Ptfe membrane syringe filter

The α-tocopherol content was evaluated using high-performance liquid chromatography (HPLC) on a Pinnacle II Silica column (Restek, USA; 5-μm particle size; 150 mm × 4.6 mm), as described previously (Fernandez-Orozco et al., 2003 (link)). Tocopherols were extracted from fresh plant tissues using pure hexane (1:10) by centrifugation at 349 × g for 5 min. The supernatant was filtered through a 0.45-μm polytetrafluoroethylene (PTFE) membrane syringe filter (VWR International, USA). The HPLC 10A system, equipped with an RF-10A fluorescence detector (Shimadzu, Japan), was used for analysis. Peaks were detected at an excitation wavelength of 295 nm and an emission wavelength of 330 nm. The mobile phase (0.5% isopropanol in hexane) was used at a flow rate of 1 ml min⁻¹. The α-tocopherol was identified according to the analytical standard. The α-tocopherol content is expressed per gram dry weight of microgreens.

Two sediment samples (1 cm-depth) were collected in each plot of all the areas with a syringe (50 mL), kept in a cooler box during transport, and stored at -20°C until storage at -80°C at the laboratory. Before extraction, sediment samples were freeze-dried and homogenized. The pigments from 0.5 g of dry sediments were extracted with 5 mL of methanol buffered with 2% of 1 M-ammonium acetate (Sigma-Aldrich, France). After 2 min in an ultrasound cold bath and at maximum power, samples were kept in the dark at -20°C for 15 min before centrifugation (High Conic Rotor, Thermo Scientific, 3220 g, 2°C, and 5 min). Supernatants were collected and the pellets were re-extracted as described above. The pooled supernatants were filtered on 0.2 μm PTFE membrane syringe filter (Ø 13 mm, VWR International, United States) and stored a few days at -80°C before High Performance Liquid Chromatography (HPLC) analysis. To prevent degradation of pigments, extractions were performed under dark conditions and samples stored on ice during handling.

Analysis of tocopherols was performed by HPLC according to Brazaityte et al.’s [25 (link)] methodology with some modifications. About 1 mg of oil was weighed in an Eppendorf tube, and then 1 mL of n-hexane with 1% of BHT was added to the tube. Afterward, the samples were filtrated through a 0.45 µm polytetrafluoroethylene (PTFE) membrane syringe filter (VWR International, Radnor, PA, USA) and were analyzed by HPLC/FLD (fluorescence detector) (Agilent Technologies, Santa Clara, CA, USA). The HPLC measurements were performed using a normal phase column (Phenomenex Luna Silica, 5 μm, 250 mm × 4.6 mm). The HPLC 10A system, equipped with an RF-10A fluorescence detector (Shimadzu, Japan), was used for analysis. Peaks were detected at an excitation wavelength of 295 nm and an emission wavelength of 330 nm. The mobile phase (0.5% isopropanol in hexane) was used at a flow rate of 1 mL min⁻¹. The α-tocopherol, γ-tocopherol, and δ-tocopherol were identified according to the analytical standard. The α-tocopherol, γ-tocopherol, and δ-tocopherol content were expressed per 100 g of oil.