BVL or BVL-7 was methylated by adding 1:1 hexane and 14% boron trifluoride/methanol and heated at 100°C for an hour. Fatty acids methyl esters were analyzed by a fully automated 6890N Network Gas Chromatography equipped with a flame-ionization detector (Agilent Technologies, Palo Alto, CA). Individual fatty acid was determined by retention time using a reference standard, GLC461 (Nu-Chek Prep, Elysian, MN).
6890n network gas chromatography
Manufactured by Agilent Technologies
The 6890N Network Gas Chromatography is a laboratory instrument designed for the analysis of complex mixtures of gases and volatile compounds. It utilizes gas chromatography principles to separate and detect the components of a sample. The 6890N is capable of precise and accurate measurements, providing analytical data for various industrial and research applications.
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4 protocols using 6890n network gas chromatography
1
Methylation and GC Analysis of Fatty Acids
2
Quantifying Ethanol Content via GC
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An Agilent Technologies® 6890 N Network Gas Chromatography (GC) system equipped with Flame ionisation detection was used to quantify ethanol content in the samples of interest using nitrogen gas as a mobile phase with a flow rate of 3.2 ml/min. A calibration curve was prepared using five different concentrations of ethanol by dilution of the specified volume of ethanol i.e. 0.2, 0.4, 0.6, 0.8, 1.0 ml into 10 ml of water. All experiments were conducted in triplicates.
3
Quantification of Phytohormones via GC-MS
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The ABA quantification and extraction were performed as reported in the literature with minor modifications [77 (link)]. Briefly, endo-ABA was extracted from 0.3 g of freeze-dried plant sample, and a chromatograph was run with Me-[2H6]-ABA as an internal standard. The fraction was then methylated with diazomethane, and 1 µL of sample was detected by GC-MS (6890 N network gas chromatography, Agilent Technologies) to determine the ABA content. ThermoQuest software (Manchester, UK) was used to monitor signal ions (m/z 162 and 190 for Me-ABA; m/z 166 and 194 for Me-[2H6]-ABA) (Table S1 ). The JA and SA were quantified using the detailed method previously reported in the literature [78 (link)] (Tables S2 and S3 ).
4
Quantitative Phytohormone Analysis Protocols
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The protocol described by [39, (link)40] (link) was used to determine the abscisic acid (ABA hereon). The ABA was extracted from the plants' aerial parts (freeze-dried plant samples, 0.3 g), and a chromatograph was run using the Me-[2H6]-ABA standard. The fraction was methylated with diazomethane for detection, and ABA was quantified using GC-MS (6890N network gas chromatography, Agilent Technologies). A software from Thermo-Quest Corp., Manchester, UK was used to monitor signal ions (m/z 162 and 190 for Me-ABA and m/z 166 and 194 for Me-[2H6]-ABA) (Table S4). The method described by [41, (link)42] (link) was used to determine the plant's jasmonic acid (JA hereon) content. In brief, a 0.1 g freezedried ground sample was extracted with acetone: citric acid (70:30 v/v) and filtered. The [9, 10-2H2] JA standard (100 ng) was added. The solvent was evaporated and washed with 0.1M potassium phosphate buffer, and pH was adjusted to 2.5. The solution was stirred with diethyl-amino cellulose, filtered, and washed with chloroform in a column fixed with sodium sulfate. The extract obtained was injected in GC-MS/SIM (6890N Network GC System and 5973 Network Mass Selective Detector; Agilent Technologies, Santa Clara, CA, USA), and the peaks obtained were noted (Table S4).
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