Db 23 capillary column

For the fatty acid isolation, 1 mL of chloroform was added to 2 mg of freeze-dried biomass in 7-mL glass tubes. The tubes were heated in a shaking water bath at 80°C for 90 min. Note that 1.5 mL of ultra-pure water and 2 mL of heptane were added to the tubes, once cooled to the room temperature, and mixed by vortexing. The tubes were then centrifuged for 5 min at 3,000 rpm to separate the phase. The upper phase containing heptane and fatty acid methyl ester (FAMEs) was recovered in a labeled screw vial. Note that 10 mg of C23:0-Me was added to the samples and used as internal standard. Finally, the samples were injected in a gas chromatography coupled with a flame ionization detector (GC-FID) from Shimadzu and a 30 m x 0.25 mm x 0.25 μm DB-23 capillary column from Agilent. FAMEs were identified by GC-FID and compared with different standards (Supelco 37 component FAME Mix and PUFA No. 3, from Menhaden). The FAMEs were quantified through the comparison against the C23:0 internal standard.

Cells were broken with 0.5 mm glass beads, and then total extracted lipids were methyl esterified with a fatty acid methylation kit (Nacalai Tesque, Kyoto, Japan). The fatty acid methyl esters were identified and quantified with a capillary gas chromatograph GC-2025 (Shimadzu, Kyoto, Japan) equipped with a DB-23 capillary column (60 m, 0.25 mm internal diameter, 0.15 μm film thickness) (Agilent Technologies, Santa Clara, CA, USA) followed by the previous method [15 (link)]. Heptadecanoic acid (Sigma-Aldrich Co., St. Louis, MO, USA) was used as an internal standard; rapeseed oil (Merck KGaA, Darmstadt, Germany) was used as a quantitative standard. The changes in the contents and compositions of lipids induced by the UV-C irradiation were also evaluated, with those at 0 min used as a control.

FAME were prepared from extracted fats according to ISO 12966-2:2011 [29 ] with the use of hexane as solvent and 2N methanolic potassium hydroxide solution for transmethylation. Thus, prepared methyl esters of fatty acids were analyzed by gas chromatography according to ISO 12966-4:2015 [30 ] on a gas chromatograph with flame ionization detector 7890B (Agilent Technologies, USA) with DB-23 capillary column 60 m long, diameter 0.25 mm and layer thickness fixed 0.25 μm phase (Agilent Technologies, Santa Clara, CA, USA) with detailed conditions described earlier by Pleadin et al. [31 (link)].

Samples for lipid analysis were taken from plants approximately 4 h into the light period, unless stated otherwise. Polar and neutral lipids were separated on silica plates (Silica Gel 60, EMD Millipore Corporation, Darmstadt, Germany) by thin layer chromatography (TLC) using acetone-toluent-water (91:30:7, by volume) and/or hexane-diethyl ether-acetatic acid (70:30:1, by volume), respectively. Fatty acid methyl esters were prepared as described by Li-Beisson et al. [13 (link)]. Separation and identification of the fatty acid methyl esters were performed on an HP5975 gas chromatograph-mass spectrometer (Hewlett-Packard) fitted with a 30 m × 250 μm DB-23 capillary column (Agilent, Santa Clara, CA) with helium as the carrier gas as described [14 (link)]. Fatty acid methyl esters were quantified using heptadecanoic acid as an internal standard using flame ionization analysis as described [14 (link)].

The composition of the oil was determined by gas chromatography with flame-ionization detection (GC-FID, model 7890A, Agilent, Santa Clara, CA, USA). The refined linseed oil sample (40 µL) was suspended in KOH/methanol solution (0.5 N) to separate the fatty acids from the triglycerides. Fatty acid methyl esters (FAMEs) were extracted from the reaction mixture with hexane and analyzed in a gas chromatograph equipped with a flame ionization detector (model 7890A, Agilent, Santa Clara, CA, USA). FAMEs were separated in a DB-23 capillary column (60 m, 0.25 mm, 0.25 µm, Agilent, Santa Clara, CA, USA) with hydrogen as the carrier gas. The composition of the different fatty acids was calculated as the ratio of the area of a given fatty acid peak to the overall area.

An Agilent 7890A Gas Chromatograph was purchased from Agilent (Folsom, CA, USA) with a DB-23 Capillary Column (30 m × 0.25 mm, 25 μm). The following HPLC condition was used: starting temperature was 170°C, ramping up to 225°C at 1°C/min and holding for 5 min, inlet temperature was 250°C, detector temperature was 280°C, carrier gas was high purity N₂, flow rate was 1.0 mL/min, injection volume was 1 μL, injection method was split injection, and split ratio was 100 : 1. The mixed reference solution was taken and injected under the above chromatographic conditions. The two solutions were each continuously injected with 5 needles, and the peak area was recorded and the relative correction factor (F_X) was calculated. The results are shown in Table 1.

We used GC-MS (Agilent 7000C, Agilent Technologies, USA) to analyze the fatty acid composition of the oil. Fatty acid methyl esters (FAMEs) were prepared by dissolving 0.1 g of oil in 2 mL of heptane and a solution of KOH (0.2 N) in methanol. Thereafter, the analyses were performed in accordance with a previously reported method (Bengana et al., 2013 (link)). GC-MS work condition: DB23 capillary column (30 m × 0.32 mm, 0.25 μm film thickness, Agilent Technologies, USA); the injection volume was 0.8 μL; linear velocity, 0.5 cm/min; split ratio of 1:30, v/v; interface, FID and the injector temperatures were 200, 280, and 250°C respectively. For each sample, three replicates were prepared and analyzed.