UNDERSTANDING BEHAVIOR OF PARTICLE STABILIZED EMULSIONS AND TRANFORMATIONS IN COLLOIDAL SYSTEMS

Practically irreversible attachment of partially wettable particles to liquid-liquid interfaces presents an effective strategy for dispersing oil as stable drops in water column in the event of an oil spill. We investigate the potentials of using carboxylterminated carbon black particles for stabilizing oil-in-water emulsions motivated by the need for developing alternative dispersants. These particles on exposure to either acid or salt, become partially hydrophobic and stabilize emulsions which remain stable for months. Once at the interface, these particles can adsorb naphthalene, a potentially toxic polycyclic aromatic hydrocarbon and reduce its transport into the aqueous phase. Stable crude oil-in-sea water emulsions are formed using these CB particles (with no added acid or salt). These attributes make these particles a viable alternative or supplement to conventional dispersants for oil spill remediation. While partially wettable particles are known to stabilize emulsions, we report a new strategy for stabilizing emulsions using very hydrophilic and hydrophobic particles which are otherwise incapable of forming emulsions alone. When such a hydrophilic and a hydrophobic particle suspension are mixed, attractive van der Waals interactions between the particles cause them to assemble at the oil-water interfaces into supraparticle aggregates that are partially wettable in both phases and stabilize emulsions. Van der Waals interaction energy between two particle types across an aqueous-organic interface provide a systematic guide to particle and liquid combinations that can be used for stabilizing emulsions using our strategy. A combination of optical microscopy, cryogenic electron microscopy and zeta potential measurements are used for these studies. Time-resolved cryogenic transmission electron microscopy (TR-cryo-TEM) is used to investigate early stages of gypsum formation from a supersaturated solution of calcium sulfate hemihydrate. Our results indicate that a multi-step particle formation model, where an amorphous phase forms first, followed by transformation into a crystalline product, is applicable even at time scales of the order of tens of seconds for this system. Addition of a small amount of citric acid significantly delays reorganization to gypsum crystals due to complexation of available calcium ions with carboxylic groups. This induces disorder, and extends the time over which amorphous phase exists. Information about phase and morphology is obtained by energy dispersive X-ray spectroscopy (EDX) and selected area electron diffraction (SAED). Complementary X-ray diffraction experiments confirm our observations. Direct imaging of transient nanoscale samples by TR-cryo-TEM is a powerful technique for fundamental understanding of crystallization, and manyother evolving systems. We have also used cryo-TEM to understand the impact of perfume raw materials (PRMs) on the evolution of microstructures in vesicular dispersions of commercial fabric softener Ultra Downy Free (UDF). While a ‘good’ perfume keeps the inherent micro structures intact, a ‘bad’ perfume triggers a series of changes which results in bilayer fragmentation and transition from multi lamellar vesicles to predominantly unilamellar structures. The behavior of bad perfume, eugenol is attributed to its interaction with the surfactant bilayer as confirmed by nuclear magnetic resonance experiments. This understanding can be exploited to screen PRMs which provide both pleasant odor over an extended period of time and also do not impact the stability of vesicular dispersions.

. Napthalene adsorption under different conditions. The reduced concentration of naphthalene in octane compared to the control case indicates that the CB particlesadsorb naphthalene from the octane. The last column is obtained by doing a mass balance for the naphthalene. ………………………………………………….. 28 x

FIGURE PAGE
Chapter 1.  Addition of HCl or NaCl make the particles partially hydrophobic and causes particleparticle agglomeration, shown by arrows in the cryo TEM images (b) and (c).
Addition of octane to the acid-or salt-mediated suspension followed by vortexing results in oil-in-water emulsions (d) and (e). The oil droplets are in equilibrium with excess water that is free of carbon black as shown in the insets (f) fluorescence image of the acid-mediated sample with pyrene-labeled octane, confirming that this is an oilin-water emulsion……………………………………………………………………..22

Particle Stabilized Emulsions
Dispersing one liquid as tiny drops in another immiscible liquid generates emulsions. This is often achieved either by adding amphiphilic molecules like surfactants or partially wettable solid particles to the system. While surfactants lower interfacial surface tension and reduce the free energy for emulsification, particles driven by their preference to reside at the liquid-liquid interfaces result in emulsion stabilization. 1 Emulsions have wide range of applications in a variety of fields including drug delivery, novel material fabrication, oil recovery, cosmetics, food, paper, paint and personal products. [2][3][4][5][6][7][8][9] Their stability against coalescence over an extended duration if time is responsible for various interesting uses.
Emulsions stabilized solely by solid particles were first observed by Ramsden 10 and Pickering 11 . Better understanding of impact of particle shape, size, wettability and interfacial particle concentration on emulsion stability has helped in advancement of this field. Detachment energy, E, for a model spherical particle from a liquid-liquid interface into either bulk phase, is given by Here R is the particle radius,  ow is the oil-water interfacial tension and θ is the contact angle through either phase. For a typical oil-water interfacial tension of 35mN/m and R = 10nm, ∆E > 500 kT for 55º < θ < 125º. This is illustrated in Fig. 1. Particles with these contact angles are partially wettable in both phases, and once at the interface, they would irreversibly attach to it and stabilize emulsion droplets. 12,13 Once at the oil-water interface, these particles can suppresses drop-drop coalescence and enhances emulsion stability. This is attributed to the fact that particles can be charged, providing repulsive barrier against coalescence between droplets, they can provide steric barriers, 8,14 bridge across neighboring emulsion drops, and can retard the thinning of intervening liquid layer as drops approach each other 15,16 because of enhanced interfacial viscosity. Intrinsic physical and chemical properties of particles can be exploited for creating emulsion drops with multifunctional propertiesan attribute that separates it from surfactant stabilized emulsions. Another frontier that is exceedingly resorting to particle based emulsification technologies for better performance and environmental preservation is dispersant development for oil spill remediation. The Deepwater Horizon oil spill at the Gulf of Mexico in 2010 resulted in a release of about 5 million barrels 9 of crude oil and about 2 million gallons of surfactant based dispersants-Corexit 9500 A and Corexit 9527 were used to contain the spill. Dispersants break the spilled oil into tiny drops so that they can remain suspended in water column for extended periods of time to be degraded by bacteria. The use of surfactants as dispersants has been primarily driven by their effectiveness in emulsification and low cost. However, inability of these surfactants in stabilizing oil indefinitely in large quantity of sea water coupled with their toxic impact 17 towards marine environment has prompted investigation of particles as dispersants.
Particle stabilized emulsions while effective, rely primarily on wettability of particles. Partially wettable particles are considered to be very good emulsifiers. 1,18 However, a consequence of this partial wettability is that these particles will have a tendency to agglomerate or form a network in the phase they are suspended in, to minimize the overall free energy. Agglomeration often causes the particles to sediment, while network formation results in a large increase of zero shear viscosity. 1,19 Generally, for creating a homogeneous dispersion of individual partially wettable particles for emulsification requires sustained input of energy up to the time a second immiscible liquid is introduced to the system. Some of these features have resulted in less usage of particles as emulsion stabilizers despite of their enormous potentials 9 for different applications which usually require less energy intensive processes. This has prompted a search for novel strategies to stabilize emulsions using particles 14 to exploit their unique properties.
A focus of this thesis is to study behavior of particles at liquid-liquid (oil-water) interfaces with the goal of developing particle based, alternative dispersants for emulsification of oil subsequent to a surface or sub-sea level oil spill. As a supplement to this, possibility of developing a new energy efficient strategy for forming particle stabilized emulsions that can be used to combine particles of different functionalities at emulsion droplet surfaces or generating novel materials is also explored.

Early Stages of Crystallization
Crystallization from supersaturated solution has been studied extensively. However, very little is known about the early stages of particle formation. Understanding these early stages is very crucial as it dictates the existence of subsequent phases and morphologies. This becomes more important when additives are to be rationally designed to modulate the crystallization process, as interaction of additive molecules with precursors are known to happen at very early stages. Classical nucleation theory predicts that the earliest particles that form have the crystal structure of the end product. On the other hand, models systems 20-23 have described formation of disordered clusters that result in reorganization to ordered crystalline domains.
Crystallization of inorganic salts [24][25][26] have shown that transitions in precursor phases occur at very early time points. Hence, it is important to understand the pathway through which crystallization proceeds. Fig. 2 illustrates some interesting questions.  Early stages of gypsum crystallization from a supersaturated solution is investigated using TR cryo TEM. For this we carefully investigate the crystallization process to capture transitions that happen over short period of time. Impact of additives on the crystallization process is also explored. This understanding is crucial not only for unraveling the steps involved in crystallization process, but also to implement these in processes which need well control of crystal or particle properties for specific uses such as in plaster and building industries, 27 scaling control in pipes, heat exchangers, desalination plants 28,29,30 and pharmaceutical industry.

Vesicles
Vesicles are formed when bilayers close back on themselves, because an open sheetlike configuration would involve a large energy along the hydrophobic edges in contact with the aqueous environment. 31 This results in encapsulation of aqueous environment. These vesicles can be uni-or multilamellar. Fig.3   Fragrance is an integral part of these consumer good products and extended duration of smell is an important product requirement as it determines acceptability by consumers. For this purpose, perfume is added to vesicular dispersions -a key constituent of many consumer products. However addition of a foreign molecule to a stable dispersion of vesicles can result in structural and morphological changes which can either destabilize a formulation by changes in rheology or decrease efficacy of product due to changes in microstructures. 33,34 Hence, understanding changes induced by different perfume raw materials(PRMs) is a key to screening 'good' and ' bad' fragrance. Efforts to understand impact of PRMs on vesicular dispersions using techniques like selected ion flow tube mass spectrometry (SIFT-MS) 35 have not yielded in complete unraveling of the dynamics involved in these processes. Insight into this process also has implication for understanding transitions induced by hydrophobic additives in self assembled surfactant based colloidal systems.  Emulsification of oil from a subsurface spill and keeping it stable in the water is an important component of the natural remediation process. Motivated by the need to find alternate dispersants for emulsifying oil following a spill, we examine particlestabilized oil-in-water emulsions. Emulsions that remain stable for months are prepared either by adding acid or salt to carboxyl-terminated carbon black (CB) suspension in water to make the particles partially hydrophobic, adding the oil to this suspension and mixing. When naphthalene, a model potentially toxic polycyclic aromatic hydrocarbon, is added to octane and an emulsion formed, it gets adsorbed significantly by the CB particles, and its transport into the continuous water is markedly reduced. In contrast to an undesirable sea water-in-crude oil emulsion produced using a commercially used dispersant, Corexit 9500A, we demonstrate the formation of a stable crude oil-in-sea water emulsion using the CB particles (with no added acid or salt), important for natural degradation. The large specific surface area of these surface functionalized CB particles, their adsorption capability and their ability to form stable emulsions are an important combination of attributes that potentially make these particles a viable alternative or supplement to conventional dispersants for emulsifying crude oil following a spill.

Introduction
Surfactants are commonly used to stabilize emulsions. Their ubiquity for this application rests on their effectiveness and their low cost. A practical example is the purpose. This is mainly driven by the fact that particles can adsorb almost irreversibly to liquid-liquid interfaces, allowing such emulsions to be stable even at extremely low concentrations of the dispersed phase. In addition, intrinsic surface, thermal, optical, electrical and magnetic properties of particles can be exploited to produce emulsions that have greater functionalities than surfactant stabilized ones. We exploit some of these features of particles in the work reported here.
Emulsions stabilized solely by solid particles were first observed by Ramsden 2 and Pickering. 3 Subsequent work investigated the relationship between the three phase contact angle and emulsion stability, 4, 5 and the effect of particle flocculation on emulsification. 6 Commercial uses of Pickering emulsions are not common, but potential application areas that take advantage of the properties of the particles are emerging, including drug delivery, 7 novel material fabrication, 8,9 oil recovery, 10 cosmetics, food, paper, paint and personal products. [11][12][13] A range of particles including silica, polystyrene, iron oxide, bentonite clay and graphene oxide [14][15][16][17][18][19][20][21][22] have been employed for emulsion formation. In each of these cases, the particle properties also play useful roles in determining the final characteristics of the emulsion.
Spontaneous entry of particles into liquid-fluid (termed oil-water for the remainder for this paper) interfaces is slow, 23 and mixing is used to accelerate this process. The energy E required to detach a particle (we assume spherical particles for this illustration) from an oil-water interface into either bulk phase is given by where R is the particle radius,  ow is the oil-water interfacial tension and θ is the contact angle through either phase. For R = 50nm,  ow = 30mN/m, and  = 90, Eq. (1) gives E ~ 5.7x10 4 kT. Therefore, once a particle is in the interface it will not detach spontaneously. The presence of particles at the oil-water interfaces suppresses dropdrop coalescence and enhances emulsion stability. This is because the particles can be charged, providing repulsive interactions between droplets, 24  to form water-in-oil emulsions. In this work, we take advantage of covalently linked surface groups that can be used to tune the CB hydrophilicity, to consistently form oilin-water emulsions. We carefully examine a model system consisting of octane, water and carbon black. We use these results to guide our work on the emulsification of crude oil, a specific application studied here.
We use optical and cryogenic scanning electron microscopy to image emulsions.
Their stability is monitored using centrifugation. Zeta potentials of the particles at different pH and salt concentrations provide key insights into the formation and stability of these emulsions. Since low molecular weight polycyclic aromatic hydrocarbons (PAH) in crude oil can partition into surrounding water, there is interest in finding methods to lower this transfer to reduce potential toxicity to marine organisms after an oil spill. In this context, we examine the adsorption of a model low molecular weight PAH, naphthalene, from the oil phase in an emulsion onto the CB particles. As a potential application of these particles, we demonstrate the successful formation of stable crude oil (taken from the Gulf of Mexico Deepwater Horizon oil spill) -in-sea water emulsions. We compare the nature and lifetimes of Corexit 9500A stabilized emulsions with those stabilized by CB and show major differences between the two.

Materials
The para amino benzoic acid (

Preparation of emulsions
We use a 0.015wt% carbon black suspension for our experiments. Two routes are used to make the emulsions. In the first, 1N hydrochloric acid is added to the suspension to lower the pH to 3.3. The surface carboxylate groups get partially protonated, and the hydrophobicity of the particles increase. Emulsions are also formed by adding sodium chloride to the CB suspension. We use 0.6M NaCl to roughly match the overall salt concentration in sea water (~3.5wt%); the Na + ions salt out some of the carboxylate groups on the surface of the CB. The protonation or the binding of sodium ions to the surface carboxylate groups results in increased particle hydrophobicity and chaining of the particles in the aqueous medium, producing a noticeable rise in the aqueous suspension viscosity. Rapid aggregation of the CB particles causes a difficulty in zeta potential measurements at 0.6M NaCl. To gain insights into the effects of salt addition through such measurements, we have used

Emulsion stability
When an oil-in-water emulsion is subjected to a centrifugal field, the denser aqueous phase collects at the end of the tube furthest from the axis of rotation, and the lighter emulsion-containing phase moves to the other end. During this process the oil droplets are forced against each other, and at a critical centrifugation rate, the first few drops start to coalesce. The pressure exerted on the oil droplets at these conditions is termed the critical de-emulsification pressure and is a reasonable measure of the stability of the emulsion under forced coalescence. 45 The critical de-emulsification pressure permits a quantitative comparison of the stability of emulsions formed under different conditions. If accelerations beyond the critical value are used, additional drop-drop coalescence take place, more oil is released, and a new equilibrium is established. The critical de-emulsification pressure, P deemulsification is calculated by measuring the volume of oil released at a particular centrifugal acceleration 46, 47 using 48 where Δ is density difference between the aqueous and oil phases, g k is the centrifugal acceleration, V oil is the total volume of oil in the emulsion, V released is volume of oil released after centrifugation, and A is the cross sectional area of the centrifuge tube.
Emulsions are centrifuged using an International Equipment Company (IEC) clinical centrifuge for 15 minutes. The centrifugal acceleration is varied between 150g and 1000g. After centrifugation, the released oil is withdrawn carefully from the top of the centrifuge tube with a syringe and its volume measured, from which the critical pressure for deemulsification is calculated using Eq. (2).

Cryogenic Scanning Electron Microscopy (cryo-SEM)
Approximately 5µl of the emulsion is placed on a cylindrical sample holder. Both are then plunged into liquid nitrogen, rapidly solidifying the emulsion. The sample is fractured with a flat-edge cold knife at -130C, then warmed to -95C for a few minutes to sublime some of the residual octane and water. Sublimation enhances surface topological details. The sample is then cooled back to -130C, sputtered with a gold-palladium composite, then moved from the preparation chamber to the imaging stage. A Hitachi S-4800 Field Emission SEM operated at 3kV and 20μA is used for imaging. The sample is maintained at -130C during imaging. All of the processes starting from the fracturing to the imaging take place under a high vacuum.

Additional characterization
A ZEISS Axioplan 2 Imaging System is used for fluorescence microscopy. into an oil phase and water phase as soon as the mixing stops. These samples are vortexed 5 times over a 72hr period, to ensure naphthalene transport away from the octane is complete, prior to measuring the concentration.  Figure 1(a). Individual CB particles are observed, as is expected from a stable aqueous suspension of hydrophilic particles (the average interparticle distance is of the order of 2µm for this suspension, so we do not see more than one particle in these cryo-TEM images at the magnification needed to see one particle, but this is a typical image seen in various portions of the TEM grid).

Results and discussion
Upon the addition of HCl or NaCl, particles start agglomerating. Cryo-TEM images of such agglomerated particles are shown in Fig. 1(b) and Fig. 1(c). We do not observe any unagglomerated particles in the acid or salt mediated cases, suggesting that CB in water + n-octane PABA modified CB in water -pH 7. carbon black (CB) particle of p-amino benzoic acid terminated, pH 7.5, 0.015% w/w suspension CB in water. Addition of HCl or NaCl make the particles partially hydrophobic and causes particle-particle agglomeration, shown by arrows in the cryo TEM images (b) and (c). Addition of octane to the acid-or saltmediated suspension followed by vortexing results in oil-in-water emulsions (d) and (e). The oil droplets are in equilibrium with excess water that is free of carbon black as shown in the insets (f) fluorescence image of the acid-mediated sample with pyrene-labeled octane, confirming that this is an oil-in-water emulsion.
changing the CB surfaces from hydrophilic to partially hydrophobic drives agglomeration, and is consistent with an observed increase in suspension viscosity.
Upon addition of octane and vortexing, shear induced in the system generates new oilwater interfaces, drives the CB into these interfaces and stabilizes the emulsion. The bottom aqueous phase looks clear and is in equilibrium with the top emulsion phase.
Examination of this bottom phase by optical microscopy revealed a carbon black concentration that is about five orders of magnitude lower than that in the original suspension, suggesting that most of the CB is at the octane-water interfaces. Optical micrographs of the emulsion droplets are shown in Fig. 1(d) and Fig. 1(e). A few drops of water disperse immediately when added to this emulsion, while drops of octane 'bead' up. When the octane is labeled with pyrene, and the emulsion imaged in fluorescence mode, the droplets are clearly visible ( Fig. 1(f)). These experiments confirm that we have produced octane-in-water emulsions.
Cryo-SEM provides a more detailed insight into the structure of the particle aggregates and their positioning at the oil-water interfaces. Fig. 2(a) shows an image of an octane droplet in an emulsion prepared after addition of acid to a 0.015%w/w CB suspension. Multiple layers of CB consisting of closely packed particle aggregates are present at the interface, more evident in the higher magnification image of the interface in Fig. 2(b). The aggregation of particles upon addition of acid implies that the entities that reside at the oil-water interfaces are not individual carbon black particles but rather clusters of a few. We see evidence of these in our cryo-TEM images. Such clusters are harder to displace from oil-water interfaces than individual CB particles because the displacement energy scales with the square of the particle size, and provide additional stability for the emulsions. Emulsions have also been formed at 0.0075% w/w CB, shown in Fig. 2(c) and Fig. 2(d). We observe incomplete coverage of the droplet surface, but the emulsions are stable. We propose that the increased interfacial shear viscosity produced by the presence of a connected network of particles at the surface prevents thinning of the intervening water layer as droplets approach each other, and is responsible for suppressing coalescence. Fig. 2(e) and The coefficients of thermal expansion of octane and water are 0.001C 1  and 0.0002C 1  respectively. 50 During rapid sample solidification, the octane shrinks in volume more than the water, creating a gap at the octane-water interfaces. These gaps are a consequence of the cooling process, and they are often observed in rapidly solidified oil-in-water emulsions. 51, 52 Quite remarkably, for an emulsion prepared in the presence of 0.15M NaCl, (Fig. 3(a) and Fig. 3(b)) the CB particles stay with the solidified aqueous phase. In contrast, for an emulsion prepared by the addition of acid, the particles stay with the octane (Fig. 3 (d) and Fig. 3(e)). If a major portion of the particle is within aqueous phase as it spans the oil-water interface, it stays with ice after freezing. Conversely, if most of the particle is in octane, it is pulled into the solidified octane upon freezing. Hydrophilic/hydrophobic balance of particles dictates particle location, and thus determines which phase they preferentially adhere to. To our knowledge, this is the first report of use of cryo-SEM to connect particle location to its hydrophilic/hydrophobic balance. Fig. 3(c) and Fig. 3(f) illustrate this concept. Since many of the features observed in these emulsions are related to the hydrophilic/hydrophobic balance of the CB particles, which in turn are related to the residual charge on the particle surface, we measured zeta potentials for the particles under different conditions, and report them in Fig. 3(g). The zeta potential of the particles in the unmodified CB suspension is -60mV. When the pH is lowered to 3.3, the zeta potential is -15mV, indicating protonation of some surface carboxylate groups on the CB particles. At 0.15M NaCl, the zeta potential changes to -42mV, caused by salting of some carboxylate groups. The isoelectric point of the CB particles is 2.9 for no salt, shifts upward to 3.5 for 0.01M NaCl and 4.0 for 0.15M NaCl, indicating specific binding of sodium ions. The salt-modified particles carry substantially more charge than those modified by a change in pH, and are thus more hydrophilic. As further support, we note that the solubility of sodium benzoate in water is ~4M at 20°C while that of benzoic acid in water is 0.02M at 20°C. 53 These measurements are consistent with the observation that more hydrophilic particles stay with the aqueous phase upon freezing.
We compare the stability of the acid-and salt-mediated emulsions using centrifugation, and report results in Table 1. The key observation is that the saltcontaining emulsions have a higher critical deemulsification pressure than those stabilized using the acid. The zeta potential measurements revealed that the saltmediated particles have more charge than those formed by addition of acid, and are therefore more hydrophilic. We propose that these particles are able to bridge octane drops across an intervening water layer, and suppress drop-drop coalescence. We show evidence of particle bridging in Fig. 4(a) and Fig. 4(b), and have observed many such particle-bridged drops in our salt-containing samples. Particle bridging is

kPa
Given the high specific surface area of CB particles, and non-specific binding capability of carbon, we have examined the absorption of naphthalene as a model polycyclic aromatic hydrocarbon from octane-in-water emulsions stabilized by CB on to the surface of the particles and report results in Table 2. The concentration of naphthalene in oil is reduced dramatically from the control case with no carbon black.
More naphthalene is adsorbed for the acid-mediated case than the salt mediated emulsions as a consequence of the greater hydrophobicity of the acid-mediated emulsions.  of experiments. First, when octane with Corexit 9500A is vortexed with sea water, no emulsion is produced. When crude oil without any added surfactant is vortexed with sea water, the emulsion is unstable, and the oil separates from the water; but we observe a few water drops dispersed within oil. When AOT, a major component of Corexit 9500A 59 is added to octane and vortexed with sea water, an emulsion is not produced. However, when AOT is added to the crude oil and the solution vortexed with sea water, a water-in-oil emulsion results, shown in the inset in Fig. 5(f). These results support our hypothesis that components of crude oil and Corexit 9500A interact to form water-in-oil emulsions. We note that for an oil spill remediation application, the presence of water droplets in the crude oil is undesirable because of the reduced calorific content, decreased ability to burn the oil, the reduction in bioremediation efficiency, the additional volume for removal, 56 and the increased viscosity. 56,60 Our experiments provide strong evidence that this CB suspension has potential for use as an alternative dispersant for subsurface oil spills. The detailed examination of octane-in-water emulsions and the successful formation of these emulsions with added NaCl provided key insights into the conditions necessary to form crude oil-in-sea water emulsions. The principle of in situ tuning of surface properties of particles to enhance their ability to form emulsions, in combination with the ability to exploit intrinsic particle properties, is a powerful strategy for making advanced materials that is being explored in our laboratory and elsewhere.

Conclusions
We demonstrate the formation of octane-in-water emulsions using carboxyl terminated carbon black particles. These emulsions are prepared either by dropping the pH, which makes the particles more hydrophobic by protonating surface carboxylate groups, or by adding NaCl, which imparts hydrophobicity due to specific adsorption and binding of the sodium ions to the particles. These two routes vary the hydrophilic/hydrophobic balance on the carbon black particles in different ways and by different amounts. We use cryo-SEM to characterize these emulsions, and examine 60. Cormack, D., Response to marine oil pollution -review and assessment.

Interfaces as a Versatile Strategy to form Stable Emulsions
(under review in ACS Nano)

Abstract
We report a conceptually new strategy for forming particle-stabilized emulsions.
We begin with stable, dilute suspensions of highly hydrophilic nanoparticles in water and hydrophobic nanoparticles in oil. When the two suspensions are mixed, attractive van der Waals interactions between the hydrophilic and hydrophobic particles cause them to assemble at the oil-water interfaces into supraparticle aggregates that are partially wettable in both phases. These aggregates effectively stabilize emulsions.
Both water-in-oil as well as oil-in-water emulsions can be formed by tuning the ratio of hydrophilic to hydrophobic particles in aggregates. Because each of the particle suspensions are stable and unaggregated, this method of forming emulsions requires less energy/volume compared to ones formed from single, partially wettable particles.
When the Hamaker constants of aqueous and oil phases are chosen to be intermediate between the Hamaker constants of the two types of particles, repulsive interactions between these particles prevent the formation of partially wettable aggregates, and emulsions are not formed. Van der Waals interaction energy between two particle types across an aqueous-organic interface provide a systematic guide to particle and liquid combinations that can be used for stabilizing emulsions using our strategy. Our analysis and experiments provide a new platform for the formation of particlestabilized emulsions and can be used to combine particles of different functionalities at emulsion droplet surfaces for generating novel materials.

Introduction
Emulsions stabilized solely by particles were first reported over 100 years ago by Ramsden 1 and by Pickering 2 . These emulsions require particles to locate at liquid-liquid interfaces. This process can be understood by examining the energy of detachment, E, for a model spherical particle from a liquid-liquid interface into either bulk phase, and is given by Here R is the particle radius,  ow is the oil-water interfacial tension and θ is the contact angle through either phase. For a typical oil-water interfacial tension of 35mN/m and R = 10nm, ∆E > 500 kT for 55º <  < 125º. Particles with these contact angles are partially wettable in both phases, and would hence lodge preferentially and irreversibly at oil-water interfaces, helping to stabilize emulsion droplets. 3,4 One consequence of this partial wettability is that these particles will have a tendency to agglomerate or form a network in the phase they are suspended in, to minimize the overall free energy. Individual nanoparticles can stay suspended because their Brownian motion dominates over sedimentation. However agglomeration often causes the particles to sediment, while network formation results in a large increase of the zero shear viscosity. 5, 6 Thus, creating a homogeneous dispersion of individual partially wettable particles requires a sustained input of energy up to the time a second immiscible liquid is added to form an emulsion. In this paper, we describe a conceptually new process for creating a particle-stabilized emulsion that relies on the in situ assembly of fully wettable particles at liquid-liquid interfaces to create partially wettable aggregates that can stabilize emulsions. This process can be applied to a broad range of particles, opening up the possibility of making emulsions and materials that have unique functionalities.
We begin with stable suspensions of highly hydrophilic particles in water and highly hydrophobic particles in oil. When these two dispersions are mixed, the hydrophilic and hydrophobic particles 'bind' at the oil-water interfaces through attractive van der Waals interactions with energies that are multiples of kT, and form partially wettable supraparticle complexes. These complexes then stabilize emulsion droplets. We perform energy calculations to guide choices of particles and liquids that will successfully form emulsions using this approach.

Preparation of Emulsions
Carbon black suspensions at 0.015wt%, and a 0.015 wt% hydrophilic iron oxide suspension are prepared in deionized water. The hydrophobic fumed silica and spherical silica particles are dispersed in toluene to form a 0.015 wt% suspension.
Different volume ratios of these suspensions are vortexed at 1200 RPM for 1 minute to form toluene-in-water or water-in-toluene emulsions.

Microscopy
A Gatan Alto 2500 sample preparation system, along with a Zeiss Sigma VP FESEM is used for cryogenic scanning electron microscopy. A Nikon Eclipse E 600 is used for brightfield optical microscopy.

Results and Discussion
We first establish the framework for the selection of particles and liquids for our experiments. The two major components to particle movement across an oil-water interface come from van der Waals forces between particles, and electrostatic interactions between a charged particle and a charged oil-water interface. [8][9][10][11][12] Using material properties that are representative of our system and at a separation distance of 0.1nm, the van der Waals contribution is ~ -30 kT, while the electrostatic contribution is 0.03kT. Hence electrostatic repulsion can be safely ignored.
Following previous work, [13][14][15][16][17] we obtain an expression for the van der Waals energy of interaction between two particles through two immiscible liquids. As the distance between particles becomes small compared to the particle dimensions, we ignore the particle curvature, and model the system as two semi-infinite flat plates interacting across two immiscible liquids, shown in Fig. 1(a). The van der Waals energy U, of a particle P1 in a medium M1 interacting with a particle P2 in medium M2 across a M1-M2 interface is given by: A i is the Hamaker constant of material 'i', d is the distance between two particles and is the fraction of the distance d occupied by M1. U < 0 implies attractive interactions between P1 and P2, while U > 0 implies repulsive interactions. In our experiments, P1 is chosen to be either hydrophilically modified carbon black or iron oxide, while P2 is hydrophobically modified silica. We chose water as M1 and toluene as M2. Toluene is selected for these experiments because it has a Hamaker constant in the higher range for oils, and can dissolve p-xylene, a high Hamaker constant solute. We note that the surface modification on each type of particle will modulate the interparticle interaction energy, but that effect is small compared to the energy of interaction between P1 and P2. We show lines of U=0 at ϕ=0, corresponding to only toluene as medium, ϕ=1, corresponding to only water as medium, and ϕ=0.5 in Fig. 1(a), and indicate the range of particle Hamaker constants where P1 and P2 will undergo either attractive or repulsive interactions. We hypothesize that emulsions can only be formed if the hydrophobic and hydrophilic particles interact attractively across oil-water interfaces, so that they assemble into partially wettable aggregates. As a general rule of thumb, this will happen if both media between the particles have Hamaker constants that are either above or below that of P1 and P2. We identify points on this plot that correspond to the properties of the particles and liquids we have used. They are all in the regions where the interparticle interaction is attractive. Such particle liquid combinations should produce emulsions. These particles will interact attractively across the water-toluene interfaces. (b) U=0 for water, toluene, water + 80%w/w PEG 200 and toluene + 90%w/w p-xylene as the two immiscible media (ϕ=0, ϕ=1). When particle Hamaker constants are in the shaded region, the interparticle interactions go from being attractive to repulsive upon adding PEG 200 and p-xylene to water and toluene respectively.

If the Hamaker constants of the water and oil phases are intermediate between A P1 and
A P2 , the particles would interact repulsively. In Fig. 1(b) we show the consequence of making the aqueous phase to be 80% w/w PEG 200, and the toluene phase 90% w/w p-xylene, 18 raising the Hamaker constants of the aqueous and oil phases to above that of silica, but below that of carbon or iron oxide. Our particles now lie in a region where the interparticle interactions are repulsive. We propose that such particle-liquid combinations will not allow emulsions to form.
Some control experiments are performed first. Toluene is mixed into the aqueous hydrophilic particle suspension or water is mixed into the toluene suspension with hydrophobic particles. Te aqueous and organic phases separate fully and emulsions are not formed in either case.
When the CB suspension in water is mixed with the suspension of hydrophobic fumed silica in toluene, a stable emulsion is formed. Fig. 2(a) shows the assembly route for hydrophilic and hydrophobic particles at oil-water interfaces to form partially wettable entities that can stabilize an emulsion. The average wettability of the particle aggregate will determine whether an oil-in-water or a water-in-oil emulsion is formed.
For a given set of particles, this can be controlled by varying the proportion of hydrophobic to hydrophilic entities in an aggregate. If the particle concentration in each of the suspensions is fixed, the volume ratio of the suspensions directly impacts the hydrophobic / hydrophilic balance of the supraparticle aggregates. Fig. 2(b) shows optical micrographs of a water-in-toluene emulsion formed by mixing a 0.015%w/w CB suspension in water with a 0.015%w/w fumed silica suspension in toluene at a ratio of 30:70 v/v. When this proportion is changed to 70:30 a toluene-in-water emulsion is formed (Fig. 2(c)). To demonstrate the versatility of this technique, we used 10nm diameter spherical iron oxide particles suspended in water at 0.015%w/w and 80nm diameter spherical silica particles suspended in toluene at 0.015%w/w, and formed W/O emulsions when mixed at a 30:70 ratio, as shown in Fig. 2(d). When the balance of the aqueous to the oil phase is changed to 70:30 for this set of suspensions, an O/W emulsion is formed, shown in Fig. 2(e). In Fig. 2(f), we show that emulsion droplets made using iron oxide can be mobilized using magnets. Experiments are also performed where volume of oil and water is kept constant, but the ratio of wt% particles in these phases is kept at either 3:7 or 7:3. When the ratio of hydrophobic to hydrophilic particle is 7:3 we get water in oil emulsion and when this ratio is 3:7 oil in water emulsion is obtained. This is applicable both for fractal, spherical particles and is illustrated in Fig. 3. (c) (a) (b) (d) Figure 3. Optical micrograph of (a) a water-in-toluene emulsion formed by mixing equal volumes of water, oil containing hydrophilic CB and hydrophobic fumed silica particles respectively at a 3:7 weight ratio, (b) a toluene-in-water emulsion formed by using hydrophilic CB to hydrophobic fumed silica at a weight ratio of 7:3. Optical micrographs of emulsions stabilized by spherical hydrophilic iron oxide particles in water and spherical hydrophobic silica particles in toluene showing (c) a water-in-toluene emulsion formed with hydrophilic iron oxide to hydrophobic silica weight ratio of 3:7 and (d) a toluene-in-water emulsion formed by using hydrophilic iron oxide to hydrophobic silica at a weight ratio of 7:3. Scale bars are 100μm. Fig. 4(a) shows a cryo-SEM image of a toluene drop stabilized by hydrophobic fumed silica and hydrophilic carbon black particles. The fracture plane is magnified in Fig. 4(b). The layer of fumed silica particles is visible in the bottom left region, while the CB particles covering the fumed silica layer is visible on the upper right. The CB and fumed silica particles form supraparticle complexes at the toluene-water interface.
Figures 4(c) and 4(d) are cryo-SEM images of oil drops stabilized by 80nm spherical hydrophobic silica and 10nm spherical hydrophilic iron oxide particle assemblies.
Assembled silica and iron oxide particles are visible at the oil-water interface. have Hamaker constants of 6.63x10 -20 J and 6.85x10 -20 J respectively, 18 each higher than the Hamaker constant for silica, but lower than that of carbon or iron oxide.
When stable suspensions of the hydrophilic and hydrophobic particles in these media are mixed, they do not form an emulsion. Repulsive interactions between the hydrophilic and hydrophobic particle prevents formation of the partially wettable supraparticle aggregates that can stabilize emulsions.
We compare the energetics of emulsion formation using single particle suspensions to that using our technique. We lowered the pH of carbon black particles to 3.0 to make them partially wettable, so they can stabilize an emulsion. 19 This change in pH protonates the surface carboxylic groups and makes the CB partially hydrophobic.
This initiates particle agglomeration. In addition, a particle network forms in the aqueous phase and increases the viscosity of the suspension. 30 vol% toluene is then added and vortexed to form an emulsion. The measured power consumption for emulsion formation over 1 minute is 22.1 watts. For our CB/silica case where we form emulsions using the same ratio of toluene to water, the power consumption over 1 minute is 7.8 watts. We suggest that the additional energy required to form emulsions in the partially wettable, single particle case is expended in breaking up aggregates or the CB network.

Conclusions
We demonstrate that when suspensions of completely hydrophilic particles in water and hydrophobic particles in toluene are mixed, attractive van der Waals interactions between these types of particles cause them to assemble into aggregates that are partially wettable in both phases. These entities are very effective at stabilizing emulsions. Both, water-in-toluene and toluene-in-water emulsions can be formed using the same sets of particles. The choice of particles and liquids is guided by calculations of van der Waals interaction between a hydrophilic and hydrophobic particle through water and oil. Our experiments suggest that the energy expended to form an emulsion using this dual particle method is lower than that required when a single particle type is used to stabilize an emulsion. This strategy for forming particlestabilized emulsions provides a powerful platform for developing new materials, with functionalities that are dictated by the types of particles in the emulsion.

Abstract
We use time-resolved cryogenic transmission electron microscopy (TR-cryo-TEM) on a supersaturated solution of calcium sulfate hemihydrate to examine the early stages of particle formation during the hydration of the hemihydrate. As hydration proceeds, we observe nanoscale amorphous clusters that evolve to amorphous particles and then reorganize to crystalline gypsum within tens of seconds. Our results indicate that a multi-step particle formation model, where an amorphous phase forms first, followed by the transformation into a crystalline product, is applicable even at time scales of the order of tens of seconds for this system. The addition of a small amount of citric acid significantly delays the reorganization to gypsum crystals. We hypothesize that available calcium ions form complexes with the acid by binding to the carboxylic groups. Their incorporation into a growing particle produces disorder, and extends the time over which the amorphous phase exists. We see evidence of patches of 'trapped' amorphous phase within the growing gypsum crystal at time scales of the order of 24 hours. This is confirmed by complementary X-ray diffraction experiments. Direct imaging of nanoscale samples by TR-cryo-TEM is a powerful technique for a fundamental understanding of crystallization, and many other evolving systems.

Introduction
While crystal growth from supersaturated solutions has been studied extensively, we use time-resolved cryogenic transmission microscopy to provide new insights into the hydration of calcium sulfate hemihydrate (CaSO 4 ·0.5H 2 O) to form gypsum (CaSO 4 ·2H 2 O). Cryo-TEM has been used previously to examine calcium carbonate formation, both in free solution 1 and by templating, 2, 3 and for studying early stages of iron oxide formation. 4 The transformation of calcium sulfate hemihydrate to gypsum has been a topic of considerable interest because of its application in the construction and medical industries, but little is known about particle shapes and phases existing at the very early stages of this process, 5 which in turn, affects the existence of subsequent phases and morphologies. Insight into this process also has implications for understanding biological mineralization. 6 Gypsum, formed by mixing calcium chloride and sodium sulfate solutions has been studied recently, 7 culminating in the formation and growth of calcium sulfate dihydrate, or gypsum, crystals. When this reaction occurs in pastes, it produces plaster, a porous material with high internal surface area containing interlocking crystals in the form of needles and plates. 8 The morphology and size of gypsum in the hardened paste affects its compressive strength. In the plaster and building industries, 9 additives (accelerators or retardants) are used to control the hemihydrate hydration rate in order to influence the morphology and size scales of the end material. Splints as well as plasters used for medical applications need rapid buildup with high final compressive strength, and hence require accelerators. 10 On the other hand, scaling inside seawater desalination plants, pipes, boilers, heat exchangers, cooling water systems and secondary oil recovery systems is caused by the precipitation and growth of inorganic salts, 5 and should be suppressed. Addition of organic, inorganic or polymeric additives such as polycarboxylates and polyphosphonates 11,12,13 are often used to reduce scaling.
Classical nucleation theory predicts that the earliest particles that form have the crystal structure of the end product. On the other hand, a two-step model 14 first allows for the formation of disordered clusters which are less constrained than crystalline phases. This is followed by reorganization to ordered crystalline domains. Another study 15 using electron microscopy has shown that the transformation of amorphous calcium phosphate spheres to crystalline spherulites occurs via a multi step surface nucleation process. Studies on model colloidal systems [16][17][18] have also indicated deviation from classical nucleation theory. Thus it is important to distinguish between classical nucleation models and the applicable crystallization mechanisms for specific systems. 19 From a practical perspective, an understanding of the precipitation/crystallization processes is essential so that additives can be rationally designed. This insight can be obtained by examining the temporal evolution of the crystal habit. Light microscopy, scanning electron microscopy and atomic force microscopy have been deployed for this purpose. 20,21 Although, their spatial resolution is often adequate, these methods typically require samples that are several microns in size. Additionally, these methods do not easily lend themselves sample 'quenching' making it difficult to capture particles in their native states at short and specific time points. Consequently, the sampled particles are usually well beyond the initial stages of transformation.
Therefore, crucial information about phase and morphology that is embedded in the very early stages of crystal growth is lost. This issue becomes even more important when the effect of additives 22-27 must be understood, as they strongly influence the very early stages of particle formation and growth. A more complete understanding of the interaction between additive molecules and initial particle formation can be greatly aided by direct probing techniques at very early time points. These interactions provide vital information about the evolution of the crystallization process in the presence of additives. This understanding will guide the choice of additives for either accelerating or retarding the crystallization process depending on the applications.
Intrinsically coupled with short time scales is the small (sub-micron) length scales for these growing particles, which requires alternate techniques for direct imaging and analysis. Conventional methods 28-32 therefore give only a late-stage picture of the influence of additives on the crystallization process. Time-resolved X-ray scattering using a strong beam source 33,34 is a viable technique for examining particle evolution at short time scales. However, this method produces data in reciprocal space, and does not produce direct real-time morphologies of particles. We

Sample Preparation for cryo-TEM
A few microliters of the solution are deposited on to a holey carbon cryo-TEM grid.
The grid is carefully blotted so that liquid bridges of the order of 100 nm in thickness are formed across the holes. The grid is then plunged into a liquid ethane reservoir cooled by liquid nitrogen. Rapid heat transfer away from the solution on the grid leads to vitrification. The wait time after filtration is managed carefully so that the sample gets vitrified at a desired time point. This entire operation is done in a controlled environment vitrification system (CEVS) maintained at 25  C and at 100% humidity to prevent water evaporation from the sample prior to vitrification.

Characterization
The grid-containing sample is transferred to a cooled tip of a Gatan 626 DH cryo transfer stage. The stage is then inserted into a JEOL JEM 2100 Transmission Electron Microscope. The sample is maintained at -175  C and low electron dosage is used for probing the samples to prevent the amorphous to crystalline phase transformation in ice as well as any potential dehydration. An Oxford Instruments Inca EDX system is used for elemental analysis. Selected area electron diffraction (SAED) is performed on regions of interest to get phase information. X-ray diffraction (XRD) patterns from our samples are recorded using a Rigaku Ultima IV diffractometer. The samples are kept in a holder and sealed using a thin plastic film to prevent drying during measurements.

Results and Discussion
To investigate gypsum crystallization, a 55mM supersaturated solution of calcium sulfate hemihydrate is vitrified at different time points. Samples vitrified approximately 10 seconds after filtration show the presence of 2nm -4nm particles, as indicated in Fig. 1. Low-dose selected area electron diffraction (SAED) for these   During the early stages, amorphous nanoparticles evolve from nanoscale amorphous clusters. 2,36,37 These nanoparticles grow. The amorphous state is favored kinetically because it is less constrained than the crystalline counterpart, but it is not one of minimum free energy. Thus, the disordered state slowly rearranges to more stable crystalline form, gypsum. A transient amorphous phase is not predicted by classical nucleation theory. In Fig. 3(c), we show an alternate multi-step mechanism for the transformation of the hemihydrate to gypsum, more closely corresponding to our observations. We have subsequently followed the evolution of morphology of these particles from 15 min to an hour and observe typical interlocking needle-like gypsum.  Optical micrographs taken at 60 minutes show no acicular structures, confirming the retardation of the crystallization process. Images taken at 24 hrs however, (Fig. 5) show acicular structures projecting from a dense internal phase similar to structures reported previously. 39

Conclusions
We

Introduction
Vesicles are often made up of single or multiple bilayers consisting of surfactants or phospholipids. 1,2 They are promising delivery vehicles for drugs and active ingredients 3-6 . Because of their similarity to biological membranes, vesicles are often used as model systems to probe the impact of biological processes on their stability and structure. In cosmetics, vesicles not only deliver encapsulated ingredients like perfume, but also counter skin dryness as the surfactants are hydrated. 7,8 Another important area that extensively uses vesicles is fabric softeners. Cationic surfactants are the chief ingredients of these vesicles. [9][10][11] During the late stage of laundry cycle, these cationic surfactants are adsorbed on negatively charged fabric which results in reduction of friction due to exposure of surfactant tails. The inability to form hydrogen bonds also aids reduction of static charge that helps in ironing clothes. Esterquats such as diethy ester dimethyl ammonium chloride (DEEDMAC) are double chained cationic surfactants used mainly as ingredients of these vesicles.
Because of ester linkages, these surfactants are readily biodegradable by hydrolysis post washing cycle. 12 Phase diagrams and thermal properties of common double-tailed charged surfactants such as didodecyl dimethyl ammonium bromide 13,14 (DDAB), dioctadecyl dimethyl ammonium bromide 15,16 (DODAB) and dioctadecyl dimethyl ammonium chloride 17,18 (DODAC) in water has shown that in concentration range of 0.15−30 wt % in water and above the main phase transition temperature of the bilayer, such cationic surfactants self assemble to form unilamellar and multilamellar vesicles.
In suspensions, these vesicles are believed to exist as a kinetically stabilized metastable phase rather than a thermodynamic equilibrium phase. [19][20][21] Fragrance is an integral part of these consumer good products and extended duration of smell is an important product requirement as it determines acceptability by consumers. Perfume raw materials (PRMs) are added to vesicular suspensions which are the key constituent of many consumer products 22  is employed to understand specific interactions that lead to these transitions. to prevent water evaporation from the sample prior to vitrification.

Cryo-TEM characterization
The grid-containing sample is transferred to a cooled tip of a Gatan 626 DH cryo transfer stage. The stage is then inserted into a JEOL JEM 2100 Transmission Electron Microscope. The sample is maintained at -175  C and low electron dosage is used for probing the samples to prevent the amorphous to crystalline phase transformation in ice as well as any potential dehydration.  Addition of 2 wt% linalyl acetate (LA) to UDF shows no structural changes in the vesicles. 10 seconds after mixing LA with UDF we see multi lamellar structures as shown in Fig. 2(a). 24 hrs after mixing we see that the multilamellar structures are preserved as shown in Fig. 2(b). Morphology and number of lamellae in these vesicles indicate that if LA is incorporated into the bilayer this does not cause any alteration of the inherent vesicular structures. When 2wt% eugenol is added to UDF we observe changes in vesicular structures.

Results and Discussion
Immediately after mixing multilamellar vesicles are observed as shown in Fig. 3(a). At 6hrs we start observing instabilities in bilayers. These are indicated by arrows in Fig.   3(b). These instabilities propagate to later stages as shown in Fig. 3(c). The external lamellae break off from the vesicles and bilayer fragments are observed as indicated by arrows at 12hrs after mixing. The inner vesicles follow similar trend. 24hrs after mixing we observe the presence of predominantly unilamellar vesicles, tubules and bilayer fragments as marked in Fig. 4(a). To confirm morphology of these structures the TEM stage is tilted by an angle of 20° into the plane of the micrograph.   is somewhat surface active, then if it gets into the bilayer during mixing, it can interact with surfactant tails of DEEDMAC resulting in bilayer breaking off due to changes in packing parameter. As exposure of surfactant tails to water is energetically unfavorable, these bilayers curl to form unilamellar vesicles. 2,24 Eugenol has an aromatic ring and a terminal hydroxyl group, which makes it more surface active that LA. In Fig. 4(c), the process of transition from multilamellar vesicles to unilamellar vesicles in UDF is illustrated in the presence of eugenol. LA on the other hand being less surface active could prefer to remain in the bilayer without any specific association owing to its hydrophobicity or can be phase separated after mixing if it doesnot manage to get into the bilayers.
During last rinsing step of washing cycle using a fabric softener, repartitioning of perfume from vesicles towards the aqueous phase is not desirable as a PRM might get away when the rinse water is pumped away. 7 If the PRM on other hand stays with surfactant, when deposited on the textile it can result in extended duration of odor which is highly desirable in these products. Hence, it is very important to understand if a PRM interacts with DEEDMAC in bilayers of UDF. For this, we employ NMR. Addition of 2 wt% or 4 wt% UDF to a 2 wt% eugenol suspension in D 2 O containing 1200 PPM calcium chloride exhibit peak broadening at chemical shifts 3.75 ppm (methoxide protons, 5.9 ppm (vinyl proton) and 6.75 ppm (aromatic protons) as shown in NMR spectra in Fig. 5. These measurements are done 3 hrs after mixing. This can be explained as association of eugenol with DEEDMAC in vesicle bilayers. Spectra are also obtained at 24 hrs after mixing for the sample where 2 wt% UDF is added to 2 wt% eugenol suspension in D 2 O. Fig. 6 shows that for chemical shifts 5.9 ppm, 6.75 ppm which correspond to vinyl and aromatic protons respectively, the integral values of peaks in question increase with time. The integrations of NMR peaks are the relative areas under the curve measured against a calibrated internal standard corresponding to a residual H 2 O peak at 4.79 ppm (calibrated to 100). Over time an Figure 6. NMR spectra tracking specific protons during the addition of 2 wt% UDF in eugenol, showing increase in integral at 24 hrs as indicated by green colored bars. This indicates that some eugenol could have been released to the aqueous environment during bilayer breaking off process.
increase in integral indicate that some eugenol leaves the bilayers and are released into water during the bilayer breaking off process as observed in TEM micrographs and proposed in our model. Fig. 7 shows NMR spectra of 2 wt% LA in D 2 O containing 1200 PPM calcium chloride when exposed to 4 wt% UDF over a period of 24 hrs.

Conclusions
We demonstrate that addition of perfume raw materials (PRM) to a vesicular structures is key to developing stable, aesthetically appealing products desired by consumers.