Gc 6850

Methanolysed PHB (methyl 3-hydroxybutanoate) was analysed via GC (GC 6850, Agilent Technologies) equipped with a 7683B Series injector coupled to an FID. A DB-WAX column (15 m × 0.32 mm × 0.50 μm; Agilent Technologies) was used for metabolite separation with helium as the carrier gas at a column flow of 1.8 ml min⁻¹. The following temperature gradient was applied: 90 °C for 1 min, 1.75 min from 90 to 230 °C, 3 min at 230 °C, 1.27 min to 90 °C and then 1 min at 90 °C. For each sample, a 1 μl volume was injected. The split ratio was 2.0 and the detector temperature was 270 °C. Peaks were confirmed using standards. Benzoic acid was used as the internal standard to correct for methodological variation.

Terpenes were extracted independently from fresh leaves of three plants after overnight incubation in hexane (2 mL per gram), supplemented with camphor as internal standard to allow for quantification, or by hydro-distillation. An Agilent GC 6850 gas chromatograph coupled with an Agilent 5973 ion trap mass detector was used for GC-MS analyses of enzymatic activities and transiently transformed tobacco leaf extracts. The instrument was equipped with a 30 m × 0.25 mm apolar capillary column DB5. Temperatures of injector and detector were 250°C. Helium was used as the carrier gas at a flow rate of 1.0 mL.min⁻¹. A volume of 2 μL of extract was injected with a split ratio of 1:2. Oven temperature settings were: 4 min at 60°C after injection followed by a 4°C.min⁻¹ temperature ramp from 60°C to 240°C. Temperature was then kept on hold at 240°C for 5 min. Molecule identification was performed using Wiley, NIST 05 and IFF-LMR mass spectra databases. GC-MS analyses in IFF analytic laboratory (Grasse) were performed on a GC-MS 6890-MS Agilent 5973. Most of the parameters were common with those applied in LBVpam except for the temperature ramp that was 60°C during 10 min, then 2°C.min⁻¹ from 60°C to 300°C. Temperature was then kept on hold at 300°C for 3 min. A volume of 2 μL of extract was injected with a split ratio of 1:110.

Symbiosis phenotypes such as nodulation frequency, plant dry weight, and nitrogenase activity were determined in M. truncatula WT plants 8 weeks post‐inoculation with S. meliloti gene deletion mutants and wild‐type control strains. Nitrogenase activity of M. truncatula was determined by measuring acetylene reduction to ethylene as previously reported. Plants were placed into 50 cm³ sealed vials to which acetylene was injected to a final concentration of 2%. Ethylene production was measured after 3 h and 5 h of incubation on an analytic gas chromatograph instrument (GC6850, Agilent Technologies). The ethylene background was monitored and systematically removed from every measurement. To determine the average ethylene production per nodule, ethylene production was normalized by nodule number and duration of acetylene incubation. Nodulation frequency was determined by manually counting all the visible nodules per plant. For dry weight determination, plant shoots were dried at 85°C for at least 20 h prior individual plant weight measurement (ABS 80‐40 N, KERN). Measurements from replicas were averaged and normalized to the S. meliloti CL150 WT reference strain. Nitrogenase activity, plant dry weight, and nodulation frequency were determined for at least 22 independent plants for each assayed S. meliloti strain.

According to the modified method of Xiong et al. [28 (link)], the fatty acid profile of beef liver paste was analyzed by gas chromatography (GC-6850, Agilent, Santa Clara, CA, USA). The nitrogen flow rate was set at 1.2 mL/min and the air flow rate was set at 450 mL/min. The column was isothermally operated at 5 °C/min at 140–240 °C and held at 240 °C for 15 min. The injection temperature and detector temperature were 260 °C and 250 °C, respectively. Hydrogen (40 mL/min) was used as the carrier gas. The isolated fatty acids were identified by comparison with the retention time of the standard solution. The results are reported in grams of fatty acids per 100 g of beef liver paste.

The viscosity of the pyroligneous acids was recorded using a Brookfield viscometer. The gas chromatography/mass spectroscopy (GC-MS) analysis of the pyroligneous acid was performed using the Agilent Technologies GC 6850 with a 5975 C mass selective detector (MS) and a 30 mm × 250 mm × 0.25 mm capillary column. The oven temperature was started at 50 °C for 2 min, was increased to 250 °C at a rate of 6 °C/min and was held at this temperature for 5 min. The injector port temperature and the detector temperature were set at 250 °C. The carrier gas, helium, was set at a flow rate of 1.0 mL/min. An injection volume of 1.0 µL was used. Components were identified by matching their mass spectra with those recorded in the NIST mass spectra library.

To evaluate permethrin concentrations, collected fabric punches will be placed in individual mylar bags and stored in an opaque container at 20°C until transport to East Carolina University. Swatches will then be transferred to individual 60 mL amber glass vials containing 40 mL acetone and soaked for 6 hours to elute permethrin. A portion of the extract (1 pL) will be analyzed directly by capillary GC with flame ionization detector using an Agilent GC 6850 in accordance with previously published protocols [58 (link)].

RBC fatty acids composition was analyzed by gas chromatography—flame ionization detection (GC-FID; GC 6850 Agilent Technologies, Santa Clara, CA, USA), as previously described (Mazzucco et al., 2010 (link)), in the Laboratory of the University of Trieste, Italy. Laboratory personnel were unaware of the clinical status of the participants (i.e., BC patients or controls, type of intervention, dietary habits). Specific fatty acids standards were used to identify fatty acid methyl esters (FAME) by retention times in erythrocyte samples. Area-under-the-curve of each selected peak was determined by highly standardized hand integration performed using commercial software (HP Chem station; Agilent Technologies, Santa Clara, CA, USA).
RBC membrane level of each fatty acid was expressed as percent ratio between area-under-the-curve of each selected FAME peak and the sum of all measured FAME peaks.
Omega 3 index was calculated as sum of the DHA + EPA in erythrocyte membranes, indicating a percentage of total erythrocyte fatty acids.