This document introduces a method for analyzing all eight naturally occurring tocopherols (T) and tocotrienols (T3) in margarine using normal-phase chromatography. This method uses fluorescence detection to avoid detecting peaks that overlap with coeluting peaks.
The document presents a high-performance liquid chromatography (HPLC) method for the analysis of tocopherols and tocotrienols in fats, oils, and foodstuffs. It highlights that normal-phase chromatography is required to separate all eight tocopherols completely, as reversed-phase chromatography cannot achieve full separation. The method utilizes a Hypersil SI 100 column with a mobile phase of hexane and 2% isopropanol.
| Column | Hypersil SI 100, 5 um |
|---|---|
| Mobile phase | hexane + 2 % isopropanol |
| Stop time | 8 min |
| Flow rate | 0.3 ml/min |
| Column temperature | 25 °C |
| Injection volume | 0.5 |
| Detector UV-DAD wavelength | 295/80 nm |
| Fluorescence emission wavelength | 330 nm |
| Sample preparation solvent | hexane |
| Repeatability (areas) | <2% |
No, tocopherols cannot be separated completely using reversed-phase chromatography.
Dissolve 20 g of sample in 15 ml of hexane.