2.2.5 Characterization of purified product
2.2.5.1 TLC analysis of purified γ-tocopherol
The conditions of TLC analysis were in accordance with the methods described in section 2.2.1.
2.2.5.2 HPLC analysis of purified γ-tocopherol
The conditions of HPLC analysis were same to the methods we mentioned in section 2.2.1.
2.2.5.3 GC/MS analysis of purified γ-tocopherol
GC7890 with 5975N mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) was employed to identified the structure of purified γ-tocopherol. And the method of Bartosińska et al. (Bartosińska et al. 2016) was used with modification. The conditions of GC/MS analysis were as follows: one microlitre of purified γ-tocopherol samples (5 µg/mL) were injected in split mode. The split radio was 25:1. The samples were separated using an HP-5 MS capillary column (30 m × 0.25 mm, 0.25μm film thickness; J&W Scientific, Folsom, CA, USA). Helium was used as the carrier gas at a rate of 1.0 mL/min (23.441 psi). The injector and detector temperature were 280 and 300 °C, respectively. Column oven temperature was set at 100 °C for 1 min, then a temperature gradient program at 50 °C/min up to 220 °C for 2 min, and 10 °C/min up to 285 °C for 15 min. MS detector 230 °C, MS quadrupole 150 °C, electron ionization energy 70 eV, and mass range 40-500 m/z.
2.2.5.4 NMR analysis of purified γ-tocopherol
All NMR spectra of purified γ-tocopherol were obtained using a Bruker 500MHz Avance Ⅲ NMR spectrometer (Bruker, Rheinstetten, Germany) using a 5 mm switchable, broadband probe. All spectra were obtained in deuterochloroform at ambient temperature (30 °C in the probe).1H-NMR spectra were obtained using a spectral width of 13889 Hz, 2 sweeps, 128 scans and a pulse angle of 30°. The13C-NMR spectra were acquired under the following conditions: combined pulse decoupling, 4 sweeps, 1024 scans and a pulse angle of 30°.