2.2. Fatty Acid Composition
Soybean oil was derivatized to fatty acid methyl esters (FAME) as described by Kiefer (Kiefer, 1997), then analyzed with GC according to AOAC Official Method 996.06 (AOAC, 2005). A gas chromatograph (GC-2010, Shimadzu) was used with a polar ionic liquid capillary column SLB-IL60 30 m x 0.25 mm x 0.20 μm (Sigma-Aldrich, St. Louis, MO), and a flame ionization detector (FID) at 280°C. Helium was used as the carrier gas with a flow rate of 1 mL/min. The column temperature was programmed from 150°C to 280°C at a heating rate of 5°C/min. A volume of 1L was injected, with a split mode of 50:1. Supelco 37-component FAME mix 10mg/mL (Sigma-Aldrich, St. Louis, MO, USA) was used as an external fatty acid standard to identify the components. Dodecanoic acid (Sigma-Aldrich, St. Louis, MO) was added as an internal standard. SLB-IL60 column allows separating oleic acid (C18:1) with the double bond in 9c or 11c position in the hydrocarbon chain, being 11c a small fraction of monounsaturated fatty acids (MUFA).
The insoluble fraction in hexane was quantified by the amount of sample used for fatty acid composition analysis. During derivatization of the triglycerides into fatty acid methyl esters, a fraction of the treated sample did not dissolve completely. The filtered (0.2 \(\mu\)m) samples were injected into the GC, and the insoluble part was quantified as reported elsewhere (AOAC, 2005).