3.5.2 HPLC analysis of purified γ-tocopherol
HPLC was used to further determine the purity of the purified γ-tocopherol (Fig. S2). Results showed that the major peak representing γ-tocopherol (relevant content: 98.86%) was observed at a retention time of 12.68 min, and the peak of β-tocopherol (relevant content: 0.96 %) was observed as a low intensity at a retention time of 11.23 min. A tiny peak (relevant content: 0.18%) was observed at retention time of 4.02 min, which was caused by the impurities in solvent (used to dissolve tocopherol) and γ-tocopherol solution.
3.5.3 GC/MS analysis of purifiedγ-tocopherol
GC/MS was used to further clarify the chemical structures of γ-T and impurities in the purified γ-T (Fig 5, Table 2, Fig S3,). One strong peak (98.74%) was observed in gas chromatographic curve (Fig S3). The ions of this peak were as follows: 43.0, 150.0, 151.0, 416.0, which was consist with other report indicating that high purity γ-T was obtained (Nagy et al. 2007). It was well known that β-tocopherol and γ-T yielded same ions (Fig 5 c, d), but γ-T owned relative high polarity, therefore, it was speculated that the peaks at the retention time of 15.33 and 15.07 were γ-T and β-tocopherol, respectively (Table 2). Two tiny peaks (relative contents: 0.07% and 0.09%, respectively) were observed at 6.32 min and 9.83 min, respectively. Based on their MS results (Table 2 and Fig 5 a, b), molecular formulas of these compounds were determined to be C16H22O4 and C23H32O2, respectively. After referring to the mass spectral library, it was found that they were dibutyl phthalate and 2,2’-Methylenebis(6-tert-butyl-4-methylphenol), respectively. And these two compounds were all used in plastic products as plasticizers and anti-aging agents, respectively.
3.5.4 NMR analysis of purifiedγ-T
NMR was used to further identified the purified γ-T. The1H-NMR and 13C-NMR spectra of purified γ-T were presented in Fig. S4 and Fig. S5, respectively. The chemical shift assignments of purified γ-T were summarized in Table 3. In 1H-NMR spectrum, three characteristic peaks (δ 4.225, 7.495 and 7.696) were observed, in addition to the characteristic peaks of γ-tocopherol. Combining the characteristic peaks, corresponding area of these peaks and the 13C-NMR spectra, it was seen that the structure of purified γ-tocopherol is consistent with that described in Fig. 1, which indicated that purified γ-tocopherol is of high purity.