Hp innowax gc column

A Varian 450-GC gas chromatograph was used to determine fatty acids as methyl esters. The chromatograph operated on the following parameters: split: 1:50; injector temperature: 250 °C; carrier gas: helium, flow rate 1 mL min⁻¹; Agilent HP-INNOWax GC column, 30 m × 0.53 mm, 1.0 µm film thickness; temperature program: 50 °C isothermal for 2 min; linear gradient of 10 °C min⁻¹ to 250 °C (20 min), isothermal 240 °C for 22 min; detector: a flame ionization detector (FID), 250 °C. To the injection port, 1 µL of the prepared sample was introduced. The retention times of standards was used to identify methyl esters of FAs.

A total of 50 mg of the fecal sample was homogenized with 50 μL of 15% phosphoric acid, 125 μg/mL of internal standard (isohexanoic acid) solution, 100 μL and 400 μL of ether for 1 min and centrifuged at 12,000 rpm at 4°C for 10 min. The supernatant was then collected for testing. GC-MS was performed using a TRACE™ 1310 ISQ LT GC-MS (Thermo Fisher Scientific, Waltham, MA, USA) with an HP-INNOWAX GC column (30 m × 0.25 mm ID × 0.25 μm; Agilent Technologies, Santa Clara, CA, USA) under the following conditions: sample volume, 1 μL; split ratio, 10:1; inlet temperature, 250°C; ion source temperature, 230°C; transmission line temperature, 250°C; quadrupole temperature, 150°C. The initial oven temperature was 90°C, which was increased to 120°C with a ramp rate of 10°C/min, then to 150°C with a ramp rate of 5°C/min, and finally increased to 250°C with a ramp rate of 25°C/min, and was held at 250°C for 2 min. Helium was used as the carrier gas at a flow rate of 1.0 mL/min. We used Thermo Chromeleon 7.2.10 (Novogene Bioinformatics Technology Co., Ltd.) to process the raw data coming out of the machine. The SCFA concentration was calculated from the respective peak areas of the sample and the internal standard, isohexanoic acid. The construction range and related information of calibration curves for GC-MS are provided in Supplementary Table 1.

Cells were collected in 80% (vol/vol) methanol, followed by repeated freezing and thawing. Insoluble material in the lysates was removed by centrifugation, and the supernatants were dried in a rotary evaporator and resuspended in 75 μl of pyridine. Metabolites were further derivatized by the addition of 25 μl of MTBSTFA containing 1% tert-butyl dimethyl chlorosilane at 60°C for 1 h. The samples were analysed using an Agilent DB-5MS column in an Agilent 7890/5975C GC/MS system (Agilent Technologies). Peaks representing each defined intensity were identified from known standards.
Formate was analysed by GC-MS as described previously (Lamarre et al, 2014 (link)). Briefly, cells were collected, and metabolites were extracted with ice-cold 80% (vol/vol) methanol, followed by centrifugation to remove insoluble material. The supernatants were dried in a rotary evaporator and resuspended in 50 μl of H₂O, and alkylation was carried out by mixing 20 μl of phosphate buffer with 130 μl of 100 mM pentafluorobenzyl bromide, vortexing for 1 min, and incubating the samples at 60°C for 15 min. Next, the tubes were cooled to room temperature, and 330 μl of n-hexane was added and vortexed for 1 min until the two layers were separated. A 200 μl volume of the top layer was transferred to glass inserts and analysed on a GC-MS system equipped with an Agilent HP-INNOWax GC column.