Abstract
A modified method for fractional determination of α, β-, γ- and δ-tocopherols by means of thin-layer chromatography (TLC) was described. The method consists of saponification of lipid in the presence of pyrogallol, extraction of unsaponifiable matter with ether containing hydroquinone, removal of interfering material by passing through neutral alumina layer and Florisil column chromatography, separation of tocopherols by TLC using a modified mixed solvent, extraction of individual tocopherol with ethanol, and spectrophotometric determination of extracted tocopherol with bathophenanthroline-FeCl3-orthophosphoric acid procedure. Absorbance was read at 534mμ and tocopherol content per g of lipid was calculated by an equation obtained from standard curves.
By introducing the bathophenanthroline procedure, the sensitivity was increased approximately 2.5-fold compared to α, α'-dipyridyl procedure. Various conditions concerning each step of this method were examined. The recovery of tocopherols added to milk fat was more than 97%.
The method was applied to determine tocopherol content of milk fat, egg yolk lipid and margarine. A major tocopherol in milk fat was α-tocopherol, but several per cent of total tocopherol was detected as γ-tocopherol. Egg yolk contained α- and γ-tocopherols in an approximate ratio of 2:1, and a trace of δ-tocopherol.
