*2.5. Experimental Design*

The objective of modeling the phase diagrams is to quantify the effect of formulation composition on the droplets size. A successful "mixture design" shows the statistical approach to obtain the relationship between the droplets size distribution and the amounts of various components. In this method, the pseudoternary phase diagrams were plotted and several points were selected within the nanoemulsion region for particle size measurement. The points (black dots) represent the nanoemulsion region in Figure 1A. Within the nanoemulsion region, a triangular area was arbitrarily selected and marked with a red triangle (Figure 1A) which shows different runs of mixture design and the enlarged version is represented in Figure 1B. Hence, Figure 1B shows the sketch diagram of Run (trial) 1 to Run 7 of mixture design. The constraint that the proportions of different components must sum to 100% should be satisfied. According to Figure 1B, the points can be chosen such as three vertexes, three halfway points between vertices and the center point. Each vertex represents a formulation containing the maximum quantity of one component, with the other two components at a minimum amount. The halfway point between the two vertices illustrates a formulation incorporating the average of the minimum and maximum quantity of the two constituents represented by the respective apex. The centre point shows a formulation containing one third of the individual component. A total of seven formulations (MM1–MM7) were opted from the nanoemulsion region for further study and their compositions are summarized in Table 1.

**Figure 1.** Pseudo ternary phase diagram showing nanoemulsion region (**A**) and distribution for each of run in a mixture design (**B**).



\* Check point batch.

### *2.6. Characterization of Moxifloxacin Nanoemulsions*

### 2.6.1. Drug Content and pH

Drug content in the prepared nanoemulsions (MM1–MM7) was determined using HPLC. The pH of formulations (MM1–MM7) were measured by a calibrated pH meter (Mettler Toledo MP-220, Greifensee, Switzerland).

### 2.6.2. Transmittance, Conductivity and Dilution Potential

The percentage of the transmittance of nanoemulsions was measured using colorimeter (Photoelectric Colorimeter 113, Systronics, Ahmedabad, India). Percentage transmission was set to zero using filter and 100% using transparent cuvette filled with water. Then, di fferent nanoemulsion samples were kept in the transparent cuvette and percentage transmission was measured. Electrical conductivity of nanoemulsions was studied using a conductometer to determine the type. Briefly, an electrode was totally immersed and fixed in the nanoemulsion (20 mL) and the temperature was raised to 1 ◦C/min steadily. The nanoemulsion was agitated with a stirrer, and the change in the conductivity was recorded. To determine dilution potential, the nanoemulsion was diluted 10 times with continuous media and the occurrence of phase separation was noted.

#### 2.6.3. Particle Size Characterization and Zeta Potential

The droplets size, size distribution and polydispersity index of nanoemulsions were analyzed employing a dynamic light scattering technique using Malvern Zetasizer (Nano ZS90, Malvern Instruments, Malvern, UK) at 25 ◦C. In brief, a few drops of respective samples were added directly to a polystyrene disposable cuvette and fixed in the direction of laser light beam. The scattered light signal was measured with a detector placed at a right angle and the droplets size were determined based on the physical properties of the scattered light such as the angular distribution, frequency shift, the polarization and the intensity of the light [14]. For the zeta potential measurement, samples were diluted with deionized water and the electrophoretic mobility values was determined at 25 ◦C using the software DTS, version 4.1 (Malvern, England, UK, 2009).
