Agilent 1260 infinity hplc chromatograph

Tocopherols were determined by HPLC with fluorescence detection following IUPAC Standard Method 2.432 [24 ]. The oil samples were dissolved in n-heptane at a concentration of 50 mg mL⁻¹ and analyzed in an Agilent 1260 Infinity HPLC chromatograph (Agilent Technologies, Santa Clara, CA, USA). The chromatograph was equipped with a quaternary pump VL (G1311C), a standard autosampler (G1329B), a thermostatted column compartment (TCC) (G1316A) and a fluorescence detector (G1321A). A silica HPLC column (LiChrospher^® Si 60, 250 mm × 4 mm i.d., 5 µm particle size) (Merck, Darmstadt, Germany) was used. The volume of the sample analyzed was 20 µL. The temperature of the TCC was set at 25 °C. The separation of tocopherols was performed using n-heptane:isopropanol (99:1, v/v) with a flow rate of 1 mL min⁻¹. The excitation and emission wavelengths in the detector were 290 nm and 330 nm, respectively. Quantification was made by external calibration using tocopherol standards.