JOURNAL OF COSMETIC SCIENCE 246 Using ultrasonic emulsifi cation to prepare nanoemulsions is a recent development phenomenon (8). Ultrasonic emulsifi cation was able to produce nanostructured drop- lets in emulsion with the advantage of less occurrence of “over processing” (9). For- mation of nanostructured droplets are controlled by the interaction between droplet disruption and droplet coalescence, the ultrasound applied excellent shear force to droplet breakup, and the rate of droplet coalescence is determined by the mixed sur- factant concentration (10). In the desired droplet size, ultrasonic emulsifi cation can reduce the surfactant concentration and energy consumption, and the emulsions were more stable compared with homogenizer or other mechanical devices (8). There are two main mechanisms during ultrasonic emulsifi cation (11). First, an acoustic fi eld produces interfacial waves to break the disperse phase into the continuous phase. Sec- ond, the formation of acoustic cavitation is used to collapse microbubbles into drop- lets of nanometric size by pressure fl uctuations. The best nanoemulsion droplets in emulsion were prepared at optimum hydrophilic– lipophilic balance (HLB) value and optimum surfactant level (12). The proper HLB values of the surfactants are important parameters for the formation of emulsion. Nanostructured droplets in emulsion are usually formulated to enhance the stability by using a mixed surfactant because of the broad chain length distribution. Paraffi n oil in water nanoemulsions have been obtained by adjusting the HLB values of the mixed surfactants Tween 80/Span 80 (2). Isohexadecane O/W nanoemulsions have been obtained in water/C12E4:C12E6/isohexadecane system at 4 and 8 wt% mixed surfactant concentration (13). Nanoemulsions containing the antioxidant astaxanthin prepared with mixed surfactant had smaller droplet size and a narrow size distribu- tion (14). The main objectives of this study were to gain a better understanding of the infl uence of mixed surfactant on the nanoemulsion droplet size by using ultrasonic emulsifi cation and also to investigate the optimum formulation for preparing D -limonene in water nano- emulsions. MATERIALS AND METHODS MATERIALS D -Limonene (RI = 1.487) was a product of Merck (Darmstadt, Germany) and used as received. Reagent grade sorbitane trioleate and polyoxyethyle ne (10) oleyl ether with an average HLB of 1.8 and 12.0 were supplied by Sigma-Aldrich (St. Louis, MO). Ethylene glycol used as a cosurfactant was obtained from Merck water was deionized and Milli-Q fi ltered. COARSE EMULSION PREPARATION Emulsions consisted of D -limonene, mixed surfactant, deionized water, and cosurfactant. All emulsions were prepared in two stages. The coarse emulsion was obtained by using Polytron (PT-MR 3000, kinematica AG, Littau, Switzerland), and then further emulsifi ed

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.