An aliquot of the BDE (5–6 mg) was separated into an apolar and a polar fraction using activated Al2O3 as stationary phase and n-hexane:DCM (9:1; v/v) and DCM:MeOH (1:1; v/v) as respective eluents. The apolar fraction, containing aliphatic hydrocarbons, was dried under a gentle stream of N2 and dissolved in n-hexane to a concentration of 1 mg ml-1 prior to analysis. Gas chromatography-mass spectrometry (GC-MS) was performed using an Agilent 7820A gas chromatograph equipped with a Phenomenex ZB-5 fused silica column (30 m × 0.25 mm i.d.; 0.25 μm film thickness). Helium (He) was used as carrier gas at a constant 1 ml min-1. The injector was at 70°C and the oven programmed to 130°C at 20°C min-1 and then at 4°C min-1 to 320°C (held 10 min). The GC instrument was interfaced with an Agilent 5975 MSD mass spectrometer operated at 70 eV and scanning m/z 50 to 650 with a cycle time of 1.7 s (resolution 1000). Straight chain and branched alkanes were assigned from retention time indices as described in Kissin et al. [37 ] and comparison with published mass spectra [38 , 39 ].
5975 msd mass spectrometer
Manufactured by Agilent Technologies
Sourced in United States
The 5975 MSD mass spectrometer is a laboratory instrument designed for chemical analysis. It is capable of identifying and quantifying various compounds in a sample by measuring their molecular masses and fragment patterns. The core function of the 5975 MSD is to provide sensitive and precise mass spectral data for analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Heneicosanoic acid, by Merck Group (3 mentions)
6890 gas chromatograph, by Agilent Technologies (2 mentions)
Zb wax column, by Phenomenex (1 mentions)
Supelco 37 component fame mix, by Merck Group (1 mentions)
Rtl dbwax method, by Agilent Technologies (1 mentions)
Agilent 6890 gas chromatograph, by Agilent Technologies (1 mentions)
6890n gas chromatograph, by Agilent Technologies (1 mentions)
Hp 5ms capillary column, by Agilent Technologies (1 mentions)
5 m guard column, by Phenomenex (1 mentions)
Zb 1701 gc capillary column, by Phenomenex (1 mentions)
Hp 5890, by Hewlett-Packard (1 mentions)
Inert cap wax, by GL Sciences (1 mentions)
G 5000, by Hitachi (1 mentions)
Polydimethylsiloxane, by Merck Group (1 mentions)
Db wax, by Agilent Technologies (1 mentions)
9 protocols using 5975 msd mass spectrometer
1
Fractionation and GC-MS Analysis of Aliphatic Hydrocarbons
2
Fatty Acid Profiling of Freeze-Dried Cells
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Freeze-dried and homogenized cell material (3 mg) was extracted and transesterfied as detailed in Mudimu et al. [40 ]. The extract was dissolved in DCM to a concentration of 0.5 mg ml-l and subjected to the analysis of FAMEs using an Agilent 7820A gas chromatograph equipped with a Phenomenex ZB-Wax column (30 m × 0.25 mm i.d.; 0.50 μm film thickness) and connected to a flame ionization detector (FID). Hydrogen was the carrier gas at 1 ml min-1. The oven temperature was programmed from 50°C (1 min) to 200°C at 25°C min-1, then to 230°C (held 18 min) at 3°C min-1. FAMEs were assigned by comparison of retention times with those of the “Supelco 37 Component FAME mix” (Sigma-Aldrich, Germany).
FAMEs not included in the standard mixture were assigned from their mass spectral characteristics using an Agilent 7820A gas chromatograph coupled to an Agilent 5975 MSD mass spectrometer. Analytical conditions were as above. Assignment of FAMEs was accomplished by comparison of mass spectra with those in the “Lipid Library” of the American Oil Chemists’ Society (http://lipidlibrary.aocs.org ).
FAMEs not included in the standard mixture were assigned from their mass spectral characteristics using an Agilent 7820A gas chromatograph coupled to an Agilent 5975 MSD mass spectrometer. Analytical conditions were as above. Assignment of FAMEs was accomplished by comparison of mass spectra with those in the “Lipid Library” of the American Oil Chemists’ Society (
3
Fatty Acid Methyl Ester Analysis in Microalgae
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Samples for fatty acid methyl ester (FAME) analyses were collected when lipid accumulation reached its peak, normally after 3–4 days of nutrient starvation. A total of 4 mL of microalgal culture was collected and centrifuged at 8,000 × g for 5 min. Biomass was collected and freeze-dried for 30 min. Lipids in the microalgal pellet were hydrolyzed and methyl-esterified in 300 μL of a 2% H2SO4 in methanol solution for 2 h at 80°C. Prior to the reaction, 50 μg of heneicosanoic acid provided by Sigma, USA was added as internal standard. After the esterification step, 300 μL of 0.9% (w/v) NaCl solution and 300 μL of hexane were added and mixed for 20 s. To separate the phase, samples were then centrifuged at 16,000 × g for 3 min. A total of 1 μL of the hexane layer was injected into an Agilent 6890 gas chromatograph coupled to a 5975 MSD mass spectrometer. The running conditions were followed using Agilent’s RTL DBWax method as described previously (Lim et al., 2012 (link)).
4
Microalgal Fatty Acid Methyl Ester Analysis
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Samples for FAME analysis were collected when lipid accumulation reached its peak, after 3 days of starvation. A total of 4 mL of microalgal culture was collected and centrifuged at 8000 × g for 5 min. Biomass was collected and dried by a vacuum pump for 30 min. Lipids in the microalgal pellet were hydrolyzed and methyl-esterified in 300 μL of a 2% H2SO4 and methanol solution for 2 h at 80°C. Prior to the reaction, 50 μg of heneicosanoic acid (Sigma, USA) was added as internal standard. After the esterification step, 300 μL of 0.9% (w/v) NaCl solution and 300 μL of hexane were added and mixed for 20 s. To separate the phase, samples were centrifuged at 16,000 × g for 3 min. A total of 1 μL of hexane layer was injected into an Agilent 6890 gas chromatograph (GC) coupled to a 5975 MSD mass spectrometer (MS). The running conditions were described by Agilent's RTL DBWax method (Brown, 1991 (link)).
5
Fatty Acid Methyl Ester (FAME) Analysis
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The condensed lipid extracts were hydrolysed and methyl-esterified for FAME analysis by GC-MS [36 (link)]. Briefly, 100 μl of extract were mixed with 500 μl of 2% H2SO4/methanol solution in a 2 ml eppendorf tube by shaking at 80°C for 2 h. In each sample, 100 μg of heneicosanoic acid (Sigma, USA) was added as an internal standard prior to the reaction. A total of 500 μl of 0.9% (w/v) NaCl and 500 μl of hexane was then added to the sample which was subsequently vortexed for 20 s and centrifuged at 16,000 × g for 3 min. The hexane layer was pipetted into an autosampler vial for FAME quantification. 1 μl of the hexane layer was injected into an Agilent 6890 gas chromatograph equipped with a 5975 MSD mass spectrometer (Agilent Technologies Australia Pty Ltd; GC/MS), for identification of FAMEs. Separation was achieved on a DB-Wax column (Application note: 5988-5871EN) with a cyanopropyl stationary phase with helium as carrier gas in constant pressure mode. Identification of FAME was based on mass spectral profiles, comparison to standards, and expected retention time from Agilent’s RTL DB-Wax method (Application note: 5988-5871EN). In the end, all FAME data were normalised in percentage of dry weight to allow the comparative analysis between different extractions.
6
Carbohydrate Derivatization and GC-MS Analysis
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The eluent for carbohydrate analysis needs a simple derivatization prior to GC-MS analysis. Briefly, 2% 0.2 mL sodium borohydride solution (dissolved in aqueous ammonia) was added into the eluent. After reaction for 20 min at 40 °C, 0.4 mL acetic acid, 0.3 mL methylimidazole and 1 mL acetic anhydride was added to the solution followed by another 10 min reaction. Finally, the carbohydrate derivatives were dissolved in 500 μL dichloromethane. The carbohydrate detection was performed on an Agilent 6890N gas chromatograph equipped with a MSD 5975 mass spectrometer (GC-MS) and electron-impact ionization (EI). A split/splitless-type injector was used for sample introduction. Chromatographic separation was carried out with a HP-5MS capillary column (30 m × 250 μm × 0.25 μm, Agilent Technology, CA, USA). The inlet temperature was 240 °C, and the oven temperature programs were as follows: the initial oven temperature was 140 °C (held for 0.5 min), ramped at 30 °C min–1 up to 190 °C (held for 5 min), and ramped at 2 °C min–1 up to 210 °C (held for 2 min). Helium was used as the carrier gas at a constant flow rate of 1.2 mL min–1. The MSD was operated in the electron impact ion (EI) mode with a source temperature of 230 °C. The electron energy was 70 eV and the filament current was 200 A.
7
GC-MS Analysis of Metabolite Extracts
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Dried intracellular and extracellular metabolite extracts were resuspended in 200 μl of sodium hydroxide (1 M). The MCF derivatization step was performed according to the method published by Han et al. [20 (link)]. All MCF-derivatized samples were analyzed using an Agilent GC7890B chromatography system coupled to a MSD5975 mass spectrometer (Agilent, California, USA) operating at 70 eV. The GC column was a ZB-1701 GC capillary column (30 m × 250 μm id × 0.15 μm with a 5 m guard column, Phenomenex, USA). GC-MS parameters and temperature programs were set according to the protocol reported in Han et al. [20 (link)].
8
Quantification of Organic Contaminants
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A total of 31 polyaromatic hydrocarbon (PAH), 10 polychlorobiphenyl (PCB), 4 polybromodimethylether (PBDE), and 13 organochlorine pesticide (OCP) compounds were quantified and concentrations of organic contaminants were expressed in µg kg -1 dry weight (dw). Briefly, PAHs were measured using a gas chromatograph (HP 6890. Agilent technologies, Palo Alto, CA, USA) equipped with a splitless injector and coupled to a MSD 5975 mass spectrometer (Baumard et al. 1999) . PCBs, PBDEs and OCPs were quantified using a gas chromatograph (HP 5890 Hewlett Packard, Palo Alto, Ca, USA) equipped with a splitless injector and coupled to an electron capture detector (Thompson & Budzinski 2000; Tapie et al. 2008 ) and confirmed using a gas chromatograph (Agilent Technologies 7890A) coupled to MS/MS (Agilent technologies 7000). Among pesticides, lindane, 2,4'-and 4,4'dichlorodiphenyltrichloroethanes (DDT), 2,4'-and 4,4'-dichlorodiphenyldichloroethanes (DDE), and 2,4´-and 4,4´-dichlorodiphenyldichloroethylenes (DDD), hexachlorobenzene (HCB), heptachlor (HC) and trans-nonachlor (TNC) were quantified. In the text, we will refer to the sum of all DDTs and their metabolites (DDEs and DDDs) as 'DDTs' and to the sum of HCB, HC and TNC as 'HCB+HC+TNC'.
More details on the methodology and quality control are described in Luna-Acosta et al. (submitted for this volume).
More details on the methodology and quality control are described in Luna-Acosta et al. (submitted for this volume).
9
GC and GC-MS Analysis of Compounds
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GC analyses were performed on a G-5000 (Hitachi) equipped with a flame ionization detector with the following conditions: a fused silica capillary column (Inert Cap-Wax, 60 m × 0.25 mm, film thickness 0.25 µm, GL Sciences) and a chiral column (CP-Chirasil-Dex CB, 25 m × 0.25 mm, film thickness 0.25 µm); column temperature program: 60 ℃ for 2 min, increasing to 220 ℃ at a rate of 4 ℃/min, then held at 220℃ for 15 min; the injector was set at 60 ℃ and the detector was set at 230 ℃ [7] ; carrier gas: helium (1 ml/min); injection volume: 1 µl; split ratio: 99 : 1.
GC-MS and SPME were performed on an Agilent 6850 series gas chromatograph connected to an Agilent MSD 5975 mass spectrometer under the following operating conditions: fused silica capillary column (DB-WAX, 60 m × 0.25 mm, film thickness 0.25 µm, Agilent Technology); SPME fiber: 100 µm polydimethylsiloxane (Supelco); the column temperature program was the same as that used for the GC analysis; ionization energy: 70 eV; carrier gas: helium (1 mL/min); injection volume: 1 µl; split ratio: 99:1. The identities of most of the separated compounds were confirmed by comparison of the retention indices (RI) and mass spectra patterns (MS library: NIST 2 and flavors). RI was accordant with past data.
GC-MS and SPME were performed on an Agilent 6850 series gas chromatograph connected to an Agilent MSD 5975 mass spectrometer under the following operating conditions: fused silica capillary column (DB-WAX, 60 m × 0.25 mm, film thickness 0.25 µm, Agilent Technology); SPME fiber: 100 µm polydimethylsiloxane (Supelco); the column temperature program was the same as that used for the GC analysis; ionization energy: 70 eV; carrier gas: helium (1 mL/min); injection volume: 1 µl; split ratio: 99:1. The identities of most of the separated compounds were confirmed by comparison of the retention indices (RI) and mass spectra patterns (MS library: NIST 2 and flavors). RI was accordant with past data.
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