The concentrations of the short chain fatty acids (SCFAs), including acetate, propionate, butyrate, and n-valerate, and branched chain fatty acids (BCFAs), isobutyrate and isovalerate in the cecal content were determined using an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmington, DE, USA). Two milliliters supernatant was prepared by reconstituting all cecal content of each animal in 0.01 M phosphate buffer solution (PBS) followed by centrifugation at 9000 g for 5 min at 4 °C. The supernatant was acidified with a 1/10 volume of 50% H2SO4 and extracted with ethyl ether. The concentrations of SCFAs and BCFAs were determined in the organic phase using an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmington, DE, USA) equipped with a polar HP-FFAP capillary column (0.25 mm × 0.25 mm × 30 m) and flame ionization detector (Agilent Technologies, Wilmington, DE, USA). Helium was used as the carrier gas. The initial oven temperature was 140 °C, which was maintained for 10 min and then raised to 165 °C at 5 °C/min, increased to 270 °C at 25 °C/min, and held at this temperature for 2 min. The detector temperature was 280 °C, and the injector temperature was 250 °C. Data handling was performed with an Agilent ChemStation (version G2070AA, Agilent Technologies, Wilmington, DE, USA).
Agilent 6890n gas chromatograph
Manufactured by Agilent Technologies
Sourced in United States, China
The Agilent 6890N gas chromatograph is a laboratory instrument designed for the separation, identification, and quantification of complex mixtures of volatile and semi-volatile compounds. It utilizes a temperature-controlled system to separate the components of a sample based on their interaction with a stationary phase within a capillary column. The instrument is equipped with a variety of detectors, such as flame ionization detector (FID) and mass spectrometer (MS), to identify and measure the concentrations of the separated components.
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Lab products found in correlation
Agilent 5973 mass selective detector, by Agilent Technologies (5 mentions)
Hp 5ms capillary column, by Agilent Technologies (4 mentions)
Hp 88 capillary column, by Agilent Technologies (2 mentions)
Agilent 5973n mass selective detector, by Agilent Technologies (2 mentions)
5973n mass spectrometer, by Agilent Technologies (2 mentions)
Agilent 7683 autosampler, by Agilent Technologies (2 mentions)
Up100h, by Hielscher (2 mentions)
Agilent chemstation, by Agilent Technologies (1 mentions)
Hp ffap capillary column, by Agilent Technologies (1 mentions)
Agilent 7683, by Agilent Technologies (1 mentions)
Pegasus 3 time of flight mass spectrometer, by Leco (1 mentions)
Combipal autosampler, by CTC Analytics (1 mentions)
Db wax column, by Agilent Technologies (1 mentions)
Milkoscan 133, by Foss (1 mentions)
Digestion system dt220, by Foss (1 mentions)
Kjeltec 8400 analyzer unit, by Foss (1 mentions)
Agilent 5973 network msd, by Agilent Technologies (1 mentions)
30 m db 5 column, by Agilent Technologies (1 mentions)
Omegawax 250 capillary column, by Merck Group (1 mentions)
Filter paper, by Cytiva (1 mentions)
59 protocols using agilent 6890n gas chromatograph
1
Determination of Cecal Short-Chain Fatty Acids
2
GC-MS/MS Analysis of Compounds
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Sample extracts were also analyzed on a 6890N Agilent gas chromatograph (Agilent Technologies Inc., Palo Alto, CA, USA) coupled to an Autospec NT high resolution mass spectrometer (EBE geometry) (Micromass, Manchester, UK), using a EI source and operating in the SIM mode.
Additionally, sensitivity and specificity for selected compounds with traditional GC-(EI)MS/MS methodology was tested. For that purpose, standards were injected on a 6890N Agilent gas chromatograph (Agilent Technologies Inc., Palo Alto, CA, USA) equipped with an autosampler (Agilent 7683) coupled to a triple quadrupole mass spectrometer, Quattro Micro GC (Micromass, Boston, MA) operating in EI mode.
Chromatographic conditions in both instruments were the same as in GC-(APCI)MS/MS analysis.
Additionally, sensitivity and specificity for selected compounds with traditional GC-(EI)MS/MS methodology was tested. For that purpose, standards were injected on a 6890N Agilent gas chromatograph (Agilent Technologies Inc., Palo Alto, CA, USA) equipped with an autosampler (Agilent 7683) coupled to a triple quadrupole mass spectrometer, Quattro Micro GC (Micromass, Boston, MA) operating in EI mode.
Chromatographic conditions in both instruments were the same as in GC-(APCI)MS/MS analysis.
3
Metabolomic Analysis of Leaf Samples
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Frozen leaf samples harvested after 4 h illumination were ground in 2 ml microcentrifuge tubes using stainless steel beads and a Retsch Mixer Mill MM 400 (Retsch, Haan, Germany) under cryogenic conditions. From these samples, metabolites were extracted, derivatized, and analyzed via gas chromatography–time-of-flight mass spectrometry (GC-TOF-MS) analyses as described before (Lisec et al., 2006 (link)). The GC-TOF-MS system consisted of a CTC CombiPAL autosampler (CTC Analytics, Zwingen, Switzerland), an Agilent 6890N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) and a LECO Pegasus III time-of-flight mass spectrometer running in EI+ mode (Leco Instruments, St. Joseph, MI, USA). Metabolites were identified by comparison with database entries of authentic standards (Kopka et al., 2005 (link); Schauer et al., 2005 (link)) using TagFinder software (Luedemann et al., 2012 ). The peak intensity of a representative fragment was normalized with that of the internal standard ribitol and sample fresh weight (FW) and referred to as relative abundance. The parameters used for the peak annotation are listed in Supplementary Table S1 according to Fernie et al. (2011) (link).
4
Liver Fatty Acid Profiling by GC-MS
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The fatty acid profile was measured by gas chromatography-mass spectrometry (GC-MS) as previously described [18 (link),59 (link),60 (link),61 (link)]. In short, snap-frozen liver tissue samples were pestled in liquid nitrogen and freeze-dried overnight. Aliquots of two to five milligrams of tissue dry weight were hydrolyzed using the fatty acid methyl ester method (FAME) according to Bode et. al. [62 (link)]. GC-MS was carried out on an Agilent 6890N gas chromatograph (Agilent Technologies, Waldbronn, Germany) equipped with a 7683B split/splitless injector with autosampler (Agilent Technologies) and coupled to a 5973 electron impact mass selective detector (Agilent Technologies) as previously described [18 (link)]. Absolute amounts of FAs were quantified by integration of the peaks in relation to the integral of methyl-nonadecanoate (74208, Merck, Taufkirchen, Germany) as an internal standard and to liver tissue dry weight.
5
Fatty Acid Extraction and Analysis
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Extraction of fatty acids was performed as previously described [30 (link)]. Firstly, 2 g of frozen tissue powder was mixed with 15 ml of n-hexane:isopropanol (3:2, v/v) and 7.5 ml of 6.7% Na2SO4, followed by centrifugation for 10 minutes. The supernatant was evaporated to dryness with nitrogen. Methanol:toluene:H2SO4 (88:10:2, v/v/v) was added to produce fatty acid methyl esters (FAMEs). After cooling, 1 ml of heptane with 0.5 g anhydrous Na2SO4 was added for FAME extraction. To detect fatty acids, an Agilent 6890 N gas chromatograph equipped with a flame ionization detector and a DB-WAX column (0.25 mm, 30 m, 0.25 m; J & W Scientific) was used. The injector and detector temperatures were 230°C. The initial oven temperature was 50°C, increased to 200°C at 25°C min−1, then increased to 230°C at 3°C min−1. Nitrogen was used as the carrier gas at 1 ml min−1. Exogenous heptadecanoic acid was added as internal standard.
6
Detailed Milk Composition and Fatty Acid Profile
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Chemical composition (fat, protein, lactose, total solids, and total solids no-fat) was analyzed using an IR spectrometer (MilkoScan 133; FOSS, Hillerød, Demark) after proper validation by Kjeldahl [22 ] and Gerber [23 ] methods. Casein content was analyzed according to the reference method, ISO 17997-1/IDF 29 [24 ] using a FOSS Kjeltec™ 8400 Analyzer Unit and a FOSS Digestion System DT220 (FOSS, Hillerød, Denmark). Fat corrected milk (FCM6%) and energy corrected milk (ECM) yield were calculated using the following formulas:
Fat corrected milk (FCM) in 6% based on the Equation (1)
where F = fat content (%) and M = milk yield in kg [3 (link)].
Energy corrected milk (ECM) yield based on the Equation (2) [25 (link)].
FA profile was performed using an Agilent 6890 N gas chromatograph equipped with an HP-88 capillary column (60 m × 0.25 mm i.d. with 0.20 µm film thickness, Agilent). Information about the temperature program and standard used are available by Mavrommatis and Tsiplakou [26 (link)]. The groups of FA were defined as follow [24 ]:
Fat corrected milk (FCM) in 6% based on the Equation (1)
where F = fat content (%) and M = milk yield in kg [3 (link)].
Energy corrected milk (ECM) yield based on the Equation (2) [25 (link)].
FA profile was performed using an Agilent 6890 N gas chromatograph equipped with an HP-88 capillary column (60 m × 0.25 mm i.d. with 0.20 µm film thickness, Agilent). Information about the temperature program and standard used are available by Mavrommatis and Tsiplakou [26 (link)]. The groups of FA were defined as follow [24 ]:
7
Solubility Parameter Determination of Alkali Lignin/PVA Composites
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The solubility parameter was measured using an Agilent 6890 N Gas Chromatograph (Agilent Technologies, Beijing, China). Mixtures of PVA and alkali lignin and 6201 red diatomaceous earth (1:10 w/w) were uniformly mixed with moderate acetone and then were dried. The blends were then packed in a solvent-rinsed stainless steel column by using a mechanical vibrator and a vacuum pump. After packing, the column was conditioned overnight in a stream of nitrogen at 130 °C. Probe solvents were injected manually using a 1 µL Hamilton syringe, with column temperatures maintained at 110, 120, 130, 140, and 150 °C. To achieve infinite dilution, the injection volume for each probe solvent was 0.5 µL. At least three injections were made for each probe solvent, and the average retention time, tR, was used for the calculations of solubility parameters of alkali lignin/PVA composites.
8
GC-MS Analysis of Organic Compounds
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GC-MS analysis: Analytical GC-MS system consisting of an Agilent 6890 N gas chromatograph and a mass selective detector (Agilent®5973 Network MSD, Agilent Technologies, Santa Clara, CA, USA) was used. Injection was done with Agilent®7683 Series Injector (Split 1:40 at 250 °C, 2.0 µL; carrier gas: helium 1.1 mL/min (60 kPa) at 110 °C; pressure rise: 6 kPa/min). The MS operated in the electron impact mode with an ionization energy of 70 eV. The oven program started with 1 min at 70 °C, the oven temperature was increased at 3 °C/min to 220 °C. Full scan mass spectra were acquired from 35–350 m/z at a rate of 4.5 scans/s and with a 5.00 min solvent delay. Chromatography was performed using a 30 m DB-5 column (J & W Scientific, Folsom, CA, USA) with 0.25 mm i.d. and 0.25 µm film thickness. The detected compounds were identified by processing of the raw GC-MS data with ChemStation G1701CA software and comparing with NIST mass spectral database 2.0 d (National Institute of Standards and Technology, Gaithersburg, MD, USA) and from retention indices and mass spectra of standard compounds. Relative amounts of detected compounds were calculated based on the peak areas of the total ion chromatograms (TIC).
9
Chicken Breast Lipid Profiling
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Lipids were extracted from chicken breast meat (1 g) with the addition of 20 μL of BHA and 15 mL of Folch’s solution (2:1 mixture of chloroform and methyl alcohol, v/v). The homogenates were filtered through filter paper (Whatman No. 1). The filtrate was vortexed with 3 mL KCl (0.88%) and incubated overnight in the dark to separate the 2 layers. The lower lipid-containing layer was condensed with N2. A 25 mg lipid sample was mixed with 1.5 mL of 0.5 N NaOH (in methyl alcohol) in glass tubes and heated to 100°C for 5 min. The mixture was mixed with 1 mL 10% boron trifluoride and heated to 100°C for 2 min. After the addition of 2 mL iso-octane and 1 mL saturated NaCl (40 g NaCl/100 mL distilled water), samples were centrifuged at 783 X g for 3 min. Iso-octane extract aliquots were injected into an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmington) equipped with an Omegawax 250 capillary column (30 m × 0.25 mm × 0.25 μm, Supelco, Bellefonte, PA). The carrier gas, flow rate, and split ratio were helium (99.99%), 1.2 mL/min, and 1:100, respectively. The analytical temperatures of the injector and flame ionization detector were 250°C and 260°C, respectively. The optimized column temperature program was as follows: initial temperature of 150°C, held for 2 min; gradual increase in temperature to 220°C at a rate of 4°C/min, held at 220°C for 30 min.
10
Fatty Acid Composition Analysis
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Fatty acid composition was determined by producing fatty-acid methyl esters (FAMEs). Total fatty acids were extracted via the chloroform-methanol (2:1, v/v) method and incubated at 95 °C for 1 h in 0.2 mL of toluene, 1.76 mL of methanol and 0.04 mL of H2SO4 [29 (link),30 (link)]. The concentration and composition of FAMEs were evaluated, as described previously [31 (link)]. Briefly, Gas Chromatography/Mass Spectrometry (GC/MS) was performed using an Agilent 6890N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with an HP-5MS column (30 m × 0.25 mm I.D. × 0.25 μm, Agilent) containing ultrapure helium as a carrier gas, and 1 μL of each sample was injected at a 10:1 split ratio. The initial oven temperature was set to 150 °C, increased to 310 °C at 8 °C per min, and held for 15 min at this temperature. Mass detection was performed as previously described for a JMS-GC System [31 (link)]. Fatty acid abundance was expressed as the percent of total fatty acids in the N2 control strain for each sample.
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