2.5. Acidity neutralization
Once the titrations for the calculation of the acid value were performed, we subjected the samples to the neutralization pro-cess, following the methodology proposed by Martins (2012). We then prepared an aqueous solution with 10% NaOH concentration (mass/volume). For each of the six treatments, we used a volume of a neutralizing solution corresponding to the acid value in NaOH grams multiplied by the total oil mass to obtain the relation of how many milligrams of solution are necessary to neutralize the free fatty acids present in each oil sample. The oils and the neutralizing solution were put in a hot plate at 70◦C with magnetic stirring for
15min and the mixture was posteriorly transferred to a glass filter funnel with paper filter for simple filtration, which took approx-imately 24h until complete draining. The retained organic phase was disposed with the filter, leaving little oil quantity.
After filtering, the oils were washed three times with 5mL of boiling water. Oil drying occurred in an oven at 85◦C for 48h. Then, we determined the acid value again.
2.6. Fatty acids identification
For the identification of fatty acids, we stored roughly 1.0ml samples of crude oil of each treatment in Eppendorf tubes properly isolated with parafilm prior to any procedure. The samples were analyzed by means of gas chromatography (GC).
To obtain the desired results, the conversion of triacylglyc-erides into esters was necessary. It was performed by means of the Hartman and Lago method of transesterification and esterifi-cation, adapted from Antoniosi Filho and Lanc¸as (2005). This way, the percentage of ester is correspondent to the percentage of fatty acid analyzed.
A Shimadzu Gas Chromatograph (GC-2010 Plus) with flame ionization detector (FID) and Select Biodiesel-Agilent column was utilized.