The extractive analytical procedure and the instrumental conditions for determine metal concentrations have been described in detail elsewhere [14 (link)]. Briefly, aliquots (about 1.0–2.0 g) of the samples were digested to a transparent solution with a mixture of HNO3-HClO4 (8:3) for Cd, Pb, Zn, Cu, Cr, and Ni determination and with a mixture of H2SO4–HNO3 (1:1) for Hg. The completely digested samples were allowed to cool and diluted with deionized water according to the method recommended by the Official Italian Agencies [15 ]. The metals content was determined by an atomic absorption spectrophotometer (Shimadzu AA 7000, Milan, Italy Zn and Ni were analyzed by flame; Cd, Pb, Cr, and Cu by using a graphite furnace (high-density tube) (GFA-7000); and Hg by using a hydride vapor generator (HVG-1) after reduction by NaBH4 (Table S1 ).
Gfa 7000
Manufactured by Shimadzu
Sourced in Japan
The GFA-7000 is a Graphite Furnace Atomic Absorption Spectrophotometer (GF-AAS) manufactured by Shimadzu. It is a laboratory instrument used for the determination of trace elements in various sample matrices through atomic absorption spectroscopy.
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6 protocols using gfa 7000
1
Atomic Absorption Spectrophotometry for Metal Analysis
2
Atomic Absorption Spectrophotometric Analysis
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An atomic absorption spectrophotometer (Model: AA-7000, Shimadzu, Japan) equipped with a GF (GFA 7000) and an autosampler (ASC 7000) was used. A cathode lamp was used and operated according to the manufacturer’s instructions for the analyses of Pb (283.0 nm, 10 mA, and slit 0.7 nm), Cd (228.8 nm, 10 mA, and slit 0.7 nm), and Cr (357.9 nm, 10 mA, and slit 0.7 nm), respectively. Nitric acid (HNO3, 69%), hydrogen peroxide (H2O2, 30%), and standards of Pb, Cd, and Cr were used for sample preparation. A microwave acid digestion system (MADS) (Ethos Easy Milestone) was used for conducting the digestion process. The standard bulk solutions were made daily by diluting respective metals stock standard solutions using 1% (w/w) (HNO3, 69%). The autosampler was used for preparing standard solutions. Deionized water (18 MW/cm) produced using an E-pure system (Thermo Scientific, United States) was used to prepare all the solutions and clean and wash all containers and glassware before use.
3
Quantifying Heavy Metals in Semen
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Semen was collected by masturbation into sterile heavy metal-free plastic containers. After that, the samples were subjected to 1,000 ×g centrifugation for 25 minutes to break the seminal plasma. Seminal plasma were added to portions of optima grade concentrated nitric acid in a glass centrifuge tube at 80°C for removing protein in fume cupboard. The resulting aqueous digests were diluted 5-fold with 2% nitric acid to provide sufficient volume of each sample to determine several heavy metals by Graphite Furnace Atomic Absorption Spectroscopy (SHIMADZU GFA-7000, AA-70000, Japan). The main parameters used for Cd determination were wavelength 228.8 nm, current 2 mA, slit width 1.2 nm, drying at 90, 105, 120°C, ashing at 300°C, and atomization at 1,300°C. The main parameters used for Cr determination were wavelength 357.9 nm, current 4 mA, slit width 0.8 nm, drying at 90, 105, 120°C, ashing at 1,100°C, and atomization at 2,400°C. The main parameters used for Cu determination were wavelength 324.8 nm, current 2 mA, slit width 0.8 nm, drying at 90, 105, 120°C, ashing at 800°C, and atomization at 1,800°C. The relative standard deviation was less than 10%. Recovery rate was between 89.4% and 91.2%.
4
Quantifying Cobalt Complexes by AAS
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Atomic absorption spectroscopy was used for determining the CoMC6*a concentrations on the basis of metal content. A stock solution of approximately 10–4 M Co(iii )MC6*a in ultra-pure metal-free water, 0.1% TFA was prepared. Three aliquots from the stock solution (50 μL each) were treated with 200 μL of 69% ultra-pure nitric acid for 2 hours at 90 °C. Mineralized samples were diluted with ultra-pure water to achieve a cobalt concentration of ∼10 μg L–1 (ppb). The cobalt concentration in the samples was read by using a Shimadzu AA700 atomic absorption spectrophotometer, equipped with the autosampler ACS-7000 and the graphite furnace atomizer GFA-7000. Co(iii )MC6*a stock solutions, analyzed for metal content, were appropriately diluted and used for determining the extinction coefficients at the Soret band maximum wavelength (410 nm), by plotting the absorbance as a function of concentration.
5
Mineral Analysis in Biological Samples
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Mineral (Zn, Cu, Fe, and Mn) content in the diets, plasma, and tissues were analyzed according to a previously reported method (1 (link), 34 (link)). Dried, ground samples (except plasma) were acid digested prior to mineral analysis using microwave-assisted digestion in an MWS 4 Speedwave device (Berghof Co., Eningen, Germany). The concentrations of Zn, Cu, Fe, and Mn were measured by flame atomic absorption spectrometry using a double-beam atomic absorption spectrometer (AAS, AA-7000 Series, Shimadzu Co., Kyoto, Japan). The Mn level in plasma and muscle was determined using a graphite furnace AAS (GFA-7000, Shimadzu Co., Kyoto, Japan) with deuterium background correction and pyrolytic-coated graphite tubes. To verify instrument accuracy a particular certificate reference material was included in each analysis (34 (link)). The analyzed mineral concentrations in the certified reference material are presented as Supplementary Material . All samples were analyzed in duplicate.
6
Spectral Analysis of Cu(II) Concentration
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The Cu(II) concentration was analyzed using an atomic absorption spectrometer (GFA-7000, Shimadzu Co., Kyoto, Japan). The data were analyzed in triplicate, and the averaged data are presented here. The experimental error of the results was within ±5%. The molecular weight distribution of the SMP samples was measured using a high-performance liquid chromatography system (Waters 1525, Waters, USA). The 2D-COS was applied in this study according to the method reported by Noda [23] .
where x and t are a spectral variable (i.e., wavelength) and an external perturbation, respectively. ỹ (x1, t) is the dynamic spectrum, and z (x2, t) is the Hilbert-transformed orthogonal spectrum.
where x and t are a spectral variable (i.e., wavelength) and an external perturbation, respectively. ỹ (x1, t) is the dynamic spectrum, and z (x2, t) is the Hilbert-transformed orthogonal spectrum.
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