# Investigation of Biochars in Terms of Vitamin E Adsorption Capacity
## 2 Materials and Methods
### Adsorption Capacity of Biochars for \(\alpha\)-Tocopheryl Acetate
#### 2.6.3 Analysis of Biochars' \(\alpha\)-Tocopheryl Acetate Adsorption  
To create a calibration line, a stock solution (21.1 mg \(\alpha\)-tocopherol) acetate in 25 mL ethanol) with 0.844 mg\({}_{\alpha\text{-tocopherol}}\)/ml\({}_{\text{ethanol}}\), as well as an intermediate dilution (0.75 mL stock solution was diluted in 25 mL ethanol) with 0.025 mg\({}_{\alpha\text{-tocopherol}}\)/ml\({}_{\text{ethanol}}\), were prepared. From the intermediate dilution, as well as the stock solution, the following standards were used: 1.266, 2.532, 5.064, 10.128, 20.256, 25.31, 42.2, 84.4, and 168.8 ug\({}_{\alpha\text{-tocopherol}}\)/ml\({}_{\text{ethanol}}\). To prepare the sample, 1 g was transferred to a cup and centrifuged at 14,000 rpm and 20 \({}^{\circ}\)C for 20 min. If necessary, the supernatant was membrane-filtered. Then, 50 \(\upmu\)L was pipetted into an amber glass vial, 950 \(\upmu\)L of ethanol was added, and the mixture was thoroughly vortexed. To analyse the \(\alpha\)-tocopherol acetate content in the supernatant, 200 mg of the homogenised supernatant was mixed with 10 mL of ethanol. Then, the samples were treated in an ultrasonic bath (RK510H, Bandelin Sonorex, Berlin, Germany) for 5 min, and were shaken overnight (150/min; SM30, Edmund Buhler GmbH, Bodelshausen, Germany). After centrifugation (3000\(\times\) g, 5 min; Heraeus Megafuge 16, Thermo Fisher Scientific, Waltham, MA, USA), aliquots of the supernatants were diluted with ethanol to achieve a suitable concentration. After membrane filtration (0.45 \(\upmu\)m; Chromafil PET-45/15 MS, Macherey-Nagel, Duren, Germany), samples were analysed by high-performance liquid chromatography (HPLC System Alliance Separations Modul 2695 with a column oven, and Photodiode array detector 2996, both Waters, Milford, CT, USA), equipped with a LiChrospher 100 RP-18 (5 \(\upmu\)m, 250 \(\times\) 4 mm, Merck, Germany). The eluent was 60% methanol and 40% acetonitrile (isocratic), the flow rate was 1 mL/min, the injection volume was 10 \(\upmu\)L, the running time was 15 min, the temperature of the column oven was 40 \({}^{\circ}\)C, and the photodiode array detector was at 210 nm. The analysis was performed using a chromatography data system with standard calibration at 210 nm (5 points, linear, correlation > 0.998, reference tocopheryl acetate). The analysis was repeated twice for each sample. The adsorption capacity \(q_{abs. }\) of the biochars of \(\alpha\)-tocopheryl acetate was determined according to Equation (2) for each processing (\(n\) = 3). The concentration of \(\alpha\)-tocopheryl acetate in the stock solution was 1.69 \(\pm\) 0.08 g/L. \[q_{abs.}=\frac{(C_{0}-C_{t})\times V_{R}}{m_{biochar}} \tag{2}\]  
* \(q_{abs. }\): absolute adsorption capacity of \(\alpha\)-tocopherol acetate (mg\({}_{\alpha\text{-tocopheryl acetate}}\)/g\({}_{\text{Biochar}}\));
* \(C_{0}\): initial concentration of \(\alpha\)-tocopheryl acetate in the stock solution (g/L);
* \(C_{t}\): concentration of \(\alpha\)-tocopheryl acetate in the stock solution (g/L);
* \(V_{R}\): volume of reaction (L), which is \(\frac{m_{biochar}}{p_{ethanol}}\pm\frac{m_{\alpha\text{-tocopheryl acetate}}}{p_{ \alpha\text{-tocopheryl acetate}}}\)
* \(m_{biochar}\): weight of biochar (g). As the moisture content varied between the biochars, the adsorption capacity \(q_{abs. }\) was also calculated based on the dry matter of the biochar, according to Equation (3), which was adapted from [PERSON] et al. [55]. The dry matter \(m_{dry\ matter}\) was calculated based on the moisture content of the biochar, which was determined by drying according to SS64 LFGB L 06.00-3 2004-07, as reported in [PERSON] et al. [56]. The adsorption capacity \(q_{abs. }\) was determined in triplicate (\(n\) = 3). \[q_{abs.}=\frac{(C_{0}-C_{t})\times V_{R}}{m_{biochar\ DM}} \tag{3}\]  
* \(q_{abs.