The samples were centrifuged (Sigma 3-18K; Sartorius AG, Göttingen, Germany) at 7500×g and 4 °C for 15 min to separate the liquid phase. A volume of 1 mL of the separated serum was taken and mixed with 1 mL of 0.2% trifluoroacetic acid (TFA) and filtered through a 0.45-µm membrane filter. The filtrate was injected into the HPLC (Thermo Finnigan Inc., San Jose, CA, USA) system coupled with an autosampler (model AS3000), a degaser (SCM 1000), a gradient pump (P4000) and a UV DAD detector (6000LP). The C18 (250 mm×4.6 mm, 5 µm, 300 Å) RP-HPLC column was used. Deionized water containing 0.1% TFA was used as mobile phase A and acetonitrile (UV grade) containing 0.1% TFA was used as mobile phase B (19 (link)). Measurements were made at 280 nm and data were evaluated with ChromQuest 5.0 software (Thermo Fisher Scientific Inc, Waltham, MA, USA).
Scm1000
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SCM1000 is a piece of laboratory equipment designed for temperature control and monitoring. It features a compact and durable design. The core function of the SCM1000 is to provide precise temperature regulation and measurement capabilities for various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
P4000, by Thermo Fisher Scientific (3 mentions)
As3000 autosampler, by Thermo Fisher Scientific (3 mentions)
Fl3000 fluorescence detector, by Thermo Fisher Scientific (3 mentions)
C18 spherisorb s5 ods2, by Waters Corporation (3 mentions)
Uv 6000 lp, by Thermo Fisher Scientific (2 mentions)
Sigma 3 18k, by Sartorius (1 mentions)
High performance liquid chromatography (hplc), by Thermo Fisher Scientific (1 mentions)
As 3000, by Thermo Fisher Scientific (1 mentions)
Lcq deca, by Thermo Fisher Scientific (1 mentions)
Surveyor autosampler, by Thermo Fisher Scientific (1 mentions)
1100 series, by Agilent Technologies (1 mentions)
6 protocols using scm1000
1
HPLC Analysis of Serum Biomarkers
2
HPLC-DAD-FD Analysis of Polyphenols
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Condensed water extracts were analyzed using HPLC-DAD-FD system. An HPLC gradient pump (P4000, ThermoFinnigan) was coupled with a vacuum membrane degasser (SCM1000, ThermoFinnigan), an AS3000 autosampler (ThermoFinnigan), a UV6000 diode array detector and a FL3000 fluorescence detector (ThermoFinnigan). Separations of polyphenols were carried out using a reversed-phase HPLC column C18 Spherisorb S5 ODS2 (Waters, 250 mm x 4 mm, 5 µm). Column temperature was set at 40 °C and injection volume was 20 μL.
Mobile phases for the determination of polyphenols in standard solutions and condensate water samples was 5% methanol-0.1% formic acid in water (eluent A) and 95% methanol-0.1% formic acid in water (eluent B). The gradient was as follows: 0-5 min, 100% A; 5-45 min, linear gradient up to 100% B; 45-55 min 100% B; 55-57 min, linear gradient up to 100% A. Post-run time was 15 min. Elution was performed at a solvent flow rate of 0.8 mL min-1. Table S1 summarized the retention time (tR), solvent used for stock solution preparation and spectra information (UV absorbance (λmax) and fluorescence (λexcitation/ λemission)) of 31 polyphenols standards separated using the above conditions. ChromQuest™ 4.2 Chromatography Data System was used to carry out HPLC-DAD/FD control, data acquisition and data analysis.
Mobile phases for the determination of polyphenols in standard solutions and condensate water samples was 5% methanol-0.1% formic acid in water (eluent A) and 95% methanol-0.1% formic acid in water (eluent B). The gradient was as follows: 0-5 min, 100% A; 5-45 min, linear gradient up to 100% B; 45-55 min 100% B; 55-57 min, linear gradient up to 100% A. Post-run time was 15 min. Elution was performed at a solvent flow rate of 0.8 mL min-1. Table S1 summarized the retention time (tR), solvent used for stock solution preparation and spectra information (UV absorbance (λmax) and fluorescence (λexcitation/ λemission)) of 31 polyphenols standards separated using the above conditions. ChromQuest™ 4.2 Chromatography Data System was used to carry out HPLC-DAD/FD control, data acquisition and data analysis.
3
HPLC Quantification of Antioxidant Compounds
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RA in the extracts was quantified using an HPLC with a diode array detector. An HPLC gradient pump (P4000, Thermo Finnigan, Thermo Finnigan Italia S.p.A., Milan, Italy) was coupled with a vacuum membrane degasser (SCM1000, ThermoFinnigan), an AS3000 autosampler (Thermo Finnigan), a UV6000 diode array detector, and a FL3000 fluorescence detector (Thermo Finnigan). Separations were carried out using a reversed-phase HPLC column C18 Spherisorb S5 ODS2 (Waters, 250 mm × 4 mm, 5 µm). The column temperature was set at 40 °C and the injection volume was 2 μL. Mobile phases were 5% methanol–95% of 5 mM sulphuric acid in water (eluent A) and 95% methanol–5% of 5 mM sulphuric acid in water (eluent B). The gradient was as follows: 0–5 min, 80% A; 5–20 min, linear gradient up to 80% B; 20–22 min, linear gradient up to 100% B; 22–45 min 100% B. Post-run time was 15 min. Elution was performed at a solvent flow rate of 0.8 mL min−1. Detection was performed in absorbance at 220, 280, and 350 nm and in fluorescence setting the excitation and emission wavelengths at 280 and 365 nm, respectively. The ChromQuest™ Thermo Finnigan 4.2 Chromatography Data System was used to carry out HPLC-DAD/FD control, data acquisition, and data analysis.
4
HPLC-DAD-FD Analysis of Polyphenols
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Condensed water extracts were analyzed using HPLC-DAD-FD system. An HPLC gradient pump (P4000, ThermoFinnigan) was coupled with a vacuum membrane degasser (SCM1000, ThermoFinnigan), an AS3000 autosampler (ThermoFinnigan), a UV6000 diode array detector and a FL3000 fluorescence detector (ThermoFinnigan). Separations of polyphenols were carried out using a reversed-phase HPLC column C18 Spherisorb S5 ODS2 (Waters, 250 mm x 4 mm, 5 µm). Column temperature was set at 40 °C and injection volume was 20 μL.
Mobile phases for the determination of polyphenols in standard solutions and condensate water samples was 5% methanol-0.1% formic acid in water (eluent A) and 95% methanol-0.1% formic acid in water (eluent B). The gradient was as follows: 0-5 min, 100% A; 5-45 min, linear gradient up to 100% B; 45-55 min 100% B; 55-57 min, linear gradient up to 100% A. Post-run time was 15 min. Elution was performed at a solvent flow rate of 0.8 mL min-1. Table S1 summarized the retention time (tR), solvent used for stock solution preparation and spectra information (UV absorbance (λmax) and fluorescence (λexcitation/ λemission)) of 31 polyphenols standards separated using the above conditions. ChromQuest™ 4.2 Chromatography Data System was used to carry out HPLC-DAD/FD control, data acquisition and data analysis.
Mobile phases for the determination of polyphenols in standard solutions and condensate water samples was 5% methanol-0.1% formic acid in water (eluent A) and 95% methanol-0.1% formic acid in water (eluent B). The gradient was as follows: 0-5 min, 100% A; 5-45 min, linear gradient up to 100% B; 45-55 min 100% B; 55-57 min, linear gradient up to 100% A. Post-run time was 15 min. Elution was performed at a solvent flow rate of 0.8 mL min-1. Table S1 summarized the retention time (tR), solvent used for stock solution preparation and spectra information (UV absorbance (λmax) and fluorescence (λexcitation/ λemission)) of 31 polyphenols standards separated using the above conditions. ChromQuest™ 4.2 Chromatography Data System was used to carry out HPLC-DAD/FD control, data acquisition and data analysis.
5
Analytical Workflow for Phytochemical Profiling
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Filter paper (Whatman No.4, England), rotary evaporator (Stuart Bibby Scientific, RE300DB), electric air‐dried oven (Memmert UN30, Zirndorf), UV–vis spectrophotometer (PerkinElmer, Lamda 950, UV/VIS, UK), blender (Panasonic), electronic analytical balance (Ohaus), freezer (Innova, IN200), electronic hot plate (VWR, Cole‐Parmer, Thermo Scientific, France), gas chromatography system (GC Clarus 690, Perkin Elemer), and reverse‐phase Purospher STAR Hibar HR RP18 end‐capped column (150 mm × 2.1 mm, 3 μm, thermostated at 30°C, Supelco) in a LC system that is composed of: a solvent degasser (SCM1000, Thermo Scientific), a binary high‐pressure pump (1100 series, Agilent Technologies), a Surveyor autosampler thermostated at 4°C (Thermo Scientific), equipped with a UV–visible photodiode array detector (UV6000 LP, Thermo Scientific) and an ion trap mass spectrometer with electrospray ionization source (LCQ Deca, Thermo Scientific).
6
HPLC Analysis of Ascorbic Acid in Rocket Leaves
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Vitamin C extraction and analysis by HPLC was undertaken as described by [44 (link)], under low light and cold conditions. Metaphosphoric acid (5 mL of 5% w/v) was added to 1 g of ground rocket leaves and the mixture was homogenized by shaking and vortexing for 5 min, keeping on ice. Samples were centrifuged at 4700 g for 10 min at 4 °C, followed by filtration through Whatman 13 mm PVDF syringe filters, pore size 0.45 μm.
HPLC was carried out using a SNA 4000 controller, an AS 300 autosampler, a P400 pump, SCM1000 degasser, and a UV 6000 LP photodiode array detector (Thermo Scientific, Waltham, MA, USA). Samples (20 μL) were analyzed isocratically over a 250 mm long 4.6 mm I.D. Phenomenex synergi polar (4 μm particle size, 80 Å pore size) column with polar “security guard” guard column, using 0.5% NaH2PO4 (pH 2.25 with H3PO4) acetonitrile (93:7 v/v) as mobile phase at a flow rate of 1.2 mL/min over 20 min. A calibration curve for ascorbic acid was obtained using standards of concentrations, 0.5 to 100 μg/mL. Each biological replicate was analyzed in triplicate. Data were analyzed using Xcalibur V 1.2. software (Thermo Scientific, Waltham, MA, USA).
HPLC was carried out using a SNA 4000 controller, an AS 300 autosampler, a P400 pump, SCM1000 degasser, and a UV 6000 LP photodiode array detector (Thermo Scientific, Waltham, MA, USA). Samples (20 μL) were analyzed isocratically over a 250 mm long 4.6 mm I.D. Phenomenex synergi polar (4 μm particle size, 80 Å pore size) column with polar “security guard” guard column, using 0.5% NaH2PO4 (pH 2.25 with H3PO4) acetonitrile (93:7 v/v) as mobile phase at a flow rate of 1.2 mL/min over 20 min. A calibration curve for ascorbic acid was obtained using standards of concentrations, 0.5 to 100 μg/mL. Each biological replicate was analyzed in triplicate. Data were analyzed using Xcalibur V 1.2. software (Thermo Scientific, Waltham, MA, USA).
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