Trace 1300 isq lt

Lipid extraction for fatty acid analysis was conducted following the method of Bligh and Dyer [33 (link)]. In brief, about 0.1 g of lyophilized biomass of each sample was extracted with a chloroform–methanol–water solution. The lipid-containing chloroform phase in the substratum was dried to powder under a nitrogen stream. Fatty acid methyl esters (FAMEs) were prepared by esterification of the powdered lipids in a KOH–methanol solution with the C17:0 ester containing cyclohexane as the internal standard. The upper layer of the mixture was separated for FAME composition analysis.
The FAME composition analysis was conducted with a GC–MS system (ThermoFisher Trace 1300-ISQLT). The GC–MS system was equipped with an electron impact ionization detector and a TR-5MS column (30.0 m × 250 μm × 0.25 μm). The injection volume was 1 μL for each sample. The temperature of the injector and detector, the column flow rate, and the split ratio were 250 °C, 1.2 mL min⁻¹, and 1:50, respectively. The running temperature was set as follows: 40 °C for 1 min, heating to 230 °C at 20 °C min⁻¹, held at 230 °C for 1 min, heating to 270 °C at 3 °C min⁻¹, and held at 270 °C for 2 min. An internal standard was used to quantify the weight (mg) of each fatty acid, and its cellular content (mg g⁻¹) was calculated in terms of its weight per gram of algal powder.

The plant extract was analyzed in a single quadrupole gas chromatography–mass spectrometry system (Thermo Scientific TRACE™ 1300 ISQ™ LT). Methanol was used as a solvent. A non-polar column TR-5MS (260F142P, dimension of 30 m × 0.25 mm × 0.25 µm with film thickness of 0.25 μm) was used as the stationary phase. The injector port was set at 250°C. The oven was initially set at 70°C and incrementally increased to 250°C. Helium was released at 1 mL/min into the oven chamber. One microliter of the sample was injected in split mode at the splitting ratio of 1:50. The mass spectrometer was set at an ionization electron energy of 70 eV. Ion source and transfer line were set at 250°C. The total running duration was 55 min. The final chromatogram and mass spectra were generated with Thermo Scientific™ Xcalibur™ software. Compounds were identified based on their retention time, chemical formula, and molecular weight from the libraries of Wiley Registry™ 10 and National Institute of Standards and Technology database.

Gas chromatography-mass spectrometry (GC/MS, TRACE1300-ISQLT, Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze the compositions and chemical concentrations of the volatile compounds in the gas sample. Chemical analysis was performed on a GC/MS system coupled with a DB-WAX-52CB (30 m × 0.25 mm × 0.25 um) capillary column. A 0.7 mL gas sample was injected into the GC/MS system using a gastight syringe. The temperature of the GC/MS inlet was set as 250 °C. The initial temperature of the oven was 50 °C, which was maintained for 3 min, then increased to 155 °C at a rate of 15 °C/min, and held for 10 min. He (99.999%) was used as the carrier gas at a flow rate of 1.2 mL/min. The compounds separated by GC were then analyzed by MS over the mass-to-charge ratio range of 40-350 amu. The temperatures of the ion source and transfer line were both set at 250 °C. The detected compounds were identified by comparing the corresponding mass spectra with the standard mass spectral library. Quantification of the detected compounds was conducted by the external standard method.