For total lipid extraction, diet samples were homogenized in chloroform and methanol 2 : 1 (v/v), mixed, and incubated at room temperature for 5 min. Then, additional volumes of 1.25 mL chloroform and 1.25 mL deionized H2O were added, and finally, following being vigorously homogenized for 3 min, samples were centrifuged at 1000 rpm for 5 min at room temperature. The chloroform layer was dried under N2, and the total extract was converted into methyl esters and was analyzed in gas chromatography (GC), coupled with a flame ionizer detector (FID), (Varian GC 3900) and fatty acid profile was determined by calculating the retention time, using a pattern of fatty acids with known retention time (Supelco, 37 Components). The addition was initiated at a temperature of 170°C maintained for 1 minute and then a ramp of 2.5°C/min to a final temperature of 240°C, which was maintained for 5 minutes. The injector and detector were maintained at 250°C and 260°C, respectively. We used a column CP wax 52CB, with a thickness of 0.25 mm, internal diameter of 0.25 μm, and length of 30 m,with hydrogen as the carrier gas at a linear velocity of 22 cm s−1.
Gc 3900
Manufactured by Agilent Technologies
Sourced in United States, Germany
The GC 3900 is a gas chromatograph that is designed for the separation and analysis of chemical compounds. It is a versatile instrument that can be used in a variety of applications, including environmental analysis, food and beverage analysis, and pharmaceutical research. The GC 3900 features advanced technology, such as a high-performance oven and precise temperature control, to ensure accurate and reliable results.
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6 protocols using gc 3900
1
Lipid Extraction and Fatty Acid Profiling
2
GC Analysis of Sugar and Acid in Juice
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Analysis of simple sugar and acid contents was performed using a GC approach following the protocol by Bartolozzi et al. [29 (link)]. In detail, 500-µL juice aliquots from each sample were added to 450 µL of imidazole buffer (pH 7.0) and stored at 4 °C until analysis. Aliquots of 500 µL of each diluted juice were dried under a continuous air flow prior to derivatization, and 1 µL of the resulting solution was injected into a VARIAN GC 3900 equipped with electronic flow control (EFC), flame ionization detector (FID), CP-Sil 5 CB capillary column (30 m long), and a CP8410 autosampler (helium as carrier gas and nitrogen as make up gas).
3
Serum Free Fatty Acid Profiling
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The serum free fatty acid profile was analyzed by gas chromatography (Varian GC 3900) coupled in flame ionization detection (FID) with a CP-8410 autosampler (Walnut Creek, CA, USA). The formation of fatty acids methyl esters (FAMEs) was performed with acetyl chloride (5% HCl in methanol) [35 ]. Samples were analyzed on a capillary column (CP Wax 52 CB, Varian, Lake Forest, CA, USA); 0.25 µm thickness, inside diameter 0.25 mm and 30 m length). Hydrogen was used as a carrier gas at a linear velocity of 22 cm/s. The temperature program was 170 °C for 1 min, followed by 2.5 °C/min of the increase until 240 °C and a final hold time of 5 min. The temperatures used were 250 and 260 °C for injector and FID, respectively. Fatty acids were identified by comparing the retention time using a known standard of FAMEs. (Supelco, 37 components; Sigma–Aldrich; Mixture, Me93, Larodan and Qualmix, PUFA fish M, Menhaden Oil, Larodan). Data were expressed as percentages of total fatty acids.
4
Quantification of Octyl Acetate in Fermentation
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The organic phase was extracted into 1 mL ethyl acetate containing 2 mM decanol as an internal standard. Samples were analysed using via gas chromatography (Shimadzu GC-2010 in the case of high cell density experiments; Varian GC-3900 in the case of low cell density experiments) using a FactorFour VF-5 ms column (95% dimethylpolysiloxane, 60 m × 0.32 mm). A significant unexpected peak was noticed in high cell density experiments, and was identified as octyl acetate via GC–MS of selected samples. The concentration of octyl acetate was calculated by comparing the areas of the 1-octanol and octyl acetate peaks.
The run programme on the Shimadzu was as follows: 1 μL injection volume, split ratio of 50, 2.4 mL min−1 H2, oven temperature held at 60 °C for 9 min, ramped at 25 °C min−1 to 180 °C, held for 1.2 min. The run programme on the Varian was as follows: 0.5 μL injection volume, split ratio of 80, 1 mL min−1 N2, oven temperature held at 60 °C for 3 min, ramped at 30 °C min−1 to 180 °C, held for 7 min.
The run programme on the Shimadzu was as follows: 1 μL injection volume, split ratio of 50, 2.4 mL min−1 H2, oven temperature held at 60 °C for 9 min, ramped at 25 °C min−1 to 180 °C, held for 1.2 min. The run programme on the Varian was as follows: 0.5 μL injection volume, split ratio of 80, 1 mL min−1 N2, oven temperature held at 60 °C for 3 min, ramped at 30 °C min−1 to 180 °C, held for 7 min.
5
Analytical Methods for Livestock Feeds
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Frozen fecal samples were defrosted, dried under mild conditions (50 °C) and milled (1.0 mm, Retsch ZM200, Haan, Germany) prior to chemical analysis. Milled feeds and feces were subject to analyses of DM, organic matter (OM), crude ash (Ash), crude protein (CP), crude fiber (CF), starch and ether extract after acid hydrolysis (EEh) according to standard procedures [25 ]. Phosphorus was determined photometrically (U5100-Spectrophotometer-Hitatchi, Metrohm, Vienna, Austria) using the vanado-molybdate method to measure color intensity at 436 nm and calcium (Ca) was determined by flame atomic absorption spectrophotometry (AAnalyst 200, Perkin Elmer, Brunn am Gebirge, Austria) following nitric acid wet digestion via microwave (MLS-ETHOS plus Terminal 320, Leutkirch, Germany). Gross energy (GE) contents were determined by bomb calorimetry (IKA-Kalorimeter C400, Bartelt, Graz, Austria). To determine fermentation characteristics, organic acids were measured in ensiled sorghum samples by gas chromatography (Varian GC 3900, Munich, Germany) according to Zhao et al. [26 (link)]. Ammonia-N concentration was determined by UV-absorption (Hitachi U5100 Spectrophotometer, Tokyo, Japan) using a commercial kit (R-Biopharm, Darmstadt, Germany). All chemical analyses were performed in duplicate.
6
Quantification of Volatile Fatty Acids
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Volatile fatty acids (VFAs), e.g. acetate and butyrate, were quantified using a gas chromatograph (GC 3900 Varian) equipped with a flame ionization detectoras previously described by Rafrafi et al ( 2013) .
The errors associated with OD, DW and organic acid measurements were 2%, 6% and 5%, respectively.
The errors associated with OD, DW and organic acid measurements were 2%, 6% and 5%, respectively.
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