Whole cell esterified fatty acid determinations were done essentially as described62 (link). Briefly, 1–2 ml S. aureus cultures (OD600=≥1) were centrifuged, washed once in 0.9% NaCl containing 0.02% Triton X-100, then washed twice in 0.9% NaCl. Cell pellets were treated with 0.5 ml of 1 N sodium methoxide. Heptane (200 μl) was then added, together with methyl-10-undecenoate (Sigma-Aldrich) as internal standard, vortexed for 1 min, and centrifuged. Fatty acid methyl esters were recovered in the heptane phase. Analyses were performed in a split-splitless injection mode on an AutoSystem XL Gas Chromatograph (Perkin-Elmer) equipped with a ZB-Wax capillary column (30 m × 0.25 mm × 0.25 μm; Phenomenex, France). Data were recorded and analysed by TotalChrom Workstation (Perkin-Elmer). S. aureus fatty acid peaks were detected between 12 and 30 min of elution.
Zb wax capillary column
Manufactured by Phenomenex
Sourced in United Kingdom, France, United States
The ZB-Wax capillary column is a high-performance chromatographic column designed for the separation and analysis of polar compounds. The column features a polyethylene glycol stationary phase, which provides excellent separation and resolution of polar analytes.
Automatically generated - may contain errors
Lab products found in correlation
Autosystem xl gas chromatograph, by PerkinElmer (2 mentions)
Totalchrom workstation, by PerkinElmer (2 mentions)
Methyl 10 undecenoate, by Merck Group (1 mentions)
N butyric, by Merck Group (1 mentions)
Trace 2000, by Thermo Fisher Scientific (1 mentions)
Ek g007 22, by Phenomenex (1 mentions)
Model 6890 gc, by Agilent Technologies (1 mentions)
Me100, by Larodan (1 mentions)
Dvb car pdms spme fiber, by Merck Group (1 mentions)
Supelco 37 component fames mix, by Merck Group (1 mentions)
Cp 3800, by Agilent Technologies (1 mentions)
Gc solution software, by Shimadzu (1 mentions)
10 protocols using zb wax capillary column
1
Quantification of Bacterial Fatty Acids
2
Measurement of Fecal Short-Chain Fatty Acids
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The faecal short chain fatty acids (SCFA) acetate, propionate and butyrate, major anti-inflammatory and immunomodulatory bacterial metabolites [12 (link)], previously implicated in the aetiology of CD [13 (link)] were measured in diethyl ether extracts by gas chromatography using a TRACE™ 2000 gas chromatograph (ThermoQuest Ltd, Manchester, UK) equipped with a flame ionisation detector (250°C) and Zebron ZB-Wax capillary column (15 m x 0.53 mm x 1 μm), made of polyethylene glycol (Phenomenex, Cheshire, UK) [11 (link)]. The carrier gas was Nitrogen (30 ml/min). Internal standard (86.1 mmol/l, 3-methyl-n-valeric acid, Sigma-Aldrich, UK) and concentrated orthophosphoric acid were added to 50 mg of freeze-dried faecal material stored in 1M NaOH. The mixture was extracted three times with 3 ml diethyl ether, centrifuged and the ether layers pooled. One microlitre of ether extract was automatically injected (230°C, splitless) into the column. The column temperature was held at 80°C for 1 min, increased at 15°C/min until 210°C and held for 1 min. The chromatograms were analysed using Chrom-Card 32 version 1.07β5 (ThermoQuest, Milan, Italy). Authentic external standards were used as calibrators (166.5 mmol/l acetic, 135.0 mmol/l propionic, 113.5 mmol/l n-butyric, Sigma-Aldrich, UK). Results were presented per mass of faecal material (μmol/g) and as proportional ratio (%) to total SCFA.
3
Quantifying Short-Chain Fatty Acids in Feces and Fermentation Fluids
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Short chain fatty acids were measured in dry faeces and fermentation fluid. Extraction and analysis were carried out according to Laurentin and Edwards [50 (link)]. SCFAs were estimated using a TRACE™ 2000 gas chromatograph (Thermo Quest Ltd, Manchester, UK) equipped with a flame ionization detector (250 °C) and a Zebron ZB-Wax capillary column (15 m × 0.53 mm id × 1 µm film thickness, catalogue No.7 EK-G007 22, Phenomenex, Cheshire, UK). EK-G007 22, Phenomenex, Cheshire, UK).
4
Gas Chromatography Analysis of Fermentation Compounds
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The compounds (acetone, butanol, and ethanol) were measured with a gas chromatograph (GC) equipped with a flame ionization detector. The system was a model 6890 GC (Agilent Technologies, Santa Clara, CA, USA) with a model 7673A automatic injector, sampler, and controller (Hewlett-Packard). Alcohol compounds were separated using a ZB-WAX capillary column (30 m, 0.25 mm inside diameter, 0.25 μm film thickness; Phenomenex Inc., PA, USA). The GC oven temperature was held initially at 40 °C for 5 min, and then raised stepwise, by 15 °C/min, until it reached 150 °C. It was then raised by 50 °C/min up to 250 °C, and held for 4 min. Helium was used as the carrier gas, with an inlet pressure of 0.065 Mpa. The injector and detector were maintained at 220 °C. A 1 μL volume of supernatant from the culture broth was injected in split-injection mode at a 1:30 split ratio. Isobutanol was used as the internal standard.
5
Fatty Acid Profiling in Bacterial Strains
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Strains were grown in THY, THY-Tween 80, THY-Plasma or THY-17:1 until OD600nm = 0.4–0.5. Fatty acids were extracted and analyzed as previously described [2 (link), 3 (link), 5 (link)]. Briefly, analyses were performed in a split-splitless injection mode on an AutoSystem XL Gas Chromatograph (Perkin-Elmer) equipped with a ZB-Wax capillary column (30 m x 0.25 mm x 0.25 mm; Phenomenex, France). Data were recorded and analyzed by TotalChrom Workstation (Perkin-Elmer). FA peaks were detected between 12 and 40 min of elution, and identified by comparing to retention times of purified esterified FA standards (Mixture ME100, Larodan, Sweden). Results are shown as percent of specific FA as calculated from their proportions compared to total peak areas (TotalChrom Workstation; Perkin Elmer).
6
Headspace SPME-GC-MS Analysis of Volatile Compounds
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Sample volatiles were analyzed on a Thermo Scientific GC Mass Spectrometer (TRACE 1300 GC, USA, and ISQ series MS).
Headspace volatiles were extracted for 20 min at 50°C, using 50/30 μm DVB/CAR/PDMS SPME Fiber (Supelco, Sigma–Aldrich, United Kingdom), following desorption for 0.2 min at 250°C in splitless mode. ZB-Wax capillary column (length 30 m, inner diameter 0.25 mm, and film thickness 1 μm) (Phenomenex Inc., Macclesfield, United Kingdom) was used with temperature programmed starting at 40°C for 2 min, 8°C/min to 240°C, and held for 5 min.
Helium was used as carrier gas at a constant pressure of 18 PSI and full scan mode scanned from m/z 35–300. Identification of volatile compounds by matching retention time of pure chemical compounds and comparing its mass spectrum against reference libraries (NIST/EPA/NIH Mass Spectral Library, version 2.0, Faircom Corporation, United States).
To determine the impact of the odour of each aromatic compound relative to its concentration the OAV was calculated (Eq. 1) as:
The odour thresholds for each compound were sourced from the literature (Leffingwell and Associates, 1985 ).
Headspace volatiles were extracted for 20 min at 50°C, using 50/30 μm DVB/CAR/PDMS SPME Fiber (Supelco, Sigma–Aldrich, United Kingdom), following desorption for 0.2 min at 250°C in splitless mode. ZB-Wax capillary column (length 30 m, inner diameter 0.25 mm, and film thickness 1 μm) (Phenomenex Inc., Macclesfield, United Kingdom) was used with temperature programmed starting at 40°C for 2 min, 8°C/min to 240°C, and held for 5 min.
Helium was used as carrier gas at a constant pressure of 18 PSI and full scan mode scanned from m/z 35–300. Identification of volatile compounds by matching retention time of pure chemical compounds and comparing its mass spectrum against reference libraries (NIST/EPA/NIH Mass Spectral Library, version 2.0, Faircom Corporation, United States).
To determine the impact of the odour of each aromatic compound relative to its concentration the OAV was calculated (Eq. 1) as:
The odour thresholds for each compound were sourced from the literature (Leffingwell and Associates, 1985 ).
7
Fatty Acid Composition Analysis of Fruit Pulp
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FAs’ composition was analyzed by FA methylation of fruit pulp [92 (link)] with minor changes. Then, 300 mg of pulp were extracted with 4 mL of methylation solution (Methanol:Toluene:2,2-Dimethoxypropane:Sulfuric acid in 39:20:5:2 proportion) and 2 mL of heptane. Tubes were transferred in a dry bath for two hours to 80 °C and then let to cool down to room temperature. Then, 1.5 mL of supernatant were analyzed by GC-FID (Varian CP-3800) using a ZB-WAX capillary column 60 m × 0.25 mm × 0.25 μm film thickness (Phenomenex). The Supelco™ 37 Component FAMES Mix (Merck, Darmstadt, Germany) was used to identify key fatty acid methyl esters.
8
GC/MS Analysis of Organosulfur Compounds
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A total of 1 µL of VGE (treated before with diethyl ether 1:1 v/v, for 24 h) was injected in the GC/MS system (Varian 3900 gas chromatograph coupled to Varian Saturn 2100 MS/MS ion trap mass spectrometer, Varian, Palo Alto, CA, USA) for the organosulfur compounds analysis.
The OSC separation was carried out in a 60 m × 0.25 mm i.d., 25 µm film thickness, Zebron ZB-Wax capillary column (Phenomenex) with a column pressure of 10 psi and supplied with a helium carrier (flow rate of 1 mL/min.). The oven temperature program was the following: start at 50 °C for 1 min, ramp to 150 °C at 1 °C/min, and hold for 1 min, while the injector temperature was 200 °C. The MS temperature conditions were trap, 180 °C; transfer line, 200 °C; and manifold, 70 °C, and the MS acquisition data. The MS acquisitions were performed by electron ionization (EI) in full scan mode, a scan time of 1 s/scan, and an emission current of 10 µA.
The mass spectrometer operated in scan mode (40–650 m/z) and the NIST MS library was used to evaluate the data and organosulfur compounds identification [7 (link)].
Peak quantification was obtained using reference standard diallyl disulfide (>95% GC), R2 = 0.9982.
Organosulfur compounds in D-VGE were detected, identified, and quantified by injecting directly the extract in diethyl ether in the GC/MS system as described above.
The OSC separation was carried out in a 60 m × 0.25 mm i.d., 25 µm film thickness, Zebron ZB-Wax capillary column (Phenomenex) with a column pressure of 10 psi and supplied with a helium carrier (flow rate of 1 mL/min.). The oven temperature program was the following: start at 50 °C for 1 min, ramp to 150 °C at 1 °C/min, and hold for 1 min, while the injector temperature was 200 °C. The MS temperature conditions were trap, 180 °C; transfer line, 200 °C; and manifold, 70 °C, and the MS acquisition data. The MS acquisitions were performed by electron ionization (EI) in full scan mode, a scan time of 1 s/scan, and an emission current of 10 µA.
The mass spectrometer operated in scan mode (40–650 m/z) and the NIST MS library was used to evaluate the data and organosulfur compounds identification [7 (link)].
Peak quantification was obtained using reference standard diallyl disulfide (>95% GC), R2 = 0.9982.
Organosulfur compounds in D-VGE were detected, identified, and quantified by injecting directly the extract in diethyl ether in the GC/MS system as described above.
9
Fatty Acid Methyl Esters Analysis by GC
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The fatty acid methyl esters were prepared according to HRN EN ISO 12966-2:2017 standard [74 ]. They were analyzed by gas chromatography (GC) with FID according to HRN EN ISO 12966-4:2015 [75 ]. Agilent Technologies gas chromatograph 7890A (Lake Forest, CA, USA) with ZB-WAX capillary column (Phenomenex, Torrance, CA, USA; 25 m x 0.25 mm i.d. the stationary phase thickness 0.25 µm) and a split–splitless injector (260 °C), and FID (280 °C) was used. The sample volume of 5 µL was injected with a split ratio of 1:40. Starting column temperature was 60 °C with 2 min holding time. The oven temperature was increased at the rate of 13 °C/min to 150 °C, then was heated to 240 °C at the rate of 2 °C/min. Helium (He; 99.9999%) was the carrier gas at a flow rate of 3 mL/min. The hydrogen flow was 70 mL/min, air flow was 450 mL/min, and the makeup gas flow (nitrogen) was 15 mL/min.
InTable 3 37 fatty acid methyl ester standard compounds were used for the identification of obtained fatty acid methyl esters (by comparison with the retention times at the same operating conditions). The results (Table 3 ) are expressed as % of individual fatty acids to total fatty acids. The method detection limit was 0.1%.
In
10
Fatty Acid Profiling of Insect Larvae
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Frozen third instar larvae were homogenized in 1% sulfuric acid in methanol and transmethylated by heating under nitrogen atmosphere (using hexane as cosolvent). The fatty acid methyl esters (FAME) formed were extracted with hexane in two steps. The dried and concentrated FAME were analyzed by a Shimadzu GC‐2010 Plus gas chromatograph (Shimadzu, Kyoto, Japan) employing a ZBWAX capillary column (length 30 m, internal diameter 0.32 mm, film thickness 0.25 µm; Phenomenex, Torrance, CA, USA) and a flame ionization detector (FID). For the temperature‐programmed runs, the sample solutions (2 µl) were injected in split mode, and helium was used as the carrier gas. The integrated and manually corrected (GCsolution software, Shimadzu) peak areas were converted to mol% by using the theoretical response factors for FID (Ackman, 1992 ) and calibrations with quantitative authentic standards (Supelco, Bellefonte, PA, USA). The FAME were identified by their mass spectra recorded by using a Schimadzu GCMS‐QP2010 Ultra with mass selective detector (MSD). The results represent the FA composition of the total lipids.
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