Once isolated, biomarker n-alkanes were analyzed first by gas chromatography (Thermo Scientific Trace 1310) with a flame ionization detector (GC-FID) fitted to a 30-m TG-5SilMS (0.25 mm × 0.25 μm) fused silica column. Sample aliquots of 1 μl were injected by programmed temperature vaporizing (PTV) injector at 320 °C through a split/splitless silcosteel inlet liner. The chromatograph oven was ramped from 60 °C (1 min) to 320 °C (20 min) at 6 °C min−1. Helium was used as a carrier at 2 ml min−1, and the detector was set to a constant temperature of 320 °C. Stable isotope measurements of biomarker n-alkanes were measured by gas chromatography combustion (δ13C) or pyrolysis (δ2H) isotope ratio monitoring mass spectrometry (GC-C-irMS or GC-pyr-irMS, respectively) with a Thermo Scientific Trace 1310 GC with split/splitless injector and TriPlus RSH liquid autosampler coupled to a Delta V Plus MS (see details in Additional file 2 : Supplementary method).
Trace 1310
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Italy
The Trace 1310 is a gas chromatograph (GC) designed for accurate and reliable analysis of complex samples. It features a high-performance oven, sensitive detectors, and advanced software for efficient data processing and reporting.
Automatically generated - may contain errors
Lab products found in correlation
Tsq 8000, by Thermo Fisher Scientific (12 mentions)
Isq lt, by Thermo Fisher Scientific (7 mentions)
Isq 7000, by Thermo Fisher Scientific (6 mentions)
Il 1310, by Thermo Fisher Scientific (4 mentions)
Tsq 9000, by Thermo Fisher Scientific (4 mentions)
Tsq 8000 evo, by Thermo Fisher Scientific (4 mentions)
Tsq duo, by Thermo Fisher Scientific (3 mentions)
Thermo fisher mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Chromeleon 7, by Thermo Fisher Scientific (3 mentions)
Zb fame column, by Phenomenex (2 mentions)
Isq lt, by Phenomenex (2 mentions)
Tr 5ms, by Thermo Fisher Scientific (2 mentions)
Tg waxms column, by Thermo Fisher Scientific (2 mentions)
As 1310, by Thermo Fisher Scientific (2 mentions)
Tr 5ms column, by Thermo Fisher Scientific (2 mentions)
Db 5ms column, by Agilent Technologies (2 mentions)
Hp 5ms capillary column, by Agilent Technologies (2 mentions)
Tg waxms a gc column, by Thermo Fisher Scientific (2 mentions)
Tsq 8000 triple quadrupole mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Gc fid, by Thermo Fisher Scientific (2 mentions)
126 protocols using trace 1310
1
GC-FID and GC-IRMS Analysis of Biomarker n-Alkanes
2
Gas Chromatography-Based SCFA Quantification
3
Quantification of Cecal Short-Chain Fatty Acids
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The content and composition of cecal SCFA were determined using gas chromatography-mass spectrometry (Trace 1310 and ISQLT, Thermo, USA) following previously published protocols (22 (link)–24 (link)). The following chromatographic settings were utilized: injection volume of 1 μL; inlet temperature of 250°C; split ratio of 4:1; ion source temperature of 300°C, and transfer line temperature of 250°C. Oven temperature program: starting temperature at 90°C, then 10°C/min to 120°C and 5°C/min to 150°C, followed by a 25°C/min, 2 min climb to 250°C. Helium was used as the carrier gas, at a flow rate of 1.0 mL/min. The following mass spectrometry conditions were used: the electron ionization source of the instruments was operated with an electron energy of 70 eV and a SIM scanning mode was adopted. Recoveries and SCFA content were calculated following the method described by Giera et al. (25 (link)).
4
Optimized GC-MS/MS Analysis for Compound Identification
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GC-MS/MS analysis was performed employing a Thermo Scientific Trace 1310 gas chromatograph coupled to a triple quadrupole mass spectrometer (TSQ 8000) with an autosampler IL 1310 from Thermo Scientific (San Jose, CA, USA). Instrumental GC-MS/MS conditions were previously optimized by the authors [24 (link)]. Separation was carried out on a Zebron ZB-Semivolatiles (30 m × 0.25 mm i.d. × 0.25 μm film thickness) obtained from Phenomenex (Torrance, CA, USA). Helium (purity 99.999%) was used as carrier gas at a constant flow of 1 mL min−1. The GC oven temperature was programmed from 60 °C (held 1 min), to 100 °C at 8 °C min−1, to 150 °C at 20 °C min−1, to 200 °C at 25 °C min−1 (held 5 min), to 220 °C at 8 °C min−1 and finally to 290 °C at 30 °C min−1 (held 7 min). The total run time was 30 min. The injector temperature was set at 270 °C working in pulsed split/splitless mode (200 kPa, held 1.2 min).
The mass spectrometer detector (MSD) was operated in the electron impact (EI) ionization positive mode (+70 eV). The temperatures of the transfer line, and the ion source were set at 290 and 350 °C, respectively. The filament was set at 25 μA and the multiplier voltage was 1950 V. Selected reaction monitoring (SRM) acquisition mode was used, monitoring 2 or 3 transitions per compound (seeTable S1 ). The system was operated by Xcalibur 2.2, and Trace Finder™ 3.2 software.
The mass spectrometer detector (MSD) was operated in the electron impact (EI) ionization positive mode (+70 eV). The temperatures of the transfer line, and the ion source were set at 290 and 350 °C, respectively. The filament was set at 25 μA and the multiplier voltage was 1950 V. Selected reaction monitoring (SRM) acquisition mode was used, monitoring 2 or 3 transitions per compound (see
5
Volatile Organic Compounds Characterization
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The D. viscosa VOCs were chemically characterized using a Thermo Fisher gas chromatograph apparatus (Trace 1310) equipped with a single quadrupole mass spectrometer (ISQ LT). The capillary column was a TG-5MS 30 m×0.25 mm×0.25μm the gas carrier was helium with a flow rate of 1 ml/min. Injector and source were settled at 200°C and 260°C, respectively. The sample (1 g of fresh plant material) was incubated for 1 minute at 40° and 1 μl of the head space was injected in split mode with a split ratio of 60. The following temperature was programmed: isocratic for 7 minutes at 45°C, from 45°C to 80°C with a rate of 10°C×min, from 80°C to 200°C with a rate of 20°C×min, then isocratic for 3 minutes 200°C. Mass spectra were recorded in electronic impact (EI) mode at 70 eV, scanning at 45–500 m/z range. Compounds identification was carried out comparing the relative retention time and mass spectra of molecules with those of the libraries (NIST 2005, Wiley 7.0 etc.).
6
Fatty Acid Extraction and Methylation Protocol
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The extraction and methylation of fatty acids in samples were conducted about the method of Banco (21 (link)). The liquid obtained from the extraction was filtered through a 0.45 μm membrane filter and tested using gas chromatography–mass spectrometry (Trace1310, Thermo, United States). The GC/MS program was as follows. Chromatographic column: TG-5MS (30 m × 0.25 mm × 0.25 μm). Temperature programming: 80°C was maintained for 1 min, then increased to 200°C at the rate of 10°C/min, then to 250°C at the rate of 5°C/min, and finally increased to 270°C at the rate of 2°C/min for 3 min; inlet temperature: 290°C. Flow rate of the carrier gas: 1.2 ml/min; split ratio: no split; ion source temperature: 280°C; transmission line temperature: 280°C; solvent delay time: 5.00 min; scanning range: 30–400 amu; energy of ionization: 70 eV.
7
Cellular Lipid Quantification by GC-MS
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To measure cellular lipid content, fatty acids were extracted and derivatized to fatty acid methyl esters (FAMEs) and subsequently analyzed by GC-MS using a previously published method [52 (link)]. Briefly, 100 μg of C17:0 TAG internal standard was added to the lyophilized cell pellets. Samples were vortex at 1200 rpm at room temperature for 1 h after addition of 500 μL of methanol solution containing 1M NaOH. The solution was then neutralized by adding 160 μL of 49% sulfuric acid. Finally, by addition of 500 μL hexane FAMEs were extracted. After centrifugation at 10,000× g for 1 min, phases were separated and 100 μL of the upper hexane phase was mixed with 900 μL hexane, 1 μL of which was autoinjected for analysis on GC-MS (Thermo Scientific Trace 1310 coupled to a Thermo Scientific ISQ LT) with a ZBFAME column (Phenomenex, length: 20 m; Inner Diameter: 0.18 mm; Film Thickness: 0.15 μm).
C16:0, C16:1, C18:0, C18:1 and C18:2 fatty acids (FAs) were considered for the calculation of lipid content per cell dry weight (g fatty acid/g dry biomass) as well as the contribution of each FA to the total FA content. To this end, dilution series of the FAME mixture standard, GLC-403 (Nu-Chek Prep, Elysian, MN, USA) was used as external standards analyzed along with samples under similar conditions.
C16:0, C16:1, C18:0, C18:1 and C18:2 fatty acids (FAs) were considered for the calculation of lipid content per cell dry weight (g fatty acid/g dry biomass) as well as the contribution of each FA to the total FA content. To this end, dilution series of the FAME mixture standard, GLC-403 (Nu-Chek Prep, Elysian, MN, USA) was used as external standards analyzed along with samples under similar conditions.
8
Quantification of Neutral Triterpenes by GC-MS
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The quantification of neutral triterpenes was carried out by a GC–MS. The instrument was composed by a TRACE 1310 and a TSQ Quantum Ultra QqQ (Thermo Scientific). Analysis was carried out by a temperature gradient, and detection was made in full-mass mode (50–450 m/z scan range). The instrumental separation conditions were injection volume: 0.5 µL, injector: PTV, splitless mode, constant temperature (280 °C), split flow: 50 mL min−1, carrier gas: He, 1.2 mL min−1, capillary column: Agilent DB-1 (30 m × 0.53 mm × 5 µm); T ramp: 0′ 150 °C, 4′ 150 °C, 12.5′ 320 °C, 20′ 320 °C. Detection parameters are listed as follows: ion source: EI 70 eV, source temperature: 250 °C. GC–MS data were managed with Thermo Xcalibur 3.0 software (Thermo Scientific). For the quantification, extracted ions were 218, 203, 426, 189 m/z. Analytical response of extracted ions (218 m/z for quantitation, 203, 426, 189 m/z for confirmation) was corrected by dividing their value by the analytical response of M-TEST (ion 124 m/z) in order to avoid any systematic error of analytical sensitivity.
9
Chlorine Isotope Analysis by GC-MC-ICPMS
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For chlorine isotope analysis a multi collector-inductively coupled plasma mass spectrometer was coupled to gas chromatography (GC-MC-ICPMS) (Horst et al., 2017 (link)). The MC-ICPMS, a Neptune (Thermo Fisher Scientific, Germany), was equipped with a gas chromatograph Trace 1310 (Thermo Scientific, Germany) coupled to FID. Triplicate samples were analyzed by manual injection of 0.05–1 mL headspace with a split ratio of 1:10 with injector kept at 250°C and a carrier gas flow of 2 mL⋅min–1. For the analysis, a Zebron ZB-1 capillary column (60 m × 0.32 mm i.d., 1 μm film thickness; Phenomenex Inc.) was utilized isothermal at 100°C for 1,2-DCA, VC and cis-DCE and 120°C for PCE analysis. The separated compounds were transferred to the plasma via a Thermo Electron TransferLine (AE2080, Aquitaine Electronique, France) heated to 250°C using an auxiliary helium flow of 5 mL⋅min–1. Instrument tuning and preparation was performed daily prior to the measurements (Horst et al., 2017 (link)). Chlorine isotope ratios were determined relative to the laboratory standards [methyl chloride, TCE-2, and TCE-6 (Horst et al., 2017 (link); Renpenning et al., 2018 (link))]. The overall analytical uncertainty was <0.5%.
10
Terpene Analysis by GC-MS
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The terpene product was measured on a Thermo TRACE 1310 gas chromatograph with a TSQ8000 mass detector (Thermo Fisher Scientific, Waltham, MA, USA) in electron ionization mode. A capillary column TR-5ms with a 1.0 mL/min Helium flow rate (30 mm × 0.25 mm ID; DF = 0.25 µm; Thermo Fisher Scientific) using splitless injection. The GC oven temperature ramp is as follows: 50 °C, 2 min, 50 °C to 210 °C with 40 °C/min; 210 °C–250 °C with 5 °C/min; 250 °C–300 °C with 40 °C/min, with a 5 min hold at 300 °C. The ion trap temperature was 280 °C. Data analysis was performed with the device-specific software Xcalibur (Thermo Scientific).
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