelles. In order to take into account the influence of both destabilization processes a global destabilization parameter, the turbiscan stability index (TSI), has been used. An optimal formulation, with minimum TSI and minimum droplet size values, was obtained for R = 0.062 and φ = 50 wt%. 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Soft Matter, 7(13), 6167-6177. Chapter 3 110 Torcello-Gómez, A., Maldonado-Valderrama, J., Jódar-Reyes, A. B., Foster, T. J., (2013). Interactions between Pluronics (F127 and F68) and Bile Salts (NaTDC) in the Aqueous Phase and the Interface of Oil-in-Water Emulsions. Langmuir, 29(8), 25202529. Torcello-Gómez, A., Maldonado-Valderrama, J., Jódar-Reyes, A. B., CabrerizoVílchez, M. A., Martín-rodríguez, A., (2014). Pluronic-covered oil-water interfaces under simulated duodenal conditions. Food Hydrocolloids, 34, 54-61. Urban, K., Wagner, G., Schaffner, D., Röglin, D., & Ulrich, J. (2006). Rotor‐Stator and Disc Systems for Emulsification Processes. Chemical engineering & technology, 29(1), 24-31. Vankova, N., Tcholakova, S., Denkov, N. D., Ivanov, I. B., Vulchev, V. D., Danner, T., 2007. Emulsification in turbulent flow. 1. Meand and maximum drop diameters in inertial and viscous regimes, Journal of colloid and interface science, 312, 363-380. Weers, J.G., 1998. Ostwald ripening in emulsions, in Binks (Ed.), Modern Aspects of Emulsions Science, Royal Sociaty of Chemistry, Cambridge, pp. 292-327. Weiss, J., Canceliere, C., McClements, D. J., 2000. Mass transport phenomena in oil-inwater emulsions containing surfactant micelles: Ostwald ripening. Langmuir, 16(17), 6833-6838. 4 Influence of emulsifier concentration on the rheology, microstructure and physical stability of D-limonene emulsions stabilized by a lipophilic biopolymer Chapter 4 113 Abstract Stable sub-micron D-limonene-in-water emulsions have been obtained by means of a single-step rotor/stator homogenising process. The formulation comprises the use of a naturally occurring lipophilic biopolymer (rosin gum) dissolved in the disperse phase and a non-ionic triblock copolymer as emulsifier (Pluronic PE9400). The addition of rosin gum highly increases the stability of Pluronic-covered emulsions by reducing the Ostwald ripening growth of the emulsion droplets. A full Ostwald ripening inhibition was obtained for rosin gum concentrations above 10 wt%. Furthermore, it has been observed that rosin gum adsorbs at the interface, reducing the interfacial tension and leading to lower droplet sizes. Emulsions were classified in three different regions as a function of Pluronic PE9400 concentration. Region I: from 1 wt% to 3 wt% the surfactant available was not enough to achieve the minimum droplet size that can be obtained under the operating conditions. Region II: from 3 wt% to 6 wt% the droplet size reached a constant value and highly stable emulsions were obtained. Region III: from 6 wt% to 8 wt% an excess of surfactant in solution provoked a depletion flocculation process, which eventually led to the creaming of the emulsion. This chapter is a contribution to the development of sustainable product engineering by scouting the applications of green solvents in model agrochemical emulsions. Chapter 4 114 4.1 Introduction D-limonene is an interesting bio-derived solvent which can be obtained from citric peels. This organic compound presents interesting applications in different fields such as cosmetics, food, pesticide applications and pharmaceutical industries (Brud, 2010, Caballero-Gallardo, 2011; Jafari, 2012; Marcuzzo, 2012; Klein, 2010; Murali, 2012). Furthermore, D-limonene is a good candidate to be used as a (bio-)solvent in the design of novel agrochemical products, replacing more pollutant chemicals (Kerton, 2009). The formulation of the disperse phase was completed with rosin gum as stabilizer. Rosin gum is a natural polymer obtained from pine trees. It is chiefly composed of 90% rosin acids (abietic acid, palustric acid, neoabietic acid, and others) and is 10% non-acidic. There exist several papers which comment on the application of rosin gum in biocompatible systems such as drug delivery (Lee 2004 and 2005, Kumar 2013) or microencapsulation (Fulzele 2004). The emulsifier function was carried out by an ABA block copolymer, Pluronic PE 9400, where A and B stand for poly(ethylene) oxide and poly(propylene) oxide respectively. These surfactants have found many industrial applications as foam and emulsion stabilizers (Alexandridis, 2000; Tadros, 2005). On the other hand, these polymeric surfactants have been employed recently in more sophisticated technological applications such as drug delivery, gene therapy and the developing of foodstuffs that provide specific physiological responses, such as control of lipid digestion or satiety (Batrakova, 2008; Kabanov, 2002; Torcello-Gómez, 2011a, 2011b, 2013 & 2014, Wulff-Perez 2012, Jódar-Reyes 2010). Emulsions are thermodynamically unstable colloidal dispersions which are destabilized by several mechanisms such as flocculation, coalescence, gravitational Chapter 4 121 confirmed the adsorption of rosin gum at the D-limonene-water interface, leading to lower emulsion droplet sizes and hindering the migration of limonene across the interface. Rosin gum (wt%) Π ΠΠ Π (mNm - 1 ) 1e-4 2 1e-3 7 1e-2 12 0.1 23 1 26 10 29 30 31 Table 1. Interfacial pressure of the D-limonene-water interface at different rosin gum concentrations at 20ºC. Figure 1. Ostwald ripening rate, kOR and sauter diameter, d3,2 as a function of rosin gum concentration. 4.3.2 Droplet size distributions and morphology of droplets Figure 2A shows the droplet size distribution of the emulsions with different surfactant concentration aged for 24 hours. First, it should be stated that all emulsions showed monomodal distributions. Moreover, droplet size distributions shifted to lower C Rosin gum [wt%] 0 5 10 15 k OR [h -1 ] 0.0 0.1 0.2 0.3 0.4 d 3,2 [nm] 400 500 600 700 800 Chapter 4 122 droplet diameters with increasing PE9400 bulk concentration and levelled off for surfactant concentrations above 5 wt%. Figure 2B shows the Sauter and volumetric mean diameters and span values of emulsions with different PE9400 concentration aged for 24 hours. For surfactant concentration above 1 wt%, submicron Sauter and volumetric mean diameters were achieved. The lowest mean diameter values were found for the 6 wt% emulsion. Despite this, it should be noted that span (droplet size polydispersity) dropped to a minimum around 4 wt%. It is stated that an increase in polydispersity highly influences the stability of the emulsion leading to an increase in the creaming rate due to higher values of the effective packing parameter (McClements, 2005). Chapter 4 123 Size [µm] 0.1 1 10 % [V/V] 0 5 10 15 20 1 wt% 2 wt% 3 wt% 4 wt% 5 wt% 6 wt% 7 wt% 8 wt% A C PE9400 [wt%] 0246810 d 3,2 , d 4,3 [µm] 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Span 1.0 1.2 1.4 1.6 1.8 2.0 B Figure 2. (A) Droplet size distribution and (B) Sauter mean diameter (d3,2), volumetric mean diameter (d4,3) and span as a function of Pluronic PE9400 concentration of emulsions aged for 24 hours. Standard deviation of the mean (3 replicates) for d3,2, d4,3 and span < 5% Figure 3 shows the time evolution for 75 days of the volume mean diameter for the eight emulsions. It is noteworthy that stable submicron emulsions were obtained at surfactant concentrations within 3-7 wt%. The increase in the d 4,3 values of the emulsions with less surfactant (1 and 2 wt%) can be ascribed to the occurrence of coalescence, enhanced by creaming of the bigger droplets in these emulsions. A slight increase in the d 4,3 value was observed for the most concentrated (8 wt%) emulsion after aging for 75 days. This increase is likely to be related to a depletion flocculation process Chapter 4 124 due to an excess of surfactant micelles in solution which cause the coalescence of emulsion droplets. C PE9400 [wt%] 1 2 3 4 5 6 7 8 d 4,3 [µm] 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Figure 3. Volume mean diameter, d4,3 as a function of Pluronic PE9400 concentration. Emulsion samples were aged for 1 day (black bars), 30 days (light-grey bars) and 75 days (dark-grey bars). Optical micrographs were acquired in order to confirm the laser diffraction results obtained so far. Emulsions were diluted (dilution ratio 1:20), so droplets were more easily observed. Figure 4 shows, by way of example, the micrographs obtained by optical microscopy with transmitted light (phase contrast mode) for the emulsions containing 4 wt% (Figure 4B) and 1 wt%, (Figure 4A) Pluronics PE9400. It is observed that 1 wt% emulsion is a more polydisperse emulsion with bigger droplets than 4 wt%, thus confirming the results shown in Figure 1. Chapter 4 125 Figure 4. Micrographs using transmitted light in phase contrast mode (objective: 40x) for diluted emulsions (dilution ratio: 1/20) containing (a) 1 wt% Pluronics PE9400 and (b) 4 wt% Pluronics PE9400. Emulsion samples aged for 24 hours. 4.3.3 Rheology Figure 5 shows the flow properties for the emulsions studied aged for 24 hours as a function of surfactant concentration. All the emulsions exhibited a trend to reach a Newtonian region at the lowest shear rate values, followed by a decrease in viscosity (shear-thinning behaviour) above a critical shear rate. This behaviour is likely to be produced due to shear-induced breaking of flocs (Chanamai, 2000). Greater tendency to flocculate has previously been associated to finer emulsions by Pal, 1996 and Barnes, 1994 (Pal, 1996; Barnes, 1994). Viscosity curves were fitted to the Cross model: 01 1 n c η η η η γ γ ∞ ∞ − − = +   +     & & (4) where η is the viscosity, η ∞ is the limiting high shear rate viscosity, η 0 is the zero-shear viscosity, is the shear rate, is the critical shear rate and n is the power law index. Chapter 4 126 The inverse of the critical shear rate gives what is commonly known as the characteristic time for flow, 1/ [s -1 ] 0.001 0.01 0.1 1 10 100 η [Pa·s] 0.01 0.1 1 10 100 1000 1 wt% 2 wt% 3 wt% 4 wt% 5 wt% 6 wt% 7 wt% 8 wt% Cross model Figure 5. Flow curves for the studied emulsions as a function of Pluronic PE9400 concentration for 24 hours of aging time at 20ºC. Continuous lines illustrate data fitting to the Cross model. Figure 5 also illustrates the fitting quality of the results obtained to the Cross model (R 2 > 0.999). The values of fitting parameters are shown in Table 2 as a function of surfactant concentration. An analysis of the fitting parameters allows the identification of three surfactant concentration regions (see figure 6). I. Concentration region I (1-3) wt%: The zero-shear viscosity and characteristic time for flow increased, while the flow index decreased with Pluronic PE9400 concentration due to the fall in droplet diameter, which resulted in greater specific surface area available for friction. Most of the Pluronic surfactant was required to cover the increasing O/W interface. This fact is associated with a tendency for the apparent viscosity to increase when the Sauter diameter decreases (Barnes, 1994). Chapter 4 127 II. Concentration region II (3-6) wt%: The zero shear viscosity, the characteristic time for flow and the flow index levelled off. Excess amounts of Pluronic in the continuous phase are in dynamic equilibrium with surfactant on the interface and in addition micelle concentration start being significant. III. Concentration region III (6-8) wt%: Mean droplet diameters levelled off, and polidispersity went clearly up leading to a striking increase in both zeroshear viscosity and characteristic time for flow. Furthermore, a marked drop in the flow index is shown, indicating the occurrence of more shear thinning emulsions. An excess of free micelles in the disperse phase is expected which is likely to lead to a depletion flocculation phenomenon influencing the rheological properties of the emulsion (Pal, 2011; Derkach, 2009). Therefore, the zero shear viscosity and the characteristic time for flow are expected to increase, whereas the flow index should decrease as shown in Table 1. This rheological change could be produced by either an enhanced viscosity of the continuous phase (due to the presence of lyotropic structures based on micelles), or the occurrence of a stronger oil network due to depletion flocculation (Palazolo, 2005; Manoj, 1998). Chapter 4 128 Figure 6. A) Zero shear viscosity, η 0 ; B) Characteristic time for flow, 1/ ; and C) flow index, n, as a function of Pluronic PE9400 concentration for emulsions aged for 24 hours. C PE9400 [wt%] 12345678 η 0 [Pa·s] 0.01 0.1 1 10 100 1000 III III A C PE9400 [wt%] 12345678 [s] 1 10 100 1000 10000 III III B C PE9400 [wt%] 1 2 3 4 5 6 7 8 n 0.0 0.2 0.4 0.6 III III C c γ & Chapter 4 129 Surfactant (wt%) η ∞ (Pa·s) η 0 (Pa·s) 1/ (s) n 1 0.014 0.2 8 0.55 2 0.015 0.8 25 0.46 3 0.015 1.6 40 0.43 4 0.025 2.2 33 0.44 5 0.028 1.8 36 0.44 6 0.045 2 30 0.44 7 0.055 4.2 50 0.37 8 0.080 184 1000 0.22 Table 2. Flow curves fitting parameters of the Cross model for the emulsions at 24 hours of aging time. The viscosity of the continuous phase prior to emulsification was measured by means of the falling ball viscometer. It is seen that increasing surfactant concentration from 2 wt% to 16 wt% causes viscosity to increase from 1.1 to 4.6 mPa·s (Table 3). Nonetheless, the increase in the viscosity of emulsions from the lowest surfactant concentration to the highest one is about four orders of magnitude (from 100 to 100000 mPa·s). Hence, the marked increase in the viscosity of the emulsions is more likely to be related to a depletion flocculation phenomenon than to the viscosity of the continuous phase. Surfactant (wt%) ρ (g/ml) η (mPa·s) 2 1.0012 1.08 4 1.0040 1.26 6 1.0065 1.51 8 1.0091 1.77 10 1.0122 2.67 12 1.0145 3.17 14 1.0170 3.72 16 1.0195 4.63 Table 3. Continuous phase density and viscosity values at 20ºC. Figures 7 A, B, and C show the flow curves as a function of aging time for three emulsions which are representative of the three different regions explained above (1 γ . c Chapter 4 130 wt%, 4 wt% and 7 wt%). Experimental data were fitted to the Cross model (equation 4) with the fitting parameters given in Table 4. Samples were taken from the upper part of the container to follow any rheological changes produced by creaming. The evolution of the rheological parameters for the emulsion containing 1wt% PE9400 pointed to the development of a more viscous and shear thinning structure at the top of the vial, which is likely to be due to the formation of a cream layer. The evolution of the flow curves for 7 wt% PE9400 emulsion showed a slower increase in the viscosity while maintaining the same flow index value. Hence, a slower formation of a serum layer is observed. Finally, the emulsion containing 4 wt% PE9400 was stable for all the aging time. Table 4. Evolution over time of the flow curves fitting parameters of the Cross model Surfactant (wt%) Storage time (days) η ∞ (Pa·s) η 0 (Pa·s) 1/ (s) n 1 0.014 0.2 8 0.55 30 0.017 0.7 77 0.48 75 0.023 10 588 0.39 1 0.025 2.2 33 0.44 30 0.022 2.3 33 0.45 75 0.022 2.5 25 0.44 1 0.055 4.2 50 0.37 30 0.056 4.6 42 0.39 75 0.065 8.2 53 0.35 1 4 7 . c γ Chapter 4 137 characteristic time for steady shear flow allowed the identification of three surfactant concentration regions. Furthermore, these emulsions only exhibited measurable linear viscoelastic (SAOS) properties from 7 wt% surfactant, when depletion flocculation started to be significant. Rheological measurements are demonstrated to be a powerful tool to assist in the prediction of destabilization processes in combination with laser diffraction and multiple light scattering techniques. 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These experimental results fitted the thermodynamic protein model. This model is based on the occurrence of adsorbed layer compression and the likely formation of a bilayer at higher interfacial coverage at the limonene–water interface. Hence, a conformational transition from 2D flat conformation to 3D brush/mushroom conformation has been proposed to take place when increasing surface pressure at both interfaces. 2. The subphase exchange technique made it possible to study the desorption behavior of pluronic PE 9400 at the air/water and limonene/water interface. The results show that the polymer is irreversibly adsorbed at the interface regardless of the interface and the conformation of the polymer. 3. The combination of laser diffraction, rheology and multiple light scattering allow a comprehensive characterization of D-limonene emulsions stabilized by Pluronic PE9400 to be made. The influence of surfactant/oil ratio (R) and disperse mass fraction ( φ ) on the droplet size distribution and physical stability of the emulsions was studied using surface response methodology. From the optimum formulation monomodal submicron D-limonene-in-water emulsions were obtained by single-step rotor/stator homogenization. 4. Addition of a lyophilic biopolymer (rosin gum) to the disperse phase yields a marked increase in the physical stability of emulsions and a reduction of the emulsion droplet size. The enhanced physical stability is ascribed to three