*2.5. Statistical Analysis*

The linear relations between the results of the microscopic measurements, laser diffraction particle size measurements, and other emulsion stability tests were investigated with bivariate correlation. For this purpose, SPSS statistical package program (SPSS ver. 13.0 for Windows, SPSS Inc., Chicago, IL, USA) was used, and Pearson's correlation coefficients were calculated. It was assumed that when the variables were continuous and approximately normally distributed without significant outliers, there was a linear relationship

between the variables. The linear correlation was assumed to be significant at *p* < 0.05, and a high correlation was determined at *p* < 0.01.

#### **3. Results and Discussion**

In this study, 10 emulsions with different formulations and/or homogenization times were prepared. Immediately after the production of these emulsions, analyses of the droplet size distribution were carried out with the laser diffraction technique, and simultaneously, microscope images of the emulsions were taken. The microscope images were processed with the help of TWS. In addition, the same analyses were repeated for six of the emulsions after one day of storage. The results of analyses performed with two different methods for the droplet size distribution are shown in Table 2. On the other hand, it is known that emulsion stability and viscosity properties are related to emulsion droplet sizes in emulsions [24,25]. Therefore, the stability and viscosity of the produced emulsions were analyzed, and the results are presented in Table 3.



For a realistic evaluation of droplet size distribution in emulsions, the largest possible number of droplets must be analyzed. It is stated in the literature that at least 2000 droplets should be analyzed to obtain reliable results in studies carried out with microscopic measurements [3]. The present study was conducted to determine the droplet size distribution with microscopy-assisted digital image analysis, and more than 2000 droplets (droplets between 3836–7850) were analyzed for each emulsion sample. Moreover, the Pearson correlation test was used to determine the relationship between the results obtained (Table 4).


**Table 3.** Turbidity and viscosity values of the emulsions.

a–f Different superscript letters indicate significant differences between samples prepared with 3 min of homogenization time with the same storage period and different oil/water ratios (*p* > 0.05). x–z Different superscript letters indicate significant differences between samples prepared with a constant oil/water ratio with the same storage period and different homogenization times (*p* > 0.05).

The results of both droplet size distribution analyses showed similar trends (Tables 2 and 4). However, the nominal values calculated by microscopy-assisted digital image analysis were slightly higher than the values obtained from the laser diffraction particle sizer. While D(90) and D[4,3] values calculated by microscopic measurements were in the range of 29.67–40.74 μm and 12.12–24.06 μm, the same parameters obtained by the laser diffraction technique were in the range of 20.90–34.10 μm and 8.46–20.70 μm, respectively (Table 2). It is known that measurement conditions (sample dilution, mixing speed, ultrasound application, etc.) have important effects on the nominal values obtained from measurements taken with a laser diffraction particle size analyzer.

The dilution and stirring processes may lead to floc disruption and result in smaller particle size values [13,26]. In this study, ultrasound was not applied during the sample injection, whereas a mixing speed of 2100 rpm was used. Although this may be the reason for the difference in nominal values between the microscopic and laser diffraction measurement results, especially for the stored emulsions, this effect seemed to be insignificant, as the other emulsion analyses showed similar trends to those observed with particle size analysis (Table 3). In general, the measured values of D(90) and D[4,3] slightly fluctuated with the storage time. According to the results, droplet size data determined by microscopyassisted digital image analysis and droplet size analysis determined by laser diffraction were positively correlated in all parameters (*p* < 0.05). Notably, D[4,3], D[3,2], and D(90) values had higher positive correlations (*p* < 0.01). It is known that D(90) is less affected by the obscuration ratio than other particle size parameters, such as D(10) and D(50), in the laser diffraction technique and yields more stable results. It is reported in the literature that D[4,3] is more sensitive to the presence of large particles in an emulsion system than the other parameters, and the differences between D[4,3] and D[3,2] generally indicate a broad or multimodal particle size distribution [1]. In the present study, the difference between D[4,3] and D[3,2] was significant according to the results of both measurement

techniques and varied in the range of 4.7–10.8 μm and 5.8–13.5 μm for microscopic and laser diffraction measurements, respectively (Table 2).


**Table 4.** The results of statistical analysis for the relations between microscopic measurements, laser diffraction, and other emulsion tests.

\* Correlation is significant at the 0.05 level (2-tailed). \*\* Correlation is significant at the 0.01 level (2-tailed).

Moreover, all of the parameters calculated from the microscopic measurements were highly correlated with the emulsion stability and viscosity results, and these relationships were even stronger than those between laser diffraction measurements and emulsion properties (Table 4). It was observed that the viscosity of the emulsions did not change significantly with storage (*p* > 0.05) (Table 3). When the effect of homogenization time was evaluated, the smallest droplet size was achieved with a 6 min process at a 10% oil ratio and with a 9 min process for an emulsion with a 30% oil ratio. These results are in agreement with the turbidity results (measurements made with both the turbidimeter and spectrophotometer), and turbidity values are high in samples with a small droplet size [2].

With the increase in the oil ratio in the produced emulsion formulation, increases in viscosity, turbidity, and droplet size values, especially those measured with the microscope (mainly D[3,2] and D[4,3] parameters), were observed (Tables 2 and 3). While no significant changes were detected in the laser diffraction measurements of turbidity, viscosity, or droplet size values of emulsions after storage, some fluctuations in the variation of droplet sizes measured with the microscope were observed (Tables 2 and 3).
