Where m is the sample oil mass (g), \(V\) is the volume of NaOH solution used in the titration (L), \(M_{\text{NaOH}}\) is the NaOH solution molarity (mol L-1) and\(\text{MM}_{\text{FFA}}\) is the average molar mass of fatty acids in Crambe oil. The average molar mass of fatty acids in Crambe oil is 317.98 g mol-1, and it was determined in previous work (Tavares et al., 2018b) performed by the same group.

2.4. Quantification of Acylglycerols

Analysis of Nuclear Magnetic Resonance (NMR) was performed to determine the concentration of acylglycerols and the methodology is described elsewhere (Tavares et al., 2018a).

2.5. Hydrolysis Reactions

The reactions were carried out in a 50 mL Erlenmeyer sonicated using an Ultronique ultrasound, microtip probe of 4 mm diameter, model disruptor, maximum power of 500 W. Additional experiments were performed in a refrigerated incubator with agitation (shaker) at room temperature and 140 rpm.
During preliminary tests, it was observed that after 5 minutes of sonication, the viscosity of the medium started to increase, affecting the cavitation phenomenon. The temperature of the mixture was increasing (more than 40°C), which could be damaging for the reaction and the equipment. Therefore, for the factorial design experiments, a time of 3 minutes was chosen. A fixed volume of 25 cm3 of water and oil solution (acetate buffer solution; 0.2 M, pH 4.2) was used. The buffer solution pH and concentration were determined based on preliminaries studies performed by the same research group (TAVARES et al., 2015).
After the reaction time, the resulting hydrolyzed oil emulsion was centrifuged for 20 minutes at 3000 rpm, and then a sample of approximately 1g of oil was collected and analyzed. The hydrolysis yield was calculated by Equation 2 .