Determination and Characterization of Water, Oil and Surfacant Induced Variations in Isotropic Domains of Plurololeique CC 497 / Labrasol System

Although microemulsions have been widely investigated and applied in various industries, existing raw materials limit applications of microemulsions in pharmaceutics and cosmetics. This work attempts to clarify the formation of isotropic systems when Labrasol/Plurololeique CC 497 combinations were used as the surfactant/cosurfactant combinations known as Km== Oil, 114, 2/3 , 111, 3/2, 4/1, 1/0. The Km== 1/1and3/2 combinations provided widest isotropic areas that included maximum of 66.67% w/w of ' mineral oil, 48.73% w/w of soybean oil and 48.82% w/w of water for Km== 1/1 , whereas for Km == 3/2 provided maximum of 54.54 %w/w of mineral oil, 28.85% w/w of soybean oil and 54.55% w/w of water. Therefore Km== l/l(HLB 10) and 3/2 (HLB 10.8) combinations were further studied to determine their response to variables such as effect of oil polarity, effect of hydrocarbon versus triglycerides, temperature and ionic strength and pH of the aqueous phase. The hydrocarbon (mineral oil) provided a wide isotropic area compared to a triglyceride (soybean oil). The effect of the mineral oil was investigated by adding a Cl8 fatty acid having no double bond ( oleic acid), two double bonds (linoleic acid), and three unsaturated chains (linoleni.._ acid). They have not produced a significant difference in the composition of isotropic areas. However at 10% concentration, they all reduced the isotropic domains significantly. Linolenic acid, which is the most polar one, is being the most effective.


Definition and Distinctions Between Macroemulsions and Microemulsions
The term microemulsion defines spontaneously formed stable disperse systems in which the average particle size of the internal droplets range from 10-200 nrn. 1 As a result, a microemulsion appears as a transparent single -phase liquid. 2 Microemulsions usually consist of three to five components: an oil phase, an aqueous phase, and a primary surfactant. ln many cases, they contain a secondary surfactant ( cosurfactant), and sometimes one or more electrolytes. These isotropic systems are usually more difficult to formulate than ordinary emulsions because their formation is a highly specific process that involves spontaneous interactions of the constituents. The type of the association structures that can form with these components at a particular temperature, depends not only on the chemical nature of each component but also on their relative concentrations. 3 While macroemulsions are ordinarily opaque or white in color, the microemulsions are transparent or translucent ~opalescent) in a state which reflects their much smaller particle size (generally less than 1/4 the wavelength of light). 4 This is one of the properties that attracted the attention of researchers from various applied sciences.
Recent studies have emphasized the stability and ease of formation of microemulsions rather than their transparency. Macroemulsions, by contrast, are generally thermodynamically unstable. Moreover macroemulsion manufacturing generally requires an application of a form of external energy, while microemulsions tend to form spontaneously.
Because they are isotropic and thermodynamically stable systems with large interfacial areas, microemulsions may be superior to macroemulsions for solubilization of poorly soluble drugs. 5 • 6 • 7 • 8 Thus, microemulsions may be considered as viable alternatives to \ macroemulsions for both cosmetic and pharmaceutical applications.

The Role of Surfactant and Cosurfactant
Surfactants are the chemicals which contain both hydrophilic and hydrophobic groups.
The hydrophobic group is not miscible with water molecules and when a small concentration of surfactant is added to water, the hydrophobic end will immediately rise to the surface in order to reduce its free surface energy. 3 This structure is called a monolayer. If the concentration of surfactant is increased and there is not enough room for all of the hydrophobic ends to escape from the water, a bilayer may form. However, if the concentration of surfactant is large enough, the bilayer may form which will not be stable. At this point, the excess surfactant molecules form micelles. In a micelle, a group of hydrophilic heads shield their hydrophobic tails from the water. Therefore, the micelle that form in the water consists of a hydrophilic shell and a hydrophobic core. 2 The concentration of the surfactant at which the micelle formed is called the critical t . (CMC) 9. 10.11 micelle concentra 1011 . Surfactant films at oil and water interfaces determine many properties of the microemulsions such as the phases, particle size and stability. The hydrophilic surfactant films tend to curve towards oil and form o/w emulsions because they will interact with water molecules more than oil molecules. The surfactants, which can reduce the interfacial tension excessively and interact with oil and water molecules spontaneously may form microemulsions. The spontaneous curvature (Ho) of the interfacial film \ depends on the hydrophilic nature of the surfactant. Spontaneous curvature (Ho) is the most important parameter to understand the structure and stability of the microemulsions which depend on the nature of the surfactant, oil, and water phases.
As shown in Fig. 1, the oil molecules penetrate to some extent between the hydrocarbon tails of the surfactant molecules. The more extensive the penetration, the deeper the curvature is toward the water side causing a reduction in H 0 . When the curvature is toward the oil , Ho is positive, when it is towards water it is negative. 12 When the spontaneous curvature of the surfactant film, Ho, is greater than zero, Ho > 0, o/w is expected, and if Ho < 0, w/o can be formed. A stable microemulsion can be formed when the spontaneous curvature (Ho) of the film is close to the inverse radius (K 1 ) of the droplet. If the spontaneous curvature is much higher than the inverse radius (Ho >> K 1 ) , the surfactant film tends to move toward a lower free energy state by decreasing with modification) 4 the droplet size and expelling the emulsified oil (or water for negative Ho) to the bulk phase. When Ho<< K 1 , the film forms larger nonspherical aggregates. Finally when Ho _ o, there is no preference for a o/w and w/o structure; therefore a bicontinuous phase can be formed. 12 In formation of microemulsions, spontaneous adsorption is an important factor. Not every surfactant can be spontaneously located at the interface and reduce the interfacial tension near or below zero values. In order to do that, the surfactant should have comparable oil and water affinities.
For example, n-alkyl polyoxyethylene ethers are able to form microemulsions by themselves. They are single-chained surfactants. Some of the double chain surfactants such as di-2-ethyl-hexyJ-sulphosuccinate (AOT) and di-octadecylarnmonium bromide (DDAB) can form microemulsions due to their favorable geometry and their ability to reduce interfacial tension. However, theories related to microemulsification are complicated. Some authors like Shinoda et al. 13 suggested the role of the critical packing parameter (CPP) and related geometry of the surfactant in forming microemulsions. Cosurfactants, due to their small molecular weight and surface activity, can easily mix with the surfactant, the water. and the oil phases. They reduce the interfacial tension fmther and adjust the viscosity of the interfacial film. In the presence of the coswfactant, the interfacial film becomes more fluid and hence can easily and spontaneously curve to form very small droplets 11 . In the presence of the cosurfactant, the HLB of the interfacial film is also changed. This is so because many authors reported the formation 6 of the microemulsion at an optimum surfactant and cosurfactant combination having an HLB which is able to solubilize both oil and water phases.

18
An optimum hydrophilic-lipophilic balance (HLB) 13  Every isotropic domain fow1d in oil-surfactant/cosurfactant and water mixtures is not a microemulsion. They m< 1y he 1.rue solution of surfactar1t and/or cosurfactan.t, mice!les in oil or in the aqueous phases, microernulsions, or liquid crystals. While changing tht surfactant/cosurfactant ratio, the HLB of the surfactant mixture is also changed and this change in HLB mfluences the structure and distribution of the oil-surfactant-water phases. 7 Winsor system was used to classify the structural changes that took place in the surfactant-oil-water mixtmes. Phase transitions involved is shown in Fig. 2. When increasing amounts of a lipophilic surfactant is added to a cosurfactant dissolved in equal amounts of oil/water mixture, a range of self assembly structures form due to changes occur in the HLB mixture. These were named as phase I, 11, and III by Winsor, Fig.2

Determination of Microemulsions by Three Phase Diagrams
A microemulsion is assumed to be the mixture of four components; water phase, oil phase, and surfactant/cosurfactant mixture. A w/o microemulsion is normally easier to produce than an o/w microemulsion. Ro Samo et al.32 suggested a simple three-step procedure for preparing an o/w microemulsion, using titration method. First. a primary surfactant that is only slightly soluble in the oil phase is dissolved in the oil phase to yield a w/o microemulsion. This mixture is added to the water phase and stirred then titrated with a cosurfactant that is more soluble in water than a surfactant to produce a clear o/w microemulsion.

'
Like macroemulsions, microemulsions may be of o/w or w/o type. The cubical bicontinuous phase is also accepted as a microemulsion. Their mean particle size and particle size distribution may differ, depending on the nature and the quantity of their components.
Some of the physical measurements that are useful in characterizing microemulsions are conductivity, light scattering, optical birefringence, ultra-centrifugation, rheology, electron microscopy, nuclear magnetic resonance, and photon correlation spectroscopy. 33 , 34 -37

Particle size analysis
The transparency of microemulsions arises from their small droplet size. Therefore it is very important to carry out particle size analysis since any variation in droplet size may 13 indicate changes in the stability of microemulsions 38 and solubilization properties of the . 1 39 part1c es.
Many techniques have been employed in the size analysis of microemulsions such as direct electron microscope imaging, light scattering method, small angle neutron scattering, and photon correlation spectroscopy. Because of the limitations of each technique, it is preferable to employ a combination of these techniques for any 1 .
. l . 30 37 and static and dynamic light scattering. 43 They all have a lower size limit of approximately 2 nm and an upper limit of approximately 1 OOnm for SANS and SA.XS and a few microns for light scattering. In general terms, all light scattering instruments contain the same components: a light source (usually a laser), a spectrometer (containing the optical components for defining the scattering angle volume), a detector (usually a photomultiplier), a signal analyzer such as a spectrum analyzer or a correlator, and a computer software for analysis are standard for dynamic measurements. 44 Gorski et al. 38 have determined the particle size of an o/w microemulsion system as around 30-100° A by using SANS and static light scattering. The microemulsion system was consisted of tetradecyltrimethylammoniwn bromide (TTABr) as a surfactant, 1hexanol as a cosurfactant, decane as an oil phase, and water.

l dynamic light scattering (DLS)
The dynamic light scattering (DLS) was used to measure the changes occmred in the particle size of the emulsions studied in this thesis. This method accurately measures the particle size and allows evaluation of the interactions that happen between different structures that may be present in the microemulsion systems. 31 15 Colloidal particles immersed in fluid and in the solution form are able to scatter a beam of the light. The scattering pattern depends on the particle sizes and on the wavelength of the light. Lord Rayleigh was introduced the equation for the scattered intensity, I, where the particles are much smaller than the wavelength of the light: where Io is the initial intensitty of unpolarized light, 0 is the angle between the incident beam and scattered beam, Np is the isolated particles per unit volume, Vis the particle ' volume, NI , No are the refractive indices of particles and medium respectively. R is the distance where the intensity I is measured from the particles and ~ is the wavelength of visible light.
In this equation, the term 1 and Cos 2 0 refer to the vertical and horizontally polarized component of the scattered light. 40 The use of lasers has ::t great advantage to study the light scattering by providing coherent, monochromatic, intense and narrow in ident beams, coupled with sensitive stable photondetection apparatus and rapid data analysis by computer. 40 The temporal variation of the intensity is measured and is represented usually through the intensity auto-correlation function. The diffusion coefficients of the particle, particle sizes, and size distribution can be deduced from such measurements. The variation of the intensity with time, contains information on the random motion of the 16 particles and can be used to measure the diffusion coefficient of the particles. The measured diffusion coefficient can then be used to determine the size of the particle. 40 • 45 • 46 Sjoblom and Friberg 41 have investigated phase transitions in the microemulsion systems containing water, pentanol , and potassium oleate by varying water content but with constant alcohol/soap ratios. They found that at low water concentrations no rise of light scattering was observed when Rayleigh radii (R) was plotted against volume fraction.
Electron microscopy of carbon replicas from freeze-fractured samples used in association with particle size measurements demonstrated the presence of molecular dispersions and absence of inverse micelles at low water content. At higher water concentrations, a slight change from a negative to positive slope of the curve was observed at approximately 15 %(w/w) for all series and the first association occured when the water molecules per srnfactant exceeded eight and the inversed micelles were formed in increasing water content of the mixture. Bond et al. 45 have investigated the structural transitions in w/o microemulsions in the three-phase diagram by following the particle size changes with light scattering. They concluded that the system had low conductivity within w/o region. In the upper region of w/o which was closer to surfactant corner, the surfactant was in excess relative to the amount of aqueous phase to be solubilized, rendering the systems to hydrated surfactant aggregates. n-butanol, and dodecane systems, the conductivity jumped from 1 Q-6 to 1 Q 4 Q-I 1 .

Electrical conductivity
Baker et al 46 investigated structural changes occmring in the system by following the conductivity-viscosity changes and particle size measurements. These explained the nature of the phase changes took place in sodium-alkyl-benzene-sulphonate-hexanol-water combination.
Lagues and Sautery 48 reported that conductivity jumps changes from 10·

1.5.l Effect of the oil phase
The need for the oil to associate with the interfacial film of the microemulsion means that the size of the oil molecules is important in determining whether a microemulsion is formed. As a rule, greater solubilization is achieved with oils with smaller molecular weight than the surfactants. 49 If the molecular weight of the oil is very large, no microemulsions form. 11 The penetration ability of oils to the o/w interface is important.

'
The hydrocarbons with shorter chain-lengths e.g. C 6 can penetrate into the C 12 surfactant chains more readily than longer alkyl chains containing 12 hydrocarbons whereas the C 14 and C 16 chains do not penetrate to the interface at all. Thus the microemulsion cannot be formed. 40 The higher the penetration ability of the oil , the more curvature of the oil-water interface occurs and this phenomena can decrease the water droplet sizes of w/o microemulsions. For examp le they range from about 4 nm for hexane to about 10 nm for dodecane. 4 J Published data o=. 1 the microemulsion technology emphasized the use of non-polar carbons such as decane 49 , heptane, hexadecane, and octadecane. 14 Studies that have been carried out with more polar triglycerides are rare. 14 • 31 Alander and Warenheim 50 have compared triglyceride behavior in the microemulsions with ordinary hydrocarbons such as mineral oil, decane, and heptane. They concluded that triglycerides, especially large triglycerides such as peanut oil, are considerably more difficult to be soluhi.lized · into microemulsions than the hydrocarbons.

19
The vegetable oils are triglycerides that are easily metabolized in the body therefore they can be used in the parenteral products. Recently by removing the active impurities such as polar components and oxidative impurities, those oils that were further purified were referred to as "superre:fined oils". Such oils are known to improve the chemical stability of the drugs incorporated in them. Six polar oils were examined including soybean oil, Miglyol 812, ethyl oleate, oleic acid, ethyl octanoate, and octanoic acid with increasing polarities in the given order. They found that the extent and composition of various solubilization regions were dependent largely on surfactant/cosurfactant ratios and the nature of the oil being incorporated. The larger isotropic domains occurred when the small oils were used. Overall, although the differences in phase behavior were observed, with different oils these differences were small, except when using the large triglycerides as oils. It appears that the size, rather than the polarity of oils investigated in this study is the main factor influencing phase behavior.
In In the pharmaceutical or cosmetic microemulsions, the aqueous phase is usually the core that contains the drug matter or the active substances. The drug may be neutral in nature, an electrolyte, a weak acid or base. All of these have the tendency to modify the ionic character of water phase. Among the surfactants that can form microemulsions the ionic ones are found to be more sensitive to the presence of electrolytes than the nonionic ones 59 . An electrolyte can penetrate into the head group of surfactants causing dehydration and shrinking of the size of the head group thus causing instability. 60 Compared to ionic surfactants, the nonionic surfactants are less prone to the changes caused by the changing pH, or to the presences of electrolytes than the ionic surfactants, ' therefore they are preferred in the pharmaceutical and cosmetic delivery systems.
Kahlweit et. al. 61 indicated that the inorganic electrolytes can be divided into two groups, the first is the lyotropic salts such as NaCl and CaCh which decrease the mutual solubility between water and surfactant. The second group comprised of the hydrotropic salts such as NaC10 4 and (C 6 H 5 ) 4 PrJ 'Nhich increase t11e mutual solubility. When NaCl was introduced to H 2 0-C 12 E 6 and H 2 0-C 12 E~ systems, the surfactant became more lipophilic.'.
In contrast, the hydrotropic NaCJ0 4 increased the hydrophilicity of the surfactant.
The effect of electrolyte concentration on the solubilization capacity in nonionic microemulsions was investigated by Wiencek and Qutubuddin. 62 The oil studied were the normal C 6 -C 16 alkanes. Neodol 91-2.5 (Shell) and DNP-8 (Emery) were used as the nonionic surfactants. The aqueous phase consisted ofNaOH and KSCN solutions of varying concentrations. It was four1d that depending up0n the type of electrolyte used, the 22 solubilization capacity of the nonionic microemulsions was increased or decreased.
Generally, typical electrolytes such as NaCl and CaCli decrease the solubilization capacity in increasing concentrations . However KSCN showed the opposite behavior.
Gan-Zuo et al. 63  In pharmaceutical and cosmetic emulsions, besides the surfactant and cosurfactant type and properties, the pH of the aqueous phase is also highly important. Garcia-Celma et al. 5 have studied solubilization of antifungal agents using a microemulsion system containing a nonionic surfactant (POE (20) sorbitan monooleate), water and isopropyl palmitate, used as the oil pliase. The s~Jected drugs w~r:c cl0tri!;w.zole, ciclopirox olamine and econazole nitrate ranging fro.rr1 sligbtly water soluble to practicaily insoluble. 23 Data obtained clearly showed that the model drugs could be successfully solubilized but the pH of the aqueous phase may effect the properties of microemulsion system and it may also influence the solubilization of weakly acidic or basic drug in the microemulsion.
De Vos et al. 64 investigated solubilization and stability of indomethacin, a weak acid, in a model transparent oil-water gel (TOW gel) as a function of pH of the water phase. The model TOW gel was composed of Cetiol""HE (polyoxyethylene (7) glyceryl monococoate) 15% w/w, an Eumulgin·· BJ (polyoxyethylene (30) cetostearyl ether) 15% w/w, isopropyl palmitate 5% w/w,and demineralized water ad 100% w/w. Indomethacin, being a weakly acidic drug is poorly soluble in the acidic environments. Although its solubility was increased in the alkaline medium, high pH hydrolyzed the drug. Solubilizing indomethacin in the TOW gels increased the stability of the drug to 300 days at pH 5.6 and 7.8. If increasing temperature causes a phase inversion of a o/w to w/o emulsion or causes a microemulsion to reveri to a rnacroemulsion within na1rnw temperature ranges, the related system is not suitable for use as a drug delivery system.  Jayakrisnan et al. 66 have formulated a microemulsion with Brij 3 5 and Arlacel 186, hexadecane oil, and isopropanol used as the cosurfactant. The solubility of hydrocortizone in the microemulsions was comparable to that in isopropanol alone.

Application of Microemulsions
However it was foimd to be six fold higher in the microemulsions containing six gram of the total surfactant, 10 ml of oil, and 3 .5 ml of alcohol at a water-to-oil ratio 0.10.
To improve the in-vitro dissolution of indomethacin, a self-microemulsifying drug delivery system (SMEDDS) has been formulated by Farah et al. 70  They are suitable products for cleansing, treating hair. eyelashes or eyebrows. The patented products assigned to L'Oreal consist of nonionic surfactants or a mixture of nonionic and cationic surfactants. The oil is described to be consisted of an amphiphilic lipid phase whose weight ratio oil-to-lipid changes from 2 to 10. The oil droplets have an average size of less than 150 nm. The nonionic an1phiphilic lipid are from the silicone surfactants and rolyolesters such as esters of polyethyleneglycol, sorbitan, ethoxylated glycero l or polyglycerols.
For example, avocado oil and ethanol are mixed with polyethylene-glycol-isosterate and \ N-stearoyl-1-glutamate disodium salt then added into the water-glycerin mixture and the system was homogenized. The result gave o/w nanoemulsion whose droplet size was approximately 63 nm. 73 Another example is an antiperspiraI1t microemuLion in the spray form which was reported to contain 0 5-30% of an oiJ phase, O. l-30%of an antiperspirant sa.l!, 0.35-30% of a nonionic hydrophilic emulsifying agent (especially an alkyl polyglucoside) and 0-7.5% of a lipophilic emulsifying agent. The dispersed-oil phase in the finished composition !tad a particle size ranging from! 0 to 100 nm.

Other fields
Microemulsion formulations are used in emulsified oils such as self-polishing products, floor waxes, aad car washes, 1111~ci1culdti ng catting oils to reduce the temperature rise 29 and provide smooth operation in the milling machines. Cutting oils consisted of a mineral oil for lubrication, diethyleneglycol as the cosurfactant, a soap (petroleum sulphonate) as a corrosion inhibitor, and water as the coolant. Pine-oil microemulsions are used as wetting and dispersing agents in the detergents. They are also used in flavour emulsions which are stabilized by a nonionic surfactant. Pesticide emulsions containing chlordane, emulsion polymers used as surface polishes also available in the market.
There have been an considerable interest in using vegetable oil microemulsions as replacements for diesel fuels, however the oil alone results in fuel injection coking and lubricant contamination clue to its high viscosity. Finally there is an increasing interest in the use of micellar systems for modifying and controlling chemical reaction systems. In polymerization processes, microemulsion have been used to encapsulate offensive products. Similar uses are reported in the acid-base equilibria, the hydrolysis of esters, the formation of metalloporphyrins, photochemical and electrochemical systems. 40

.o OBJECTIVES
Working with polyglycolysed glycerides (Labrasol) and polyglyceryl oleate (Plurololeique CC 497), as the surfactant/cosurfactant mixtures, water, mineral oil and soybean oils, the objective of this study was to determine the isotropic regions on the three phase diagram and investigate the effects of temperature, pH, ionjc strength and type of ions on the isotropic phase boundaries.
Such combinations may provide multiple uses in cosmetic and especially in pharmaceutical technology since they are easy to manufacture, remain stable during the shelf-life and maintain their original characteristics during administration.
The specific objectives of this study were as follows: a. To determine an optimum ratio of Polyglycolysed glycerides (Labrasol) to Polyglyceryl oleate (Plurololeique CC 497) that provides the largest isotropic domain with mineral oil, soybean USP, purified soybean oils, and water. Hence Labrasol is used as a surfactant and Plurololeique CC 497 is used as cosurfactant.
b. To find out the effects of oil type (mineral oil, soybean USP and purified soybean oils) on these microemulsion domains.

31
T o mechanistically determine the effect of unsaturation of the C 18 fatty acids on the c. microemulsion regions. This will be carried out by adding oleic, linoleic and linolenic acids to mineral oil and investigating their effect on the microemulsion domains.
d. To find out the effects of ion type, ionic strength and pH on soybean and mineral oil microemulsions by adjusting the ionic strength of the aqueous phase to 0.06 and 1 ionic strengths with NaCl and CaCli. (The pH effect will be studied by adjusting the pH of the aqueous phase to 1.5 with hydrochloric acid buffer (USP), to pH 5.5 with distilled water and to pH 7.4 with phosphate buffer, USP).
e. To investigate the effect of temperature on the soybean and mineral oil microemulsions by exposing the systems studied to 20, 25, 32 and 37 °C temperatures. 32

Materials
The materials used in this study are listed in Table 3.1.1. The properties of surfactants, oils and fatty acids are given in more detail in 3 .1.1, :_;. l .2 and 3 .1. 3.  It is a powerful water so luble solubilizer for the pharmaceutical and cosmetic preparations.
It can also be usee as an enhancer in oral liquid and capsule formulations. It shows no oral irritation, but has a slight irritating effect on the ocular membrane.
It is a useful surfactant for microemulsion formulations. Its critical micelle concentration (CMC) is 0.3% (w/w) . lt is used in combination with a hydrophobic co-emulsifier to b ·1· l . 1 .  It is a high visco£ity emuisifier ai~d used as a major coestirn~nt of the microemul sions that can be administered orally or topically. It is also a good suspending and stiffening agent.
It can improve the dissolution of most drugs and vitamins which can be filled into capsules. It is a GRAS substance and therefore can be safely used in the pharmaceutical and cosmetic applications. It has been found to be the best co-emulsifier with Labrasol to . l . Light mineral oil is a colorless. clear and odorless liquid, insoluble in alcohol or water.
Mineral oils are mainly used in topical drug and cosmetic preparations. They are excellent moisturizers and emollients as well as suitable lipophilic bases in which active ingredients can be delivered. Formulas for baby oils, creams aad lotions, bath oils, lipsticks and lip 35 gloss, sunscreen and hair products contain Mineral Oil, USP or Light Mineral Oil NF, in the concentrations ranging from less than 1 % to approximately 99%. 55 It is likely that in most people, acute chronic exposure to mineral hydrocarbons either orally or topically may produce carcinogenesis. Because of the toxicity of mineral oil, vegetable oils been receiving increased attention. 78

soybean and superpurified soybean oils
Soybean oil is obtained from soybeans by solvent extraction using petroleum hydrocarbons or, to a lesser extent, by expression using continuous screw press . 79 operat10ns .
In addition to the difficulty of emulsification, the triglycerides can also become rancid due to the presence of unsatmated fatty acids. Consequently, antioxidants usually have to be added to 1he soybean oil. Superpurified soybean oil contains no polar impurities and hence enhances the stability of dosage forms . It is colorless, odorless and tasteless and is \ commonly used in parenteral formulations . CH2(CH 2 ) 6 COOH. 81 The purities of oleic, linoleic and linolenic acids used in this study were -99%, greater than 99% and-99% respectively. 82

Instruments
The instruments used in this thesis study are listed in Table 3 Solutions obtatned were kept in a constant temperature water bath.  . . ___ _________ L ____ · _L _________ ~ 38 The water phase was also kept at 20°C in a water bath and was added to the oil/surfactant mixture with a syringe, until a transparent phase was obtained. Water added was weighed and recorded after each addition and percent of each constituent in the mixture was calculated. The mixture was replaced in the water bath after thorough mixing and observed visually for the turbidity development. If it did not turn turbid, it was accepted as an isotropic phase. Only those mixtures which formed clear solutions were considered to be acceptable isotropic systems. Those were further analyzed by polarized microscopy to determine whether they contained any liquid crytal phases. Each titration was carried out three times.

\
The pH, ionic strength and temperature effects were studied by using surfactant/cosurfactant ratio of Km = 3/2 and 111 , which provided the largest isotropic areas on three phase diagran1s. The pH effect was studied by adjusting the pH of the aqueous phase to 1.5 with hydrochloric acid buffer (USP), to 5.5 with distilled water and to 7.4 with phosphate buffer (USP). The ionic strengt..hs were adjusted to 0.06 and 1 µ with NaCl and CaCb. The temperature effect was investigated at 20, 25 , 32 and 37°C.
The effects of fatty acids were studied by adding adding 1 % and l 0% of oleic, linoleic and linolenic acids to light mineral oil respectively and investigating their effect on the microemulsion domains at 25 °C. The temperature effect was investigated at Km = 3/2, which provided the widest isotropic areas on three phase diagrams. The effect of oil type (hydrocarbon versus. triglycerides) was also studied by using surfactant/cosurfactant ratio of Km= 3/2 at 25°C. The oils used were mineral oil, soybean oil, and superpurified soybean oil. 39

.3.2 Characterization of Isotropic Domains
The main purpose of this study was to construct isotropic domains using light mineral oil and soybean oils. A few light scattering and conductivity results conducted, provided some information about the isotropic phases obtained at different compositions.

light scattering
The measurements of scattered light intensity were made on a light scattering instrument, Model BI-9000 AT (Brookhaven Instruments, Holtsville, NY). The scattering intensities at 90° were recorded by a photomultiplier. Dust particles were removed from the solutions by passing it through a 0.45 ~tm Millex-HV filter. Microemulsion samples were prepared in test tubes (Fisher Scientific, Pittsburgh, PA), which were then tightly sealed by plastic caps and placed in a water bath at 20°C before the measurements. Three replicates were measured for the particle size determination. The particle sizes were directly obtained from the instrument's output.

Effect of Surfactant and Cosurfactant ratios
The .~  (points g,h,i in Fig. 4.1.2), the system is likely to be a w/o emulsion. The low conductivity values obtained (3.12-5.98*10-7 Q-1 ) strengthen this fact. In this region the particles measured are around 80-100 nanometers. These points correspond to point g to i in fig. 4.1.1.D indicating that at the oil/smfactG.Ilt corner, the system is in the form of w/o emulsion.
The second isotropic phase obtained in the middle is also investigated by measuring the conductivity and particle sizes at the smfactant/oil/water combinations of j, k, l in Fig.   4.1.1.D. Along this region the particle size increases from 143 to 180 nanometers. At the ' same time, the conductivity readings increase ten times from 8.76*10-6 to 1.55*10-5 n-1 , which may indicate particle aggregation. This region is likely to be an o/w microemulsion. When water content increases above 48.82% (w/w) water the system turns turbid and forms an o/w emulsions. As shown in Fig. 4.1.1.d, the largest isotropic phase occurs at Km of 3/2 (HLB 10.8), where the system is able to solubilize the maximum amount of water (54.55%).
The surfactant/cosurfactant combination corresponding to (Km) of 3/2 also provides the largest isotropic area. The system is enlarged in Fig. 4 .1.1.E. The changes in the particle size and conductivity of the isotropic phase occurring at at Km = 3/2 combination were also investigated along the points a, b, c, d, e, and f.   At a high Plmololeique CC 497 ratio of Km= 1/4,Fig. 4.1.4.(b), there is a very thin isotropic region that appears between 9. 75% oil to 29.24% oil concentrations. When Labrasol concentration is increased (Km= 2/3), more oil at the smfactant-oil line can be solubilized (58 .82% oil), Fig. 4.1.4.( c ). The isotropic regions obtained with soybean oil are smaller than the ones that are obtained with mineral oil. However at Km= 111 , the same pattern of phase separations similar to Fig. 4.1.1 ( d) occurred. The maximwn amount of water that this system can solubilize is 34.81 %. The particle size and 'Onductivity was s1udied along the a-f line, Fig. 4.1.6. The conductivity still remains very low (2-8*10-7 n.-1 )and the particle size remains around 182 nanometers.
This microemul'lion is likely lo be a w/o type. Both properties remain constant upon .~ increasing water content. Since the particle size remains constant up to 20% water concentration region, it is expected that the number of the water droplets in oil mcrease until the whole system reverses into a macroemulsion.

Effect of Oil Type and Polarity
Jn this section, the influence of oil polarity (soybean oil, superpurified soybean oil and mineral oil) on microemulsion formation was investigated. By using a C12E10 surfactant, Malcolmson and Lawrence 14 found that with the decreasing molecular weight of the oils, the formation of microemulsions was favored. These findings were also supported by the study of Aboofazeli et al. 19 who found that the smaller oils such as ethyl octanoate, ethyl oleate are preferential to soybean oil in the formation of a microemulsions . With the surfactant combination used in this study, the size of the oil appears to be: less sensitive than its polarity.
The superpurification of soybean oil has not affected the isotropic regions. According to the manufacturer, in the soybean oil (USP), the maximum polar impurities were 2.3-4.8% \ whereas in the superpurified soybean oil, this amount was reduced to Jess than 1 % Oleic acid has one, linoleic has two, and linolcnic acid has three double bonds. Their effect in the boundaries of the isotropic zone were studied by adding individual acids to mineral oil at 1-· 10% concentration.
At 1 % concentration, all fatty acids have the same effect on the microemulsion domains of the mineral oil, Fig. 4 whereas oleic acid has the least impact.
Incorporation of linolenic acid reduced the solubilization of the oil in the system. It appears that for C 18 fatty acids, more than the double bonds that they have in their structure, the amount incorporated in the oil phase influences microemulsion formation.
The same figure demonstrates that at 1 % concentration, the fatty acid concentration does not influence the system, whereas at 10%, they interfere with the formation of isotropic domains immensely, because they increase the polarity of mineral oil. Therefore it is concluded that the concentration of the polar groups is the main factor influencing isotropic phase formation. For both oils, when Km is 1 /1 (HLB 10), the system is extremely temperatme sensitive between 20-25°C, Fig. 4 3.1 and 4.3.2. This is the same regardless of the oil type used.
The hydrophilicities of Labrasol and Plurololeique CC 497 must be primarily influenced within this temperature range. When the temperature is increased to 25°C, the wide region of water continuous phase disappears entirely in the mineral oil systems in Fig.   4.3.1. This must be due to the dehydration ofLabrasol, knowing that when the temperature increases the hydrophilicity of the surfactant decreases. 3 • 13 Similarly, the small isotropic region occurring in the middle of the three phase diagram of soybean oil shown in Fig. 4   insensitive between 20-37°C indicating that this phase may be a micellar dispersion of the surfactant/cosurfactant in the oils studied, although there is about 7% water solubilized at this corner.
At compositions corresponding to Km = 3/2, Fig. 4 ' At Km = 3/2, the amount of Labrasol increases and therefore the hydrophilicity of the mixture increases. It can be concluded that within this particular mixture at this surfactant concentration, increasing the hydrophilicity reduces the temperature sensitivity of the surfactant mixture. Even in the 32-37°C region, water uptake is not significantly reduced (from 54.55% at 20°C to 44.44% at 37°C for mineral oil microemulsion and from 34.81 % at 20°C to 32.52% at 37°C for soybean oil microemulsion ).
The compositions given in Fig.4.3.3 appear to be highly useful for topical applications because of their long term stability. They will not revert to emulsions provided that they remain within the same oil-surfactant-water composition.

Effect of Aqueous Phase
The effect of the inorganic electrolytes on the isotropic domains was investigated by adjusting the ionic strength(µ) of the aqueous phase to 0.06 and 1 with NaCl and CaCh.
Effect of ions on the phase behavior of mineral oil and soybean oil systems was investigated at Km= 1 /1 (Fig. 4.4.1 and 4.4.2) and 3/2 ratios (Fig. 4 Kahlweit et al. 79 classified NaCl within a group that effectively caused salting out of the nonionic surfactants. However, within the region of the ionic strengths used, NaCl and CaCh are not destructive for our systems,  It is interesting to see that NaCl reduces the isotropic areas more effectively than CaCh, . This can be explained as the result of the smaller size of sodium which readily dehydrates the polar heads of the surfactant than that of CaCh. 60 As the ionic strength of the solution was decreased by the addition of NaCl and CaCh, the effective polar pai1 of the surfactant shrinks and becomes less bulky than the hydrophobic part.

'
The HLB moves towards lower regions and as the result, the o/w structures are not favored 13.18.61,62. The pH effect was studied at Km = 3/2 surfactai1t combination, for both mineral oil and soybean oil systems. For the mineral oil microemulsions. the pH does not appear to be effective at 3/2 surfactant combination between pH of 1. 5 to 7.4,Fig. 4.4.5. This finding offers an advantage for Labrasol-Plurololeique CC 497 systems for topical formulations.
It is even more encouraging because the buffer systems used contained different acid/salt combinations. HCl, USP was used for pH 1.5, ai1d phosphate buffer was used for pH 7.4.
Their effects were compared against that of distilled water. The excess W ions do not influence the original isotropic nature of the mineral oil systems. Soybean microemuision, shown in Fig. 4.4.6 aiso demonstrates the same pH stability. 73 This property may be specifically useful for small-volwne intradermal or intramuscular injection dosage forms . ..

SUMMARY AND CONCLUSIONS
Although microemulsions have been widely investigated and applied in various industries, existing surfactant systems available for microemulsification have limited their applications in the pharmaceutic~ and cosmetics. This work determined the boundaries of Labrasol and Plurololeique CC 497 combinations in microemulsion systems that may be useful as pharmact!utical and cosmetic delivery systems. It investigated the effects of surfactant/cosurfactant ratios, mineral oil and soybean oils, fatty acids, pH, electrolytes (NaCl and CaCh), and temperature on the solubilization of ' oil and water phases.
The system provides the largest isotropic regions with mineral oil and soybean oil when a surfactant/cosurfactant co;n bination of Km= 312 (HLB of 10.8) is used. The Km of 1/1 (HLB =l 0) is aJs0 found to be a useful combination. At HLB 10.8, the temperature sensitivity decreases and the critical temperature moves from 20-25°C to 32-37°C. Therefore, this surfactant/cosurfactant ratio gives an optimum HLB to be used in the microemulsion formulation. At this combination, the isotropic regions become less sensitive to the presence of NaCl and CaCh up to ionic strength of 1 (µ=l). The microemulsions prepared tolerate pH changes of the aqueous phase from 1.5 to 7.4.
Mineral oil system provides a wider selection of oil-surfactant-water combination that can be used in microemulsion formulations than soybean oils systems. The latter is limited to solubi!ization of 28l;lo oi l and 34% water. All the systems are free-flowing in nature and su itable for injection. 77 We also demonstrated that the presence of polar fatty acids (oleic acid, linoleic acid, and linolenic acid) in a nonpolar oil (mineral oil in a low concentration (1 %)) has not signigficantly affected the boundaries of the isotropic domains. Phase changes occur when their concentrations are increased to 10%, which shrinks the isotropic zones. This effect may be related to the concentration and number of double bonds present in the fatty acid system. There is a trend showing that increasing double bonds in the fatty acids reduces the microemulsion domains.