2.2.2 Adsorption isotherms
Pre-weighted 0.8 g of activated silica gel (200-300 mesh) were added into 20 mL γ-tocopherol solutions with different initial concentrations, the conical flasks with cover were shaken (25 °C, 120 rpm) until adsorption equilibrium was reached. The initial and equilibrium concentrations of γ-tocopherol solutions were analyzed by HPLC. The adsorption capacity of γ-tocopherol on silica gel was calculated according to the following equation (1). Langmuir and Freundlich models were employed to describe the adsorption behaviors.
\begin{equation} Q_{e}=\frac{\left(C_{0}-C_{e}\right)\times V}{M}\text{\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ }\left(1\right)\nonumber \\ \end{equation}
Langmuir equation:
\begin{equation} Q_{e}=\frac{Q_{m}K_{L}C_{e}}{1+K_{L}C_{e}}\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (2)\ \nonumber \\ \end{equation}
Freundlich equation:
\begin{equation} Q_{e}=K_{F}\ C_{e}^{\frac{1}{n}}\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (3)\nonumber \\ \end{equation}
Where, Qerefers to the adsorption capacity (mg/g silica gel). C0and Ce stand for the initial concentration and equilibrium concentrations of γ-tocopherol (mg/mL), respectively.V refers to the volume of the initial sample solution (mL), andM represents the weight of the tested adsorbent (g).Qm stands for the theoretically calculated maximum adsorption capacity (mg/g). KL is the Langmuir constant. KF and n are both the Freundlich constants.
2.2.3 γ-tocopherol purification process
The pre-weighed mixed tocopherol sample was dissolved in three mL of hexane to form a homogeneous solution which was then loaded onto a chromatographic column (length and diameter, 60 × 3 cm) packed with silica gel (200-300 mesh). Elution solvent was comprised of n-hexane and ethyl acetate. Elution flow rate was controlled by a constant flow pump, and a UV detector (wavelength, 290 nm) was employed to monitor the elution processes. Elution fractions were collected in test tubes and analyzed by using TLC.
Elution fractions containing only γ-tocopherol were combined into a single mixture and the organic solvents were removed using a rotary evaporator (IKA, Germany) under reduced pressure. The amount of purified γ-tocopherol was calculated using mass balance, and the purity of γ-tocopherol was analyzed by HPLC according to the method above mentioned. The recovery yield and purity of γ-tocopherol were calculated according to the following equations, respectively.
\begin{equation} P\left(\%\right)=\frac{C\times V\times 10^{-6}}{W}\times 100\%\ \text{\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ }\left(4\right)\text{\ \ }\nonumber \\ \end{equation}\begin{equation} R(\%)=\frac{W\times P}{L\times B}\times 100\%\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (5)\ \ \nonumber \\ \end{equation}
Where C is the concentration of purified γ-tocopherol (µg/mL).V is the volume of the purified γ-tocopherol solution (mL).W is the mass of purified γ-tocopherol (g), P represents the purity of γ-tocopherol (%), and B refers to the γ-tocopherol content of mixed tocopherols (g/100g), and L is the loading mass of column chromatography (g).