TOCOPHEROLS:  ANTIOXIDANT COMPOUNDS

 Principle

Tocopherol content is determined by high-performance liquid chromatography coupled with a UV-VIS diode array detector and fluorescence detector.

The chromatographic analysis is performed under isocratic conditions with a THF / n-heptane mixture. UV detection is carried out at 298 nm, while fluorescence detection occurs at 298 nm (exc.) / 344 nm (em.).
 

 

Method adapted from the NF EN ISO 9936 standard ("tocopherols and tocotrienols/chromatography")

TOCOPHEROLS:  ANTIOXIDANT COMPOUNDS

 Equipment & Reagents

• Glassware
• High-performance liquid chromatography coupled with a UV-VIS diode array detector and fluorescence detector (HPLC/DAD/Fluo)
• HPLC column: LiChrospher 100 Diol 250 x 4.6 mm, 5μm
• Mobile phase: THF/n-heptane (3.85/96.15, v/v), with previously filtered solvents (0.45 μm)
• Flow rate 1 ml/min
• UV detection: 298 nm – fluorescence detection: 298 nm (exc.) / 344 nm (em.)
• Standards: mixture of α-, β-, γ- and δ-tocopherols and α-tocopherol solution at 100 mg/L of n-heptane.

Method adapted from the NF EN ISO 9936 standard ("tocopherols and tocotrienols/chromatography")

TOCOPHEROLS:  ANTIOXIDANT COMPOUNDS

 Experimental procedure

• Accurately weigh about 1 g of fat in a volumetric flask of 10 mL.
• Make up to 10 mL with an HPLC-quality n-heptane.
• Mix in a vortex for precisely 1 minute, then in an ultrasound bath for 30 seconds.
• Filter the sample with a 0.45 μm filter and inject 20 μL of the filtrate into HPLC.
• To identify the different tocopherol isomers in the fat, inject 20 μL of a sample containing the 4 isomers (α, β, γ, δ) and determine their respective retention times.
• To quantify the different isomers in the fat, perform an external calibration with a stock solution of standard α-tocopherol at different concentrations (0 to 100 mg/L). To do this, inject 20 μL of the α-tocopherol sample at each concentration. 

 ≈ 45 minutes / sample

Method adapted from the NF EN ISO 9936 standard ("tocopherols and tocotrienols/chromatography")