Agilent 5975c msd

The floral volatile analysis was performed by placing the whole flower in a glass bottle for 30 min, as explained previously [7 (link),55 (link)]. Polydimethylsiloxane (PDMS) fiber was inserted into the bottle for 30 min to adsorb volatile compounds followed, then injected into a GC-MS system (Agilent). The GC–MS system with Agilent 7890A GC and Agilent 5975C MSD was provided with an Agilent DB-5MS capillary column (30 m × 0.25 mm), and helium gas was provided as a carrier. The flow of helium gas was kept constant at 1 mL/min. Initially, the GC injection port temperatures were kept at 40 °C for 3 min, which was followed by an increase in temperature of 5 ℃/min to 250 °C. The chromatographic running time was 30 min. The relative quantification of volatiles was calculated using the Agilent ChemStation data analysis application based on the peak area ratio and the quantity of the internal standard.

GC-MS was performed with an Agilent 6890 Plus gas chromatograph interfaced with a single-quadrupole Agilent 5975C MSD (Agilent Technologies, Palo Alto, CA). The electron energy was 70 eV and the ion source temperature was 230°C. Each sample (2 μl) was injected in split mode (10:1) at 280°C and separated through a MXT-1 cross-linked dimethylpolysiloxane capillary column (30 m × 0.25 mm inner diameter, 0.25 μm film thickness, Silcosteel-treated stainless steel; Restek, Bellefonte, PA). The oven temperature was held initially at 260°C for 3 min, ramped to 320°C at 10°C/min, increased to 330°C at 2°C/min (held for 8 min), and finally increased to 380°C at 30°C/min and held for 3 min. The carrier gas was ultra-high-purity helium at a column head pressure of 75.8 kPa (14.2 psi; column flow: 1.1 ml/min at an oven temperature of 260°C). For quantitative analysis, the characteristic ions of each compound were determined as their TMS derivatives. Peak identification was achieved by comparing the retention time and matching the height ratios of the characteristic ions (Table 1).

Total lipids were extracted from mid-log-phase promastigotes (3 × 10⁶ to 7 × 10⁶ cells/ml) by following the method of Folch et al. (74 (link)). An internal standard, cholesta-3,5-diene (formula weight, 368.34), was provided at 2.0 × 10⁷ molecules/cell during extraction. Lipid samples were dissolved in methanol at 1.0 × 10⁹ cell equivalents/ml. Equal amounts of each lipid extract in methanol were transferred to separate vial inserts, evaporated to dryness under nitrogen, and derivatized with 50 μl of BSTFA plus 1% TMCS-acetonitrile (1:3), followed by heating at 70°C for 30 min. GC-MS analysis was conducted on an Agilent 7890A GC coupled with Agilent 5975C MSD in electron ionization mode. Derivatized samples (2 μl each) were injected with a 10:1 split into the GC column with the injector and transfer line temperatures set at 250°C. The GC temperature started at 180°C and was held for 2 min, followed by 10°C/min increase until 300°C and then held for 15 min. To confirm that the unknown GC peak retention time matched that of the episterol standard, we also used a second temperature program started at 80°C for 2 min, ramped to 260°C at 50°C/min, held for 15 min, and increased to 300°C at 10°C/min and held for 10 min. A 25-m Agilent J & W capillary column (DB-1; inner diameter, 0.25 mm; film thickness, 0.1 μm) was used for the separation.

The flower developmental process from squaring stage to senescence stage was divided into six stages starting at 10:00 AM with 12-h intervals (Fig. 1a). The headspace collection and GC-MS analysis were performed as described previously [46 (link)]. The whole flower of each stage was enclosed in a 500-ml glass bottle with the addition of 1.728 μg ethyl caprate as internal standard. After equilibrium of volatiles for 30 min, a polydimethylsiloxane (PDMS, with 50/30 μm divinylbenzene/Carboxen) fiber (Supelco) was inserted into the bottle to adsorb volatiles for 30 min. Then, trapped floral scent compounds were analyzed by a GC-MS system with Agilent 7890A GC and Agilent 5975C MSD. The instrument was equipped with an Agilent HP-5MS capillary column (30 m × 0.25 mm) and helium as a carrier gas at a constant flow of 1 ml/min. The oven temperature was initially maintained at 40 °C for 2 min, followed by an increase to 250 °C at a rate of 5 °C/min, and held at 250 °C for 5 min. The volatiles were identified by comparing the mass spectra and retention times with authentic standards. Quantification was based on peak areas and the quantity of internal standard using Agilent ChemStation Data Analysis Application. Analysis of variance was performed by SPSS software using Duncan test (P = 0.05).

The collection of headspace floral volatiles and GC–MS analysis were carried out as previously described (Yue et al., 2014 (link); Ke et al., 2021 (link)). In short, entire flowers from each stage were placed in a 500 mL glass bottle, and 1.728 μg (microgram) ethyl caprate was added as an internal standard. The glass bottle was stilled for 30 min, and then a polydimethylsiloxane (50/30 μm divinylbenzene/carboxen) fiber (Supelco) was injected into the glass bottle to trap volatiles for 30 min. Thereafter, the adsorbed volatile compounds were analyzed using a GC–MS system with an Agilent 7890A GC and Agilent 5975C MSD as previously explained (Yue et al., 2015 (link), 2021 (link)).

Isotopic fractions of DIC in the liquid media were measured based on a modified headspace method [49 ]. 3 ml of culture liquid were collected from the bioreactor with a syringe and directly filtered through a sterile 0.45 µm filter (Whatman, cellulose acetate) and 26 G needle into a 60 ml bottle containing 1 ml 6 M HCl and crimp sealed with a rubber stopper. Prior to adding the liquid sample, bottles and HCl were flushed with either 100% N₂ or Ar gas to void the headspace of background CO₂. Samples were equilibrated with the acid in the bottles for at least 1 hour at room temperature to drive all DIC into the gas phase. 50 µl of the bottles headspace was then injected with a gas tight syringe (Hamilton) into a gas chromatograph (Agilent 6890 equipped with 6 ft Porapak Q columns) at 80 °C with helium as a carrier gas at a flow rate of 24 ml min⁻¹, coupled to a mass spectrometer (Agilent 5975 C MSD; Agilent, Santa Clara, CA) to determine the isotopic fractions of ¹²CO₂ and ¹³CO₂.
Reactor headspace gas samples were collected manually using a gas tight syringe and needle (Hamilton) through a rubber septum in the reactor headplate and directly injected into the GC-MS as described above.

Isotopic fractions of dissolved inorganic carbon (DIC) in the liquid medium were measured based on a modified headspace method (47 (link)). One milliliter of liquid culture was collected from the batch incubations with a syringe, directly filtered through a sterile 0.45-μm-pore filter (Whatman; cellulose acetate) and a 26G needle into a vial (12 mL) (Exetainer; Labco, Ltd., United Kingdom) containing 340 μL 6 M HCl, and crimp sealed with a rubber stopper. Prior to addition of the liquid sample, vials with HCl were flushed with 100% N₂ gas to void the headspace of background CO₂. Samples were equilibrated with the acid in the bottles for at least 1 h at room temperature to drive all DIC into the gas phase. Fifty microliters of the bottles’ headspace was injected into a gas chromatograph (Agilent 6890 equipped with a 6-ft Porapak Q column) coupled to a mass spectrometer (Agilent 5975C MSD; Agilent, Santa Clara, CA) with a gas-tight syringe (Hamilton, Reno, NV, USA). The gas chromatograph was set at 80°C with helium as a carrier gas at a flow rate of 24 mL min⁻¹ to determine the isotopic fraction of ¹³CO₂.