Vf 5m

IR-spectra: Perkin-Elmer FT-IR Paragon 1000; MS: Hewlett Packard MS-Engine; electron ionisation (EI) 70 eV, chemical ionisation (CI) with CH₄ (300 eV); NMR: JNM-Eclipse+400 (400 MHz) (¹H: 400 MHz, ¹³C: 100 MHz), and Avance III HD 400 MHz Bruker BioSpin (400 MHz); melting points: Büchi Melting Point B-540 (not corrected); flash column chromatography (FCC): silica gel 60 (230–400 mesh, E. Merck, Darmstadt); GLC-MS: Shimadzu GC-17 A (carrier: He, oven temperature program: 100–280°C, 10°C / min, capillary column: Varian VF-5 ms 30 m × 0.25, split injector T = 250°C, detector T = 260°C).

GC-MS analysis was carried out on a Perkin Elmer Clarus 500 GC-MS and gas chromatography-free induction decay (GC-FID) (Waltham, MA, USA) equipped with a Varian VF-5ms (Factor Four of Superchrom) capillary column (60 m × 0.25 mm, film thickness 0.25 µm), according to previous methods [38 (link)] with some modifications. Injector temperature was 250 °C, detector temperature 300 °C, while the column temperature was held at 50 °C and programmed to 170 °C at a rate of 6 °C/min then increased to 270 °C at a rate of 8 °C/min and then kept isothermally at 270 °C for 10 min; helium was used as the carrier gas with 1 mL/min flow; ionization energy was 70 eV and mass ranged from 35 to 450 amu. The components were identified by comparison of their mass spectra to those reported in the Wiley and NIST/NBS (National Institute of Standards and Technology/National Bureau of Standards) libraries. Furthermore, the Linear Retention Indices (LRIs) were calculated and compared with the available retention data reported in the literature. The peak areas of the FID signal were used to calculate the relative percentages of the components without the use of an internal standard and any factor correction. All of the analyses were carried out in triplicate.

Vitamin E (α-, β-, γ-, δ-tocopherol; Tc) content was measured using GC/FID chromatography with squalene as internal standard. For the investigation, 0.5 g of dry alga was mixed with 10 mL of 10% NaCl, 20 mL of hexane, 10 mL of methanol, and 10 µL of squalene solution (1.2 mg/mL) for 24 h at room temperature, and then sonicated for 1 h. Hexane phase was separated by centrifugation and evaporated to dryness. The remains were dissolved in 25 µL of hexane, and 2 µL of the prepared sample was placed in an injection chamber. The conditions of the analysis were as follows: Injection chamber temperature 250 °C; split 1:50; passive gas: He, flow-5 mL/min; column Varian VF-5 ms, 30 m × 0.53 mm; temperature: isothermal 110 °C/0.2 min, linear gradient 30 °C/min to 140 °C (1 min), linear gradient 10 °C/min do 230 °C (9 min), isothermal 230 °C for 6 min, linear gradient 10 °C/min to 300 °C (10 min) isothermal 300 °C for 7 min; detector: FID, detector temperature: 340 °C. The limit of the detection was equal to 0.1 mg/100 g.

Hydro distillate extract from a cell suspension after 40 days was diluted with n-hexane (GC grade, 2 μL:1 mL) and injected (1 μL) using an auto-sampler injector (Model Combi Pal, Varian) to the GC–MS (GC, Model CP-3800, Varian, Walnut Creek, CA, USA) linked with a mass spectrometer (MS, Model Saturn 2200, Varian) using a VF-5ms fused silica capillary column (5% phenyl- dimethylpolysiloxane, 30 m × 0.25 mm i.d., film thickness 0.25 μm, Varian, Palo Alto, CA, USA). The electron impact (EI) ionization detector was used with an ionization energy of 70 eV. Helium was a carrier gas (99.99%) with a constant rate (1 mL/min). The injector and mass transfer line temperatures were 240 and 300 °C, respectively. The oven temperature was held at 50 °C for 1 min, raised to 230 °C for 50 min at 30 °C/min, finally raised to 290 °C for 5 min at 10 °C/min and held isothermal for 6 min. The sample split injection ratio was 1/500, with a total time of 54.3 min. The identification of components was based on matching with a mixed standard (n-alkanes (C6–C26)) and the Wiley and National Institute of Standards and Technology (NIST) electronic library.