NANOEMULSION OF D-LIMONENE IN WATER SYSTEM 247 by ultrasound process. The concentration of D -limonene was in 10 wt%, while the HLB values of mixed surfactant varied from 2 to 12. The mixed HLB values were calculated as follows: HLBmix = HLBS S% + HLBP P%, where HLBS, HLBP, and HLBmix were the HLB values of sorbitane trioleate, polyoxyethylene (10) oleyl ether, and mixed surfac- tants, and S% and P% are the mass percentages of sorbitane trioleate and polyoxyethylene (10) oleyl ether in the mixed surfactants, respectively. The HLB number of the surfactants was considered to be the algebraic average of HLB of the individual surfactant. The ratio of D -limonene to mixed surfactant was expressed in terms of So ratio. The cosurfactant concentration was fi xed in 1%. ULTRASONIC PROCESS Ultrasonic process was performed by using a 20 kHz sonicator 3000 (Misonix Inc., Farm- ingdale, NY) with a 20-mm-diameter tip horn. The tip of the horn was symmetrically placed in the coarse emulsion, and the experiment was started at various preset ultrasonic nominal powers (6–51 W) for 30–300 s controlled by the software of the device. Each experiment was triplicated. DROPLET SIZE DETERMINATION Emulsion droplet size was determined by dynamic light scattering using Nanotrac 150 (Microtrac, Inc., Montgomeryville, PA). To avoid multiple scattering effects, all emul- sion samples were diluted to 10% with deionized water before the measurement. Infor- mation about emulsion droplet size was obtained via a best fi t between light scattering theory and measured droplet size distribution. A refractive index of 1.487 was used for D -limonene. Emulsion droplet size results are an average of three measurements and are quoted as the mean diameter (MD). The MD is calculated using the volume distribution data and is weighted to the smaller droplets in the distribution. This value is related to population or counting of droplets. œ ( MD= ( i i i i V d2) V d3) TRANSMISSION ELECTRON MICROSCOPIC ANALYSIS The morphology of the D -limonene nanostructured droplets in emulsion was visualized by using the transmission electron microscope (TEM). Samples (50 μl) were added to 200-mesh formwar-coated copper TEM sample holders (EM Sciences, Hatfi eld, PA). The samples were then negatively stained with 50 μl of 1.5% (w/v) phosphotungstic acid for 10 min at room temperature. Excess liquid was blotted with a piece of Whatman fi lter paper. The TEM samples were observed with JEOL JSM-1200EX II TEM (Peabody, MA) equipped with 20 μm aperture at 67 kV.

JOURNAL OF COSMETIC SCIENCE 248 RESULTS AND DISCUSSION THE EFFECT OF ULTRASONIC APPLIED POWER In this study, nanoemulsion droplets in D -limonene in water system were prepared by two steps. The fi rst step was to prepare a coarse emulsion with droplet size around 20 μm. Then, ultrasonic process was used to further decrease the droplet size. There are two main mechanisms of emulsion droplet formation. First, droplet disruption is controlled by the type and amount of shear force applied to droplets as well as the droplet resistance to deformation, which is determined by the surfactant (9,10). The other is droplet coales- cence. The rate of droplet coalescence is related to the droplet stability of emulsion, which is determined by the ability of the mixed surfactant to rapidly adsorb onto the surface of newly formed droplets. This pathway is governed by surfactant surface activity and con- centration. In our study, ultrasonic emulsifi cation was used as the source of shear force and the results of applied power on droplet size of emulsion are included in Figure 1. The results showed that the droplet size reached a minimum size at applied power of 18 W. A similar trend between emulsion droplet size and applied power has been observed by oth- ers for emulsions made with fl axseed oil and Tween 40 (10). Increasing the applied power of ultrasound can provide more shear force to decrease the size of emulsion droplet. THE EFFECT OF ULTRASONIC TIME The nanoemulsion droplet size under different ultrasonic time are shown in Figure 2. The result showed that the droplet size were around 200 nm during ultrasonic time of 120 s, but increase in the ultrasonic time would not obviously change in emulsion droplet size. Ultrasonic time was related to the thermodynamic equilibrium of emulsion system. Ul- trasonic time affects the rate of adsorption of surfactants to the droplet surface and the droplet size distribution of newly formed droplet. Figure 1. The effect of ultrasonic applied power on the emulsion droplet size of D -limonene in water emul- sion system (So ratio= 0.4 ultrasonic time= 120 s).