The analysis was performed as previously described by Zhao et al.20 (link) with some modifications. One gram of stool was homogenized with 5 ml of distilled water for 3 min. The pH was adjusted to 2–3 with 5 m HCl, and the samples were left at room temperature for 10 min, with occasional stirring. The suspension was centrifuged for 20 min at 5000 r.p.m., and the supernatant was removed. Ethyl butyric acid-2 was added as an internal standard at a final concentration of 1 mm . The detection and quantification of the SCFAs were performed in a gas chromatograph (Agilent Technologies, Santa Clara, CA) equipped with a FID detector and a Stabilwax capillary column (Restek, Bellefonte, PA). The separation of the SCFAs was carried out in a temperature range of 100–200 °C with an initial heating at 100 °C for 0.5 min and a rise to 180 °C at 8 °C min and held at 200 °C for 5 min. Nitrogen was used as a gas carrier and the temperature of the injector was 200 °C. For calibration, aqueous solutions of acetic, propionic, butyric, isobutyric, valeric, and isovaleric acids (Restek) were used.
Stabilwax capillary column
Manufactured by Restek
Sourced in United States, Japan
The Stabilwax capillary column is a high-performance, fused-silica column designed for the separation of polar compounds. It features a Carbowax-type stationary phase for excellent peak shape and resolution of polar analytes.
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5 protocols using stabilwax capillary column
1
Quantitative Analysis of Gut Short-Chain Fatty Acids
2
GC-MS Analysis of Essential Oils
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GC-MS analyses were carried out using a Perkin Elmer Clarus 500 GC equipped with a flame ionization detector and coupled with a Clarus 500 mass spectrometer. A Stabilwax capillary column (Restek, Bellefonte, PA, USA) was used with helium as carrier gas (1.0 mL/min). GC oven temperature was kept at 60°C for 5 min and programmed to 220°C at a rate of 5°C/min, and kept constant at 220°C for 30 min. Mass spectra were acquired over 40–500 amu with ionizing electron energy 70 eV. In all, 1 μL of the EO was diluted in 1 mL of methanol and 1 μL of the solution was injected into the GC injector at 280°C. The identification of compounds of EOs was performed by comparing mass spectra with those reported in Nist and Wiley libraries. Linear retention indices were calculated after injection of C8–C30 aliphatic hydrocarbons mixture under the same conditions described above and compared with available linear retention indices data in the literature.
3
Fatty Acid Profiling of Seed Samples
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About 1 g of finely chopped seed samples were taken for initial extraction with chloroform and methanol (2:1) followed by agitation at room temperature for 1 h and centrifuged for 5 min at 3500× g. Fatty acid profiling was performed at the School of Agriculture and Food sciences, University of Queensland laboratory. The GC-MS (Shimadzu QP2010, Shimadzu Coporation, Tokyo, Japan) was used at oven temperature of 100 °C, injector temperature 250 °C, total program time was 39 min, and helium used as the carrier gas. The inlet pressure used for gas chromatography was 0.4 kPa, at linear gas velocity of 42.7 cm/s, column (Restek stabilwax capillary column; 30 m × 0.25 mm ID × 0.5 µm film thickness) flow 1.10 mL/min with a split ratio of 1:1 and injection volume of 0.2 µL. For mass spectrometry, the ion source temperature used was 200 °C, the interface temperature was 250 °C and the mass range was 35–500 atomic mass units. Identification of the compounds was done by comparing their retention times and mass spectra with corresponding data from a standard food industry FAME Mix (Restek Corporation, Bellefonte, PA, USA).
4
Lactose and Ethanol Quantification
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Before analyses, the samples were centrifuged at 12,000 rpm for 5 min at room temperature. The concentration of lactose was determined by the Nelson-Somogyi method [25, 26] . The concentration of ethanol was determined by gas chromatography with a flame ionization detector (GC-FID), using a Shimadzu GC-2025 system (SHIMADZU Co., Kyoto, Japan), Stabilwax capillary column (30 m, 0.25 mm i.d., 0.25 µm d.f.; Restek Co., Pennsylvania, USA) at a split ratio of 1:25, and helium as the carrier gas at 35 cm/s. The injector and FID were heated to 230°C. The column temperature program was as follows: 50°C for 5 min, then raised at 15°C/min to 200°C and held for 3 min. n-Amyl alcohol (0.01% v/v) was used as the internal standard and 1 µL samples were injected.
5
Garlic Oil Fatty Acid Profile Analysis
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The fatty acid profile of garlic oil was determined by gas chromatography by injecting the mixture of the methyl esters derived from the fatty acids of the samples. The methylation process consisted of reacting samples of 10 mg of oil with 0.5 mL of a 5% solution of sodium methoxide in methanol. The reaction was carried out by heating at 65 °C for 30 min and stopped by adding 0.5 mL of a saturated solution of NaCl. The methyl esters were extracted with 1 mL of hexane (HPLC grade) and subsequently analyzed on a Varian gas chromatography (model 3400) equipped with a split injection system set at 250 °C and a flame ionization detector set at 300 °C. A Stabilwax capillary column (Restek Corp.) of 60 m long × 0.25 mm and 0.25 µm stationary phase film was used. Hydrogen was used as a carrier gas. The column temperature was increased from 150 °C to 200 °C at a rate of 10 °C/min, then to 250 °C at a rate of 3 °C/min, and finally, it was kept at 250 °C for 20 min. The individual components were identified by comparison with the retention times of the fatty acid methyl esters of a standard mixture. Each sample was analyzed in duplicate. Each reported quantity corresponds to the percentage of each fatty acid concerning the 100% of fatty acids present in the sample [51 ].
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