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Optimization of Nano-emulsion Preparation by Low-Energy Methods

in an Ionic Surfactant System

Isabel Sole,, Alicia Maestro, Carmen Gonzalez, Conxita Solans, andJose M. Gutierrez*,

Departament dEnginyeria Qumica, UniVersitat de Barcelona, 08028 Barcelona, Spain, and Departamentde Tecnologia de Tensioactius, Instituto de InVestigaciones Qumicas y Ambientales de Barcelona, CSIC,

Barcelona, Spain

ReceiVed May 15, 2006. In Final Form: June 21, 2006

The low-energy emulsification method Emulsion Inversion Point (EIP) was used to prepare O/W nano-emulsionsin the W/potassium oleate-oleic acid-C12E10/hexadecane ionic system. This method had not practically been usedin ionic systems up to now. The resulting droplet sizes, much smaller than those obtained with the high-energyemulsification methods, depend on the composition (formulation variables) and preparation variables (addition andmixing rate). Phase diagrams, rheology measurements, and experimental designs applied to nano-emulsion dropletsizes obtained were combined to study the formation of these nano-emulsions. To obtain small droplet sizes, it isnecessary to cross a direct cubic liquid crystal phase along the emulsification path, and it is also crucial to remainin this phase long enough to incorporate all of the oil into the liquid crystal. When nano-emulsion forms, the oil isalready intimately mixed with all of the components, and it only has to be redistributed. Results show that the smallerdroplet sizes are obtained when the liquid crystal zone is wide and extends to high water content, because in this case,during theemulsification process, thesystem remains long enough in theliquid crystal phase to allow theincorporationof all of the oil. Around the optimal formulation variables, the liquid crystal zone crossed during emulsification iswide enough to incorporate all of the oil whatever mixing or stirring rate is used, and then the resulting droplet sizeis independent of preparation variables. However, when the composition is far from this optimum, the liquid crystalzone becomes narrower and the mixing of components controls the nano-emulsion formation. High agitation ratesand/or low addition rates are required to ensure the dissolution of all of the oil into this phase.

1. Introduction

Nano-emulsions, also known as miniemulsions,1-5 ultrafineemulsions,6,7 emulsoids,8-10 unstable microemulsions,6,11 andsubmicrometer emulsions,12,13 area type of emulsion with dropletsof very small diameters, typically in the range 20-500 nm.Because of this small droplet size, they may appear transparentor translucent. Contrary to microemulsions, nano-emulsions arethermodynamically unstable systems; however, they may have

a long kinetic stability. All of these characteristicproperties haveled to an increased use of nano-emulsions in many differentapplications related to chemical, pharmaceutical, and cosmeticfields.2,4

For nano-emulsion nonequilibrium systems, external energyis required for their formation. There exist two main methodsfor thepreparation of nano-emulsions: dispersion or high-energymethods, and condensation or low-energy methods.14 Thedispersion or high-energy methods involve an energy input thatis achieved by high-shear stirring, high-pressure homogenizers,or ultrasound generators.5 However, to obtain small droplet-sized nano-emulsions, a great amount of mechanical energy is

needed, making this preparation route unfavorable for industrialapplications. The condensation or low-energy methods makeuse of the phase transitions that take place during the emulsifica-tion process as a result of a change in the spontaneous curvatureof thesurfactant. This changeof curvaturecan be achieved throughtwodifferent routes: the composition can be kept constant whilethe temperatureis changed(Phase Inversion Temperaturemethod,PIT),15 or temperature is maintained constant and compositionis changed (Emulsion Inversion Point method, EIP).16

The preparation of nano-emulsions stabilized with nonionicsurfactants has been widely reported, as much by means ofdispersion methods17,18 as by condensation methods.19-21 In thelow-energy methods and for oil-in-water nano-emulsions (O/

* Corresponding author. E-mail: [email protected]. Universitat de Barcelona. Instituto de Investigaciones Qumicas y Ambientales de Barcelona.(1) Ugelstad, J.; El-Aasser, M. S.; Vanderhoff, J. W. J. Polym. Sci., Polym.

Lett. Ed. 1973, 11, 503-513.(2) El-Aasser, M. S.; Lack, C. D.; Choi, Y. T.; Min, T. I.; Vanderhoff, J. W.;

Fowkes, F. M. Colloids Surf. 1984, 12, 79-97.(3) El-Aasser, M. S.; Lack, C. D.; Vanderhoff, J. W.; Fowkes, F. M.Colloids

Surf. 1988, 29, 103-118.(4) El-Aasser, M. S.; Miller, C. M. InPolymeric Dispersions: Principles and

Applications; Asua, J. M., Ed.; Kluwer Academic Publishers: Dordrecht, TheNetherlands, 1997; pp 109-126.

(5) Sudol, E. D.; El-Aasser, M. S. InEmulsion Polymerization and EmulsionPolymers; Lovell, P. A., El-Aasser, M. S., Eds.; John Wiley & Sons Ltd.:Chichester, U.K., 1997; pp 700-722.

(6) Nakajima, H. In Industrial Applications of Microemulsions; Solans, C.,Kunieda, H., Eds.; Marcel Dekker: New York, 1997; Vol. 66, pp 175-197.

(7) Nakajima, H.; Tomomasa, S.; Okabe, M. Premier Congres Mondial DelEA mulsion; Paris, France, 1993, Paper no. 1-11-162.

(8) Lachampt, F.; Vila, R. M. Am. Perfum. Cosmet. 1967, 82, 29-36.(9) Lachampt, F.; Vila, R. M. Parfums, Cosmet., SaVons1967,10, 372-382.(10) Lachampt, F.; Vila, R. M. Parfums,Cosmet.,SaVons 1969, 12, 239-251.(11) Rosano, H. L.; Lan, T.; Weiss, A.; Whittam, J. H.; Gerbacia, W. E. F.

Phys. Chem. 1981, 85, 468-473.(12) Benita, S. In Submicron Emulsions in Drug Targeting and Deli Very;

Florence,A. T., Gregoriadis, G., Eds.;HarwoodAcademicPublishers: Amsterdam,1998; Vol. 9.

(13) Benita, S.; Levy, M. J. Pharm. Sci. 1993, 82, 1069-1079.

(14) Solans,C.; Esquena, J.; Forgiarini,A. M.; Uson, N.; Morales, D. Izquierdo,P.; Azemar, N.; Garca-Celma, M. J. In Surfactants in Solution: Fundamentalsand Applications; Mittal, K. L., Shah, D. O., Eds.; Surfactant Science Series;Marcel Dekker: New York, 2002.

(15) Marszall,L. InNonionicSurfactants; Shick, M. J.,Ed.; SurfactantScienceSeries; Marcel Dekker: New York, 1987; Vol. 23, pp 493-547.

(16) Shinoda, K.; Saito, H. J. Colloid Interface Sci. 1968, 26, 70.(17) Walstra, P. In Encyclopedia of Emulsion Technology; Becher, P., Ed.;

Marcel Dekker: New York, 1983; Vol. 1, p 57.(18) Tomamasa, S.; Kochi, M.; Nakajima, H. J. Jpn. Oil Chem. 1988, 37,

1012.(19) Morales, D.; Guti errez, J. M.; Garc a-Celma, M. J.; Solans, C. Langmuir

2003, 19, 7196-7200(20) Izquierdo, P.;Esquena, J.;Tadros,T.; Dederen,C.; Garca,M. J.;Azemar,

N.; Solans, C. Langmuir2002, 18, 26-30.(21) Uson, N.; Garcia, M. J.;Solans,C. ColloidsSurf., A 2004, 250, 415-421.

8326 Langmuir2006, 22, 8326-8332

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W), the change of curvature necessary to obtain nano-emulsionsis achieved by changing the hydration degree of the polyoxy-ethylene chains of polyoxyethylene-type nonionic surfactants,changing the temperature (PIT method) or the water content(EIP method). Inverse nano-emulsions (W/O) have also beenobtained through the EIP method.21 In all cases, it has beenfound that small droplet-sized nano-emulsions can be obtainedthrough condensation methods in nonionic systems if during theemulsification process and near the zone where nano-emulsions

are formed, all of the oil (for O/W nano-emulsions19,20

) or allof the water (for W/O nano-emulsions21) is dissolved in a singlephase, which can be a bicontinuous microemulsion (D) or alamellar liquid crystal (LR).19,20 When, for example, an excessof water is added to a lamellar liquid crystal, the increase of thehydration degree of the polyoxyethylene chains increases thespontaneous curvature, and, as a result, a flat structure is nolonger stable and it is disrupted into small droplets. In theequilibrium, all of the oil cannot be contained inside these smalldroplets, and more droplets cannot form because their numberis limited forthe amountof surfactant available. Next, theexcessof oil should be separated into another phase. However, smalldroplets with a certain kinetic stability can be obtained if the EIPmethod is used. In the lamellar liquid crystal, all of the oil is

already closely incorporated into the system. When an excess ofwater is added, theliquid crystal is disrupted. Next, small dropletsof a diameter quite similar to the thickness of the hydrophobiclayer are formed and encapsulate the oil, which is thenincorporated into the nano-emulsion. Of course, these dropletsare slightly bigger than those corresponding to the equilibrium,and, as a result, their surface is under tension because theircurvature is a bitlower than the spontaneous one, and the systemwill tend to the separation of phases. However, the kinetic ofdestabilization can be very slow, and they can, in many cases,be considered as pseudo-stable. A similardiscussion can be madewhen the PIT method is used.

The preparation of nano-emulsions stabilized with ionicsurfactants by condensation methods has not practically been

studied. In this case, the PIT method cannot be used to changethecurvature of thesurfactant, as the behaviorof ionic surfactantsis not affected by temperature. Referring to the EIP method,nano-emulsions have been obtained by Taylor et al.22,23 in ionicsurfactant systems with an alcohol as a cosurfactant by dilutionofan O/W microemulsion with an excess ofwater.In this method,during the dilution process, the alcohol suffers a process ofdiffusion from the microemulsion droplets into water, whichcauses a slight change in the surfactant curvature and a deficitof surfactant in the interface, and so the system is thermody-namically unstableand nano-emulsions are obtained. In thisway,nano-emulsions of 10-20 nm of diameter have been formed inthe ionic system water-SDS/hexanol or pentanol-hidrocar-bure,22,23 but only very diluted nano-emulsions (around 98% of

water) present a relative stability.Another EIPlow-energymethod forobtaining nano-emulsions

in ionic systems was presented in a recent work,24 wherepreliminary results werepublished.In thisnew method, thechangein surfactant curvature is achieved by progressive neutralizationof a fatty acid with an alkaline aqueous solution. The resultingfatty carboxylate acts as the ionic surfactant stabilizing thenano-emulsion formed. However, nano-emulsions are not obtained ifthe fatty carboxylate is the unique surfactant used, and it needsto be combined with a nonionic surfactant. Only in this case did

the adequate phases to form nano-emulsions cross through theemulsification path. In contrast to the works published withnonionic surfactants, in this case a single phase of flat structure(Dor LR) is not present along the emulsification path and nearthe nano-emulsion zone, but a cubic liquid crystal with all of theoil dissolved appears instead. Nano-emulsions with droplet sizearound 20 nm can be obtained. The ionic system studied wasW/potassiumoleate-oleicacid-C12E10/hexadecane. Potassiumhydroxidesolutions were added to themixtures formedby water,

hexadecane, C12E10, and oleic acid, with this last componentpartially neutralized into potassium oleate during the emulsifica-tionprocess. We recently reported thepreliminary results referringto the formation of O/W nano-emulsions in this ionic system;however, in that work only oneoleic acid/C12E10 ratiowas studied.

In the present Article, the formation of nano-emulsions in theionicsystem W/potassium oleate-oleicacid-C12E10/hexadecanehas been studied in a wider range of composition variables.Moreover, a deeper study of the mechanism of formation ofthese nano-emulsions is presented, which tries to elucidate thetransition from cubic liquid crystal to nano-emulsions. Theequilibrium phases along the different emulsification paths forseveral final compositions have been determined to establish therelationship between the droplet sizes of the nano-emulsions

formed with the nature of the phases present during theemulsification process. Furthermore, in this work the influenceof formulation or compositionvariables and preparationvariables(mixing and stirring rate) has been studied, and they have beenoptimized through an experimental design.

Experimental design is a usefultool that allowsthe evaluationof the effect of different variables collectively, in contrast to theconventional and classical method of one variable at a time, inwhich onlyone variableis studiedwhile theothers aremaintainedconstant and the combined effect of all of the variables is nottaken into account.25-27

2. Experimental Section

2.1. Materials. Oleic acid (extra pure), polyoxyethylene lauryl

ether (98% purity) with 10 mol of ethylene oxide per surfactantmolecule (C12E10), and potassium hydroxide were obtained fromSigma-Aldrich. Hexadecane (99% purity) waspurchased fromMerck.Water was deionized and further purified by Milli-Q filtration.

2.2. Determinationof Equilibrium Phases. All components wereweighed, sealed in ampules, and homogenized with a vibromixer.Thesamples were kept in a thermostaticbath at 25 C to equilibrate.The phases were identified under polarizedlight, visually, and usingsmall-angle X-ray scattering (SAXS) and polarizing microscopy.Rheology was used as a complementary technique.

2.3.Small-Angle X-rayScattering (SAXS). SAXS measurementswereperformedon small-angle scattering equipment(Rigaku Nano-Viewer, Rigaku Co.) equipped with a CCD detector at an appliedvoltage and filament current of 40 kV and 20 mA, respectively.

2.4. Rheological Measurements.All rheological measurementswere performed in an ARES7 rheometer (Rheometric Scientific)using a cone-plate geometry. Dynamic frequency sweep measure-ments were performed in the linear viscoelastic regime, which waspreviously determined by dynamic strain sweep measurements.

2.5. Nano-emulsion Formation. Nano-emulsions were preparedby adding potassium hydroxide solutions to hexadecane-oleicacid-C12E10 mixtures.The addition wascarried outunder controlled mixingand addition rates. The final concentration of water was 80% w/win all cases.

(22) Taylor, P.; Ottewill, R. H. Colloids Surf., A 1994, 88, 303-316.(23) Taylor, P.; Ottewill, R. H.Prog. Colloid Polym. Sci.1994,97, 199-203.(24) Sol e, I.; Maestr o, A.; Pey, C. M.; Gonzalez, C.; Sol ans, C.; Gutierrez, J.

M. Colloids Surf., A, in press.

(25) Box,G. E.P.; Hunter, J. S.Estadsticapara InVestigadores. Introduccional diseno de experimentos, analisis de datos y construccion de modelos; Reverte,1999.

(26) Montgomery, D. C. Design and Analysis of Experiments; Wiley: NewYork, 2001.

(27) Prat, A.; Tort-Martorell, X.; Grima, P.; Pozueta, L.Metodos Estadsticos.Control y mejora de la calidad; UPC: Barcelona, 1997.

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2.6. DropletSize, Polydispersity, and Stability Determinations.Nano-emulsion droplet size and polydispersity were determined byphoton correlation spectroscopy (PCS) using a Malvern ZetasizerZS instrument at 25 C. All of the samples were diluted with waterbefore the measurement. Stability was assessed by measuring thedecrease in transmission using TURBISOFT equipment MA2000.

2.7. Effect of Selected Variables on the Nano-emulsionProperties. The experimentaldesign used in this study has been theresponse surface methodology (RSM),24 which consists of a groupof mathematical and statistical techniques that allows one to model

a responseof interest,suchas nano-emulsion droplet size, asa functionof selected variables. In a second step, experimental design has alsobeen used to optimize the formulation variables.

Response surface methodology (RSM) was applied for modelingemulsion droplet size and polydispersity as a function of selectedvariables. Three different central composite designs (CCD), withtwovariables, werecarriedout to generate 11 treatmentcombinations.In two of them, oil/surfactants and oleic acid/C12E10 ratios werechosen as independent variables, and studied at different ranges,while thepreparationconditions were hold constant;and in the thirdone,mixingand addition rates were selected as independentvariablesat a constant composition. Five levels of each variable were studiedin each design.

Response surfaces are represented by a second-order polynomialregression model, eq 1, to generate contour plots:

whereYdenotes a nano-emulsion property, such as droplet size orpolydispersity, andx1andx2are the independent variables studied.Statgraphics software was used to obtain the combination of valuesthat draw the surface response.

3. Results and Discussion

To study the emulsion formation in the ionic system W/potas-sium oleate-oleic acid-C12E10/hexadecane, the equilibriumphases of the different emulsification paths used have beenidentified. While in a preliminary previous work24 only one oleicacid/C12E10 ratio was studied, just to indicatethat nano-emulsionscould be formed in this systemusing low-energy methods, in the

present Article a deeper and more systematic study is presented,and the phases found for four oleic acid/C12E10ratios between20/80 and 50/50 are shown.

Two variables of the system were fixed. First, the final waterconcentration of the nano-emulsions prepared was fixed at 80%,as it is a water concentration that allows one to have a relativelyhigh percentage of dispersed phase with a reasonable stabilityof the droplets. Second, through preliminary assays, theneutralization grade of the oleic acid at the final point (80%water) wasfixed to thatcorresponding to a stoichiometricrelationbetween the oleic acid and the KOH.

The study was carried out in a hexadecane/[oleic acid/C12E10]ratio range (oil/surfactant, O/S) of 30/70 to 60/40. Ratios lowerthan 30/70 would require a too high amount of surfactant to form

the nano-emulsions, and ratios higher than 60/40 do not lead tothe formation of nano-emulsions. Above this ratio, a whiteemulsion that quickly separates into two phases is obtained.

3.1. Equilibrium Phases. Theequilibriumphases in therangeof O/S ratios between 30/70 and 60/40 at four oleic acid/C12E10ratios of 20/80, 30/70, 40/60, and 50/50 are shown in Figure 1.In all of the cases, a W/O microemulsion (Om) is present in thefurthest zone from the water corner. By increasing the waterconcentration, this phase first coexists with a lamellar liquidcrystalline phase, and, subsequently, a region of pure lamellarliquid crystal (LR) is observed, identified by SAXS [Figure 2]and polarizing optical microscopy [Figure 3]. The characteristicdistancedof this phase increases with water content, becausewater places itself in thehydrophilicsheet and increases itswidth.

All three regions extend to higher water concentrations andhigher O/S ratios with the increase of the oleic acid/C12E10 ratio.In this way, while in the lowest oleic acid/C 12E10ratio studieda multiphasic region is observed, at the highest O/Sratios studied,it becomes narrower anddisappears, by increasing theoleic acid/C12E10 ratio. Moreover, the characteristic distance d of LRincreases with both oleic acid/C12E10 and O/S ratios.

At water contents further from the lamellar phase zone, thereis a region in which this lamellar phase coexists with a cubicliquid crystal of Pm3n structure, and, subsequently, the pure

cubic crystal is obtained, with a clear tendency of the cubiccrystal characteristicdistance dwith water content not observed.The SAXS pattern is shown in Figure 2. In the biphasic zone thediffraction corresponding to the lamellar liquid crystal stillappears, but itsintensity decreases with the water concentration,indicating that the amount of LR diminishes. However, newdiffractions appear in this range with ratios corresponding to aPm3n cubic crystal. The biphasic LR+Pm3n zone becomesnarrower with the increase of the oleic acid/C12E10ratio, untildisappearing at a ratio of 50/50. The cubic phase region alsobecomes narrower with the increase of the oleic acid/C12E10ratio, but extends to higher O/S ratios. The characteristic distancedof this cubic crystal increases with both oleic acid/C12E10andO/S ratios.

Y) b0 + b1x1 + b2x2 + b11x12 + b22x2

2 + b12x1x2 + (1)

Figure 1. Equilibrium phases determination of the system water/oleic acid-potassium oleate-C12E10/hexadecane at different oleic

acid/C12E10ratios (25 C). The concentration of the alkaline waterysolutions in the vertexes corresponds to a stoichiometric relationbetween the oleic acid and the KOH at the final point (80% water).

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Finally, in the nearest zone from the water corner a two-phaseregion in which an O/W microemulsion (Wm) coexists with anoil phase (O) is present. It is the zone where nano-emulsionswereformed. The cubicliquid crystalline phasehas beenidentifiedas the determinant phase to obtain small droplet-sized nano-emulsions, as will be shown below in the text. Because of this,this phase was thoroughly investigated, and further experimentswere carriedout bothby SAXS and by rheological measurements.

First,its microstructurewasstudied as a function of temperatureusing SAXS. Figure 4 shows that at 25 C a peak ratio of 1:1/

2:2/5 was obtained, confirming the existence of Pm3ncubic liquid crystal. A change of the characteristic distance dwas not observed when the temperature increased, indicatingthatdis not appreciably affected by the decrease in hydration

of thenonionicsurfactant. A phase transition wasdetectedvisuallyat35 C, observed through theloss of theextremelyhigh viscosity,transparency, and optical isotropy that the system presented atlower temperatures. This corresponds to the appearance of alamellar phase (LR) in equilibrium with Pm3n. The systembecomes a homogeneous lamellar liquid crystal (LR) at 50 C.At this temperature and higher ones, the samples are opticallyanisotropic and not so much transparent, obtaining diffractionpatterns with a peak ratio of 1:1/2, which corresponds to thepresence of a lamellar liquid crystal. As it happened with cubiccrystal, dof LR was constantin the range of temperaturesstudied,indicating that, like with Pm3n, the change of hydration gradeof the nonionic surfactant does not affect the microstructuralparameters of the lamellar liquid crystal.

Figure 2. SAXS patterns of samples with an oil/surfactant ratio of30/70 and an oleic acid/C12E10 ratio of 30/70 at different waterconcentrations.

Figure 3. Polarizing optical photomicrograph of a sample in thelamellar liquid crystalline phase LR exhibiting the typical textureof a lamellar liquid crystal: focal-conicunits and extendedstructures.Composition: oleic acid/C12E10 ) 30/70; O/S ) 40/60; 35% water.Temperature) 25 C.

Figure 4. Change of SAXS patterns with temperature for a cubicliquid crystalline sample. Composition: oleic acid/C12E10 ) 30/70;O/S ) 40/60; 55% water.


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In a secondstep, theeffectof thetemperature on theviscoelasticbehavior of the cubic crystal was studied [Figure 5]. The changeof tendency of rheological parameters also reflects the phasetransition from cubicliquid crystal to lamellar liquid crystal. The

loss modulus G

seems to remain constant and the storagemodulus G slightly decreases, with the subsequent smoothincrease of the diphase angle delta, within the homogeneousdirect cubic phase region. In the biphasic region, G and Gdecrease with temperature and delta progressively raises,indicating that the proportion of LRincreases, and, finally, theloss and storage moduli increase in parallel with the resultingconstant value for the delta parameter when the sample becomesa homogeneous lamellar phase.

3.2. Formation of Nano-emulsions. Nano-emulsions wereprepared according to the low-energy emulsification methoddescribedabove. Thismethod permitsone to obtainsmall droplet-sized nano-emulsions without the need of high-energy inputs.The droplet size obtained through this method was compared to

the droplet size obtained with two high-energy emulsificationmethods, in which the energy input was achieved by applyingmechanical shear produced by high-shear stirring (ultra-turrax)and by an ultrasound generator.

All of the components were weighted and subjected to high-shear stirring or the ultrasound generator for 15 min and 2 heach. The results obtained are shown in Figure 6 and comparedto those obtained through the addition method. After 15 min ofmechanical energy input, both using the ultra-turrax and theultrasoundgenerator,the droplet sizes obtained were muchbiggerthan that obtained by the addition method. If the exposure timeto ultrasound is increased to 2 h, droplet size is reduced to 78nm, but it is still far from the 27 nm obtained by the additionmethod. Longer ultrasound exposition times would probably

reduce the droplet size, but they would imply a too costlyemulsification. Referring to the use of the ultra-turrax, only aslight decrease in the droplet size is observed if the exposuretime is increased to 2 h. Moreover, another problem is added inthis case, which is the high amount of foam formed during theprocess, which led to a very difficult handling. So, the low-energy method seems to be the best alternative, from the pointof view of size of nano-emulsions required,handling, andenergyconsumption.

Nano-emulsions were then formed by the addition method, inwhich potassium hydroxide aqueous solutions were added toO/Smixtures. If water withoutKOH is added to theO/Ssolutions,and potassiumoleate is therefore not formed, emulsionsobtainedquickly separate into two phases. In the phase diagram without

KOH, one can observe that, while with KOH the pure cubicliquid crystal zone extends to high water content values (around

70% or more), in this case the pure liquid crystal zone is muchnarrower and separate oil appears around 40-45% water, farfrom the nano-emulsion formation zone (diagram not shown).This fact corroborates the idea that small nano-emulsions areobtained throughthe addition methodif,duringthe emulsificationpath and near the nano-emulsion zone, a zone of liquid crystalor D appears with all of the oil dissolved.

So, KOH solutions were used for emulsification. Theconcentrationof these potassiumhydroxidesolutions was differentdepending on the emulsification path chosen to have stoichio-metric quantities of oleic acid and KOH at the final point wherenano-emulsions were formed (80% water content). Because ofthe fact that each path has a different amount of oleic acid, adifferent amount of potassium hydroxide is needed. The ionic

surfactant potassium oleate was progressively formed along thedifferent emulsification paths by neutralization of the oleic acidwith KOH.

Experimental designs were used to optimize nano-emulsioncharacteristics as a function of composition and preparationvariables in the system W/potassium oleate-oleic acid-C12E10/hexadecane.

3.2.1. Study and Optimization of Formulation Variables.

Central compositedesigns werecarried out to study the influenceof theformulationvariables O/S ratio andoleicacid/C12E10 ratio,to optimize the droplet size of the O/W nano-emulsions formedin thesystem W/potassium oleate-oleicacid-C12E10/hexadecaneas a functionof these variables. Central composite design permitsone to obtain a quadratic equation for the droplet diameter:

The preparation conditionswere maintainedconstant at a stirringrate of 750 rpm and addition rate of 1.6 mL/min.

Two central composite designs were done at different rangesof the variables studied. First, a wide range of the variables wasstudied [Table 1]. Theresults ofthisstudyshowthatthe quadraticequation that approximates experimental response as a functionof O/S ratio and oleic acid/C12E10 ratio in terms of the significantvariables is as follows:

Figure 5. Dynamic temperature ramp test (strain 1%, ) 10 s-1)for a cubicliquid crystalline sample. Oleic acid/C12E10 ) 30/70;O/S) 40/60; 55% water. Figure 6. Droplet diametersof nano-emulsions obtained for different

emulsification methods. US ) ultrasonic emulsification; UT )ultraturrax emulsification. Composition: oleic acid/C12E10 ) 30/70; O/S ) 40/60; 80% water. Temperature ) 25 C.

droplet diameter (nm) ) a + b(O/S) +

c(oleic acid/C12E10) + d(O/S)(oleic acid/C12E10) +

e(O/S)2

+ f(oleic acid/C12E10)2


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wherea ) 289.6 ( 82.6; b ) 150.3 ( 33.3; c ) -1255.2(259.4; f ) 1004.1 ( 204.0, with the terms (O/S)(oleic acid/C12E10) and (O/S)2 having no significant influence on dropletsize. Figure 7a shows the response surface developed by the

model. It can be seen from this graphic that there is an optimaloleic acid/C12E10ratio, which minimizes the droplet size. Thisoptimum, around oleic acid/C12E10 0.65, approximatelycorresponds to the composition where the cubic liquid crystalextends to higher water content (see Figure 1, oleic acid/C12E10) 40/60). The optimal value for the oleic acid/C12E10ratio does

not depend on the O/S ratio. Onthe other hand, a linear influenceon droplet size is observed for the O/S ratio, as the term (O/S) 2

is not present in the equation. In this case, the smaller is the O/Sratio, the smaller is the droplet size obtained, due to the fact thatthe presence of more surfactant allows the stabilization of moresurface and, as a result, thepresenceof smaller droplets.Moreover,when O/S decreases, the liquid crystal zone extends to higherwater content values, favoring the formation of small dropletsin the zone of nano-emulsions.

In a second step, a central composite design in which the

range of O/S ratio andoleic acid/C12E10 ratio wasreduced [Table2], maintaining the same preparation variables (750 rpm; 1.6mL/min), was carried out. In this case, the experiments wererepeated to obtain a more accurate description.The equation thatdescribes the droplet size is as follows:

where a ) -15.7 ( 5.6; b ) 297.6 ( 78.7; c ) -375.2 ( 225.2;d) -364.4 ( 124.0; f ) 581.3 ( 151.7, in which the term(O/S)2 continues to not have significance but the crossed term(O/S)(oleic acid/C12E10) is now significant. This fact seems to

indicate that in the wider range the points were too separated tonotice the influence of this term, which becomes evident whena smaller range is studied in more detail. The response surfacedeveloped is represented in Figure 7b. It can be seen from thisgraphic that thereis also an optimal oleic acid/C12E10 ratio, whichminimizes the droplet size, but, in this case, the response surfaceis accurate enough to observe that the minimum goes to higheroleic acid/C12E10ratios as the O/S ratio increases, as the crossedterm in equation indicates. This fact seems logical, as, when theoleic acid/C12E10increases, the zone of pure cubic liquid crystalnecessary for the proper formation of nano-emulsions movestoward higher O/S ratios (see Figure 1).

Referring to the O/S ratio, a linear influence is also observedin this case, but the tendency depends on the oleic acid/C 12E10

Table 1. Experimental Field for a Design Matrix: FormulationVariables and Nano-emulsion Properties Measureda

runoleic acid/

C12E10 O/Sdroplet

size (nm) polydisp.

1 0.89 1.34 154 0.272 0.63 1.50 144 0.213 0.63 0.96 32 0.164 0.25 0.96 167 0.115 0.63 0.96 34 0.236 0.63 0.96 32 0.17

7 0.36 0.59 20 0.718 1.00 0.96 246 0.449 0.63 0.43 15 0.27

10 0.89 0.59 23 0.3211 0.36 1.34 162 0.13

a Agitation rate ) 750 rpm; addition rate ) 1.6 mL/min.

droplet diameter (nm) )

a + b(O/S) + c(oleic acid/C12E10) + f(oleic acid/C12E10)2

Figure7. Response surfaces for two different ranges of thevariablesstudied (wider one (a) and narrower one (b)): Droplet diameter asa functionof composition variables. Stirring rate ) 750 rpm;additionrate ) 1.6 mL/min.

Table 2. Experimental Field for a Design Matrix: FormulationVariables and Nano-emulsion Properties Measureda

run block oleic acid/

C12E10 O/Sdroplet

size (nm) polydisp.

1 1 0.47 1.20 100 0.202 0.63 0.96 38 0.113 0.84 0.96 88 0.204 0.41 0.96 50 0.145 0.47 0.73 36 0.156 0.78 0.73 43 0.26

7 0.63 1.30 72 0.178 0.63 0.63 34 0.199 0.63 0.96 39 0.12

10 0.63 0.96 39 0.0911 0.78 1.20 57 0.1712 2 0.47 1.20 105 0.2013 0.63 0.96 38 0.1014 0.84 0.96 90 0.2215 0.41 0.96 54 0.1416 0.47 0.73 37 0.1317 0.78 0.73 43 0.2418 0.63 1.30 65 0.1619 0.63 0.63 32 0.1420 0.63 0.96 38 0.1321 0.63 0.96 38 0.1122 0.78 1.20 55 0.16

a Agitation rate ) 750 rpm; addition rate ) 1.6 mL/min. Blocks 1and 2 are the originaland the repeated experiments,respectively, as theywere replicated.

droplet diameter (nm) ) a + b(O/S) +

c(oleic acid/C12E10) + d(O/S)(oleic acid/C12E10) +

f(oleic acid/C12E10)2


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ratio, due again to the crossed term. At a low oleic acid/C 12E10ratio, the smaller is the O/S ratio, the smaller is the diameter. Asthe oleic acid/C12E10ratio increases, the decrease in droplet sizewith the decrease in O/S ratio is not so sharp, until at the highestoleic acid/C12E10ratio studied the droplet size decreases whentheO/S ratio is increased. These results canbe explainedthroughthe equilibrium phases observed along emulsification paths

[Figure 1]. Atlow oleic acid/C12E10 ratios, the cubic liquid crystalextends to higher water content when O/S decreases. However,if the fourth diagram in Figure 1 is observed (oleic acid/C12E10)50/50), it does not happen; on the contrary, at the lower O/Sratio tested, the cubic liquid crystal zone disappears and, as aresult, the droplets obtained are bigger.

3.2.2.Study and Optimization of PreparationVariables.Centralcomposite design was also used to optimize the preparationvariables mixing rateand stirring rateof the O/Wnano-emulsionsformed in the system W/potassium oleate-oleic acid-C12E10/hexadecane at a constant composition.

First, a composition was selected. If the optimal compositionischosen, that is, thecomposition that gives theminimumdropletsize, there is no variation in the droplet size when the addition

and/or the stirring rate are changed. This fact can be explainedbecause, for the proper formation of nano-emulsions, enoughtime is required to incorporate all of the oil into the liquid crystalandto achieve theequilibriumin this zone duringthe emulsifica-tion time. It would imply low addition rates and/or high stirringrates. However, around thecompositionwhere optimumis found,the cubic phase zone crossed along the emulsification path iswide enough to allow the incorporation of all of the oil into thiszone during the addition time whatever addition rate or stirringrate is used, so a small droplet size is always obtained. However,when a composition was chosen where the cubic phase regioncrossed alongthe emulsificationprocess is narrow, the preparationvariables affected the droplet size obtained because they weredecisive to achievetheequilibrium in thisphase.The composition

chosen to carry out the central composite design was acid oleic/C12E10 ) 0.6 and O/S ) 1.3 [Table 3]. Experimental designpermits oneto obtaina quadraticequation forthe droplet diameter:

However, taking into account only the significant parameters at95% of confidence level, the equation is reduced to:

wherea ) 137.4( 13.2;b ) 9.3 ( 1.3;c ) -0.102( 0.018.Figure 8 shows the response surface developed by the model

for mixing and addition rates. As no quadratic term appears inthe equation, droplet size has a linear dependence with bothvariables, stirring rate and mixing rate, which are independent.The smallest droplet-sized nano-emulsions are obtained at highmixing rates and low addition rates. This means that mixing iscontrolling the process along this emulsification path, and theincorporation of all of the oil in the cubic liquid crystal, due tothe extremely high viscosity of this phase, can only be achievedat the highest agitation rates and the slowest addition rates.

4. Conclusions

In the W/potassium oleate-oleic acid-C12E10/hexadecaneionic system, the low-energy addition method (EIP) was usedfor the formation of O/W nano-emulsions. Droplet sizes as lowas 17 nm can be obtained, much lower than those reached byhigh-energy emulsification methods. For this system, to obtainsmall droplet sizes, it is necessary to reach the equilibrium in thecubic liquid crystal phase crossed during the emulsificationprocess,with all of the oil incorporatedintothis phase. Extendingthis conclusion to other systems, direct or flat phases without oilin excess permit one to obtain these good nano-emulsions. Next,when a nano-emulsion forms, theoil hasonly to be redistributed.However, this condition is not enough. It is also necessary thatthe liquid crystal zone is near the zone where the nano-emulsionis formed.

Experimentaldesigns arevery usefulto quickly find theoptimalconditions for the formation of nano-emulsions. With thesemathematical tools, we can reduce the number of experiments,and, moreover, the combined effects of the different variablesare taken into account. For the system studied, experimentaldesigns indicate that the best nano-emulsions appear when the

liquid crystal zone is wide and extends to high water content.Moreover, in this system, due to the particularly high viscosityof the cubic liquid crystal, the mixing of components controlsthe nano-emulsion formation, and, as a result, smaller dropletsare obtained if high agitation rates and slow addition rates areused. However, no influence of preparation variables appearsaround the composition where the optimum appears, as at thiscomposition the cubic liquid crystal zone is wide enough toreach the equilibrium in this phase during the emulsificationtime whatever addition rate or stirring rate is used.

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Table 3. Experimental Field for a Design Matrix: PreparationVariables and Nano-emulsion Properties Measureda

runadd rate

(mL/min)mix rate

(rpm)droplet

size (nm) polydisp.

1 3.0 700 101 0.212 3.0 700 104 0.223 4.4 594 118 0.214 3.0 700 101 0.215 1.6 806 77 0.186 3.0 850 81 0.2

7 1.6 594 90 0.208 5.0 700 114 0.219 4.4 806 86 0.20

10 3.0 550 114 0.2111 1.0 700 68 0.15

a Formulation variables: oleic acid/C12E10) 0.6 and O/S ) 1.3.

droplet diameter (nm) ) a + b(addition rate) +

c(mixing rate) + d(addition rate)2

+

e(addition rate)(stirring rate) + f(mixing rate)2

Figure 8. Response surfaces: Droplet diameter as a function ofpreparation variables.

droplet diameter (nm) )

a + b(addition rate) + c(mixing rate)


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