L-arginine oxidation reactions by graphene–haemin were carried out in the presence of 200 μM L-arginine and 5 mM H2O2 in a pH 7.4 PBS buffer. L-arginine oxidation reactions by graphene–haemin–GOx were carried out in the presence of 200 μM L-arginine and 5 mM glucose in a pH 7.4 PBS buffer. The product was characterized using FT-IR, GC-MS, DAF assay and chemiluminescence. For FT-IR spectroscopy, the gas-phase FT-IR spectrum of the headspace gas was taken after 2-h reaction. For GC-MS measurement, the headspace gas was injected into an Agilent 6890-5975 GC-MS with a 30 m Rt®-Q-Bound column (Resteck Co, Columbia, MD) at an operating oven temperature of 45 °C under 14.6 p.s.i. helium carrier gas. For the DAF assay, 10 μM DAF-2 was added to the reaction solution. The excitation wavelength was 448 nm. At each time interval, florescence spectra were obtained from an average of five accumulations. Peak intensities of 515 nm were also monitored continuously for reaction catalysed by graphene–haemin–GOx conjugates. For chemiluminescence, the solution after 2-h reaction was bubbled with Ar and the products were measured via a chemiluminescence NO Analyzer™, Model 280 (Sievers Instruments, Boulder, CO). In situ measurements were also carried out.
Agilent 6890 5975 gc ms
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 6890/5975 GC-MS is a gas chromatography-mass spectrometry (GC-MS) system designed for analytical applications. It combines a gas chromatograph with a mass spectrometer to provide separation, identification, and quantification of chemical compounds in complex samples. The system is capable of analyzing a wide range of volatile and semi-volatile organic compounds.
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4 protocols using agilent 6890 5975 gc ms
1
Graphene-Haemin Catalyzed L-Arginine Oxidation
2
Quantification of Glucosinolate Hydrolysis Products
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Glucosinolate hydrolysis products were analysed as described previously (Ahuja et al., 2011 (link)). The seedlings were crushed with a glass rod in MQ H2O in a 2ml screw-top vial with a PTFE/silicone septum. The mixture was left for 10min at ambient temperature for hydrolysis. A mixture of 0.5ml hexane:dichloromethane (3:2) with an internal standard (12 μg butyl-isothiocyanate) was injected through the septum into the vial, and the sample was vortexed for 50 s. After centrifugation at 3 100rpm for 2min, the solvent phase was pipetted into a 2ml screw-top vial with a PTFE/silicone septum, and concentrated under nitrogen flow to a volume of 50 μl. Agilent 6890/5975 GC-MS (Agilent Technologies Inc., Palo Alto, CA) was used for all analyses. Mass spectra were acquired in EI mode, and a mass range of m/z 39–250 was recorded. The compounds were quantified as described previously (Ahuja et al., 2011 (link)).
3
GC-MS Metabolite Derivatization Protocol
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Samples were dried in a SavantTM SpeedVac plus SC210A (Thermo Scientific) overnight and stored at –80 °C before derivatization. Dried samples were redissolved in 80 µl of methoxyamine hydrochloride in pyridine (20mg ml–1), derivatized for 90min at 30 °C, further treated with 80 µl of N-methyl-N-(trimethylsilyl)trifluoroacetamide for 30min at 37 °C, and finally transferred to 1.5ml autosampler vials with glass inserts prior to GC-MS. Separations were performed on an Agilent 6890/5975 GC-MS (Agilent Technologies) equipped with a HP-5MS capillary column (30 m×0.25mm internal diameter, film thickness 0.25 µm) (Agilent Technologies). Sample volumes of 3 µl were injected with a split ratio of 15:1. Injection and interface temperature were set to 230 and 250 °C, respectively. The GC temperature program was held isothermically at 70 °C for 5min, ramped from 70 to 310 °C at 5 °C min–1, and finally held at 310 °C for 7min (run time: 60min). The MS source was adjusted to 230 °C and a mass range of m/z 70–600 was recorded (EI mode).
4
GC-MS Analysis of Metabolomics Samples
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An Agilent 6890/5975 GC/MS (Agilent Technologies Inc., Palo Alto, CA, United States) was used to analyze all samples. GC separations were carried out using a Supelco SLB 5 ms capillary column (30 m × 0.25 mm and film thickness 0.25 μm). Sample volumes of 1 μl were injected with a split ratio of 15:1. Injection temperature was set at 230°C, and the interface was set to 250°C. The carrier gas used was He at a constant flow rate of 1 ml/min. The GC temperature program was held isothermically at 70°C for 5 min, ramped from 70 to 310°C at a rate of 5°C/min, and finally held at 310°C for 7 min (analysis time: 60 min). The MS source was adjusted to 230°C, and a mass range of m/z 70–700 was recorded. All mass spectra were acquired in electron impact ionization (EI) mode (70 eV). Upon visual inspection of GC/MS chromatograms using Agilent ChemStation software (Agilent Technologies, Waldbronn, Germany), raw data was subjected to peak detection, baseline correction, alignment of mass signals, and peak height integration using the data alignment software MetAlign (Wageningen UR, Netherlands). Metabolomics raw data have been deposited to the GNPS database (Global Natural Products Social Molecular Networking, Wang et al., 2016 (link)) with the identifier: MassIVE MSV000086585. The complete dataset can be accessed here— doi: 10.25345/C5Z19Z.
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