2.4. Determination of tocopherol concentration in base algae oil
The concentration of tocopherol in base algae oil during storage time was determined by normal phase HPLC. For HPLC analysis, weighted oil sample (approximately 0.1 g) was dissolved in 5 mL hexane. The solution was passed through a 0.45 μm filter after vortexing. A 20 μL aliquot of the solution was separated using a Shimadzu (10A, Shimadzu Co., Kyoto, Japan) HPLC system with a 250 mm × 4.6 mm i.d., 5 μm, ZORBAX Rx-SIL analytical column. The mobile phase of the HPLC system consisted of hexane:isopropyl alcohol (99:1 v/v) using isocratic gradient at a flow rate of 1 mL/min. Column temperature was maintained at 40 °C. Detection of tocopherol was conducted using both a Shimadzu diode array detector (DAD) at 295 nm, and a Waters 474 scanning fluorescence detector at an excitation wavelength of 290 nm and an emission wavelength of 330 nm. Calibration curves were calculated by linear regression analysis of the florescence peak surface area versus the concentration of the tocopherol standard.