Lichrosorb si 60

Optical rotation was measured using a JASCO DIP-180 digital spectropolarimeter. IR spectra were recorded using a Nicolet 510P FT-IR spectrometer. UV spectra were measured in MeOH using a Shimadzu UV-1601PC spectrophotometer. The NMR spectra were recorded in CDCl3 at room temperature on a Varian Mercury plus 400 NMR spectrometer with residual solvent resonance as an internal reference. The 2D NMR spectra were recorded using the standard pulse sequences. For EI-MS and HR-EI-MS, Finnigan TSQ-700 and JEOL SX-102A spectrometers were used, respectively. TLC was performed on silica gel 60 F254 plates (Merck, Darmstadt, Germany). Column chromatography was performed on silica gel (230 400 mesh ASTM, Merck). HPLC was performed using a Hitachi L-7000 chromatograph with a Bischoff RI detector (Leonberg, Germany). A normal phase column (LiChrosorb Si 60, 7 μm, 250 × 10 mm, Merck) was used for isolation.

The tested samples were subjected to saponification. Next, the sample was injected from the upper layer (unsaponifiable substances) for injection by HPLC. Tocopherols were identified qualitatively and quantitatively by HPLC liquid chromatography (Waters 600 Asc. Milford, MA, USA) in a system consisting of a pump Waters 600, column LiChrosorb Si 60 (200 × 4.6 mm, 5 μm, Merck, Darmstadt, Germany) and fluorimetric detector (Waters 474 Asc. Milford MA, USA). The mobile phase was a mixture of n-hexane with 1,4-dioxane (96:4 v/v). The flow rate was 1.0 mL/min. Detector was set at excitation λ = 290 nm and emission λ = 330 nm. The concentration of individual tocopherols homologues was calculated on the basis of a previously performed calibration curve [44 (link)]. Results were expressed as milligram of each tocopherol per 100 g of dry mass.