Cell growth was monitored by measuring optical density at 600 nm (OD600). Glucose was determined using a glucose analyzer. Extracellular metabolite concentrations were determined by HPLC [33 (link)]. The mobile phase consisted of 5 mM H2SO4 with the flow rate at 0.4 ml/min, and the column temperature was set at 65 °C. To determine the enantiomeric distribution of 2,3-BD, the fermentation samples were saturated with sodium chloride and extracted with ethyl acetate. The isomers in the extracts were then analyzed by GC-FID (Hewlett Packard) equipped with a HP-chiral 20b column as described previously [57 (link)]. The oven temperature program was as follows: 40 °C (2 min), increased to 75 °C (4 min) at 5 °C min−1, followed by a ramp of 1 °C min−1 to 80 °C (2 min), and finally, 15 °C min−1 to 230 °C (4 min). The intracellular NADH and NAD+ levels were determined by NAD/NADH Quantitation Kit (SIGMA).
Gc fid
Manufactured by Hewlett-Packard
Sourced in United States
The GC-FID (Gas Chromatography-Flame Ionization Detector) is a versatile analytical instrument used for the separation and detection of volatile organic compounds. It utilizes gas chromatography to separate the components of a sample, and a flame ionization detector to quantify and identify the separated compounds.
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8 protocols using gc fid
1
Metabolic Analysis of 2,3-Butanediol Fermentation
2
Phthalate Quantification in Liquid and Bioreactor Samples
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For liquid samples, 0.5 mL of the sample (reaction mixture) was mixed with 0.5 mL of hexane. For bioreactor experiments, 0.5 g of SMC fragments was mixed with 0.5 mL hexane. The mixture was vortexed for 10 s and centrifuged at 12,000× g for 10 min. The organic phase was collected and filtered using 0.22 μm filters and applied to GC analysis. Samples were analyzed by gas chromatography with a flame ionization detector, GC-FID (Hewlett-Packard, Palo Alto, California, CA, USA) and a J&W DB-1701 GC column (Agilent Technologies, Santa Clara, California, CA, USA). The initial column temperature was set at 45 °C; the temperature increased by 10 °C/min to 260 °C and then increased by 2.5 °C/min to 340 °C. Injector and detector temperatures were set at 270 and 290 °C, respectively. Nitrogen was used as both a carrier gas (flow rate 5.0 mL/min) and a makeup gas (flow rate 20.0 mL/min). The recovery percentages for BBP, DBP, DCP, DEP, and DEHP were 97.1, 98.4, 93.3, 96.3, and 94.9%, respectively. The remaining percentages of phthalates were calculated using the formula: [%] = (residue phthalate concentration/initial phthalate concentration) × 100.
3
Air Benzene Monitoring in Northeast Thailand
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Air benzene monitoring was done by personal sampling with an active sampler with a low flow rate control pump and using a coconut charcoal sorbent tube following the standard method of National Institute Occupational Safety and Health (NIOSH) number 1501 [17 ]. The sampling was carried out during the dry season (November–April) of Northeast Thailand. The single measurement by personal sampling was done in the 8 hour working period of each worker. Temperature ranged from 21.9 °C to 35.5 °C, humidity was 52% to 94.6%, and the wind velocity range was 0.63 to 5.75 km/hour. Benzene concentration was analyzed by gas chromatography with flame ionization detector (GC-FID) (Hewlett Packard 1996, Germany).
4
Fatty Acid Composition Analysis
5
Fatty Acid Composition Analysis Protocol
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For the fatty acid composition analysis, 10 g of each sample was mixed with 100 mL of a chloroform and methanol (2:1) solution, extracted at room temperature for 24 h, and then concentrated under reduced pressure, following the method described by Folch et al. [18 (link)]. After methyl esterification of the extracted lipids with a 14% boron trifluoride–methanol (BF3-MeOH; Sigma-Aldrich) solution, they were analyzed with a GC-FID (Hewlett Packard) coupled to an HP-88 column (100 m × 0.25 mm, 0.2 μm; Agilent Technologies). The column temperature was maintained at 140 °C for 5 min, heated at 4 °C per min, and maintained at 240 °C for 20 min. The inlet temperature was maintained at 260 °C, and the Agilent Flame Ionization Detector (FID; HP 6890 Plus; Agilent Technologies) was kept at 270 °C. N2 gas was used as the carrier gas at a flow rate of 1 mL/min, and the split ratio was adjusted to 1/50. A sample volume of 1 μL was injected, and the fatty acid peaks were confirmed by comparing the retention times of the methyl esters with those of a standard 37-component fatty acid methyl ester (FAME) mix (CRM47885; Supelco, St. Louis, MO, USA). The analyzed individual fatty acid contents were obtained by calculating the area ratio of each fatty acid to the total fatty acid area and expressed as a percentage of each fatty acid.
6
Fatty acid analysis from MCF7 cells
7
Gammarid Lipid Extraction and FAME Analysis
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After lipid extraction of adult gammarids (n=24), lipids were dried down and reconstituted in 1 mL of methanol with 2.5% sulfuric acid containing 5 µg of heptadecanoic acid methyl ester as internal standard. The mixture was incubated at 80°C for 1 h. Then, 1.5 mL of water was added. Fatty acid methyl esters (FAMES) were extracted using 750 µL of hexane and separated in a 15 m × 0.53 mm Carbowax column (Alltech Associates, Deerfield, IL, U.S.A.) on a GC-FID (Hewlett-Packard 5890 series II). The oven temperature was programmed for 1 min at 160°C, followed by a 20°C per min ramp to 190°C and a second ramp of 5°C per min to 210°C, and then maintained at 210°C for a further 6 min. FAMES retention times were determined by comparison with those of standards and quantified using heptadecanoic acid methyl ester as standard.
8
Aromatic Profile Analysis of Beer Samples
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For the sample preparation, 1 ml of each beer sample was filtered using 0.45 µm pore size cellulose ester filters (Teknokroma, Barcelona, Spain) and 100 µl of internal standard was added (concentration 500 mgl -1 ) to each.
Aromatic profiles of the beer were determined by GC-FID using an Agilent Technologies 6850 gas chromatograph (GC System Network) equipped with an integrated flame ionization detector (GC-FID) (Hewlett-Packard, Palo Santo, CA, US). A DB-624 column (60 m × 250 mm × 1.4 mm) was used, calibrated with compounds as external standards: acetaldehyde, methanol, 1-propanol, diacetyl, ethyl acetate, 2-butanol, isobutanol, 1-butanol, acetoin, 2-methyl-1-butanol, 3-methyl-1-butanol, ethyl lactate, isobutyl acetate, 2,3-butanediol, isoamyl acetate, 2-phenylethyl acetate and 2-phenylethyl alcohol. Similarly, 4-methyl-2-pentanol was used as an internal standard (all compounds of Fluka, Sigma-Aldrich Corp., Buchs SG, Switzerland). The temperature of the injector was 250 °C and the detector was 300 °C. The column temperature was 40 °C for the first 5 min, progressively increasing at 10°C/min intervals to 250 °C, with the latter remaining constant for 5 min. Hydrogen was used as the carrier gas. A microliter per sample was injected into the gas chromatograph. The limit of detection was 0.1 mg/l.
Aromatic profiles of the beer were determined by GC-FID using an Agilent Technologies 6850 gas chromatograph (GC System Network) equipped with an integrated flame ionization detector (GC-FID) (Hewlett-Packard, Palo Santo, CA, US). A DB-624 column (60 m × 250 mm × 1.4 mm) was used, calibrated with compounds as external standards: acetaldehyde, methanol, 1-propanol, diacetyl, ethyl acetate, 2-butanol, isobutanol, 1-butanol, acetoin, 2-methyl-1-butanol, 3-methyl-1-butanol, ethyl lactate, isobutyl acetate, 2,3-butanediol, isoamyl acetate, 2-phenylethyl acetate and 2-phenylethyl alcohol. Similarly, 4-methyl-2-pentanol was used as an internal standard (all compounds of Fluka, Sigma-Aldrich Corp., Buchs SG, Switzerland). The temperature of the injector was 250 °C and the detector was 300 °C. The column temperature was 40 °C for the first 5 min, progressively increasing at 10°C/min intervals to 250 °C, with the latter remaining constant for 5 min. Hydrogen was used as the carrier gas. A microliter per sample was injected into the gas chromatograph. The limit of detection was 0.1 mg/l.
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