Solutions were analyzed with an Agilent 1260 series HPLC coupled with an Agilent single stage quadrupole mass spectrometer, equipped with a Jet StreamTM electrospray ionization source (Agilent Technologies, USA). Chromatographic separation was achieved on an Agilent Zorbax C18, 3.0 × 150 mm column with 5 μm particle size, maintained at 30 °C. The mobile phase consisted of 45 mM ammonium formate buffer, pH 3.5 (A) and 80% acetonitrile with 0.01% formic acid (B) with a linear gradients (40–100% B over 8 min, 100–40% B over 1 min, held at 40% B for 3 min) at a flow rate of 0.5 mL/min. The sample volume injected was 100 µL and the auto sampler was set at 4 °C. The mass spectrometer was run in positive and negative ion ionization modes, with dual polarity scans from 100–600 m/z. Instrument parameters of the mass spectrometer were: fragmentation voltage 70 V, gas flow 12 L/min, gas temperature 250 °C, sheath gas flow 10 L/min, sheath gas temperature 325 °C, nebuliser 35 psi, capillary voltage 2500 V, and nozzle voltage 500V. Absorbance detection was at 276 nm (bandwidth 16 nm) with quantitation by integration of peak areas with Agilent Open Lab CDS Chemstation software. Unirradiated samples were used for single-point calibration.
1260 series hplc
Manufactured by Agilent Technologies
Sourced in United States
The 1260 series HPLC is a high-performance liquid chromatography system designed and manufactured by Agilent Technologies. It is used for the separation, identification, and quantification of various chemical compounds in complex mixtures. The 1260 series HPLC system consists of essential components such as a solvent delivery system, an auto-sampler, a column compartment, and a detector, which work together to provide accurate and reliable analytical results.
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Lab products found in correlation
Zorbax eclipse xdb c18, by Phenomenex (4 mentions)
Pre column securityguard, by Phenomenex (4 mentions)
Absoluteidq p180 kit, by Biocrates (4 mentions)
Qtrap 4500, by AB Sciex (4 mentions)
6120 quadrupole mass spectrometer, by Agilent Technologies (4 mentions)
Accq tag 3.9 150 mm, by Waters Corporation (2 mentions)
1525 binary pump, by Waters Corporation (2 mentions)
2998 photodiode array detector, by Waters Corporation (2 mentions)
Jp u 3010 uv spectrophotometer, by Hitachi (2 mentions)
Jms 700 double focusing b e configuration instrument, by JEOL (2 mentions)
Eclipse xdb c18, by Agilent Technologies (2 mentions)
8452a diode array spectrophotometer, by Hewlett-Packard (2 mentions)
C18 column, by Agilent Technologies (2 mentions)
Rezex roa organic acid h column, by Phenomenex (2 mentions)
Kinetex c18 100a column, by Phenomenex (2 mentions)
Alliance 2796 separations system, by Waters Corporation (2 mentions)
2996 photodiode array detector, by Waters Corporation (2 mentions)
Optizen nanoq, by Mecasys (2 mentions)
Avance 600 mhz, by Bruker (1 mentions)
Avance 400 mhz spectrometer, by Bruker (1 mentions)
64 protocols using 1260 series hplc
1
HPLC-MS Analysis of Irradiated Samples
2
Peptide Fractionation Using HPLC
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Tryptic digested peptides (100 µg) were fractionated using a poly-sulfonylethyl column A size 200 × 2.1 mm, 5 µm, 200 Å column attached to the 1260 series HPLC (Agilent, Santa Clara, CA, USA). The separation was initiated, at a constant flow rate of 0.3 ml/min, with 100% buffer A (5 mM KH2PO4, pH 2.72, 25% acetonitrile) for 25 min. This was followed by a gradual increase in buffer B (5 mM KH2PO4, pH 2.72, 350 mM KCl, 25% acetonitrile) concentration from 0 to 45% over 70 min.
3
Targeted Metabolomics Profiling by LC-MS/MS
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AbsoluteIDQ p180 kit (Biocrates Life Sciences AG, Innsbruck, Austria) was used for the targeted analysis of 188 metabolites and their ratios. An Agilent Zorbax Eclipse XDB C18, 3.0 × 100 mm, µm with Pre-Column SecurityGuard, Phenomenex, C18, 4 × 3 mm was used on a 1260 series HPLC (Agilent, Santa Clara, CA, USA) in tandem with a QTRAP 4500 (ABSciex, Framingham, MA, USA) mass spectrometer. The protocol is set out in the user’s manual of the AbsoluteIDQ p180 kit. Briefly, lyophilized samples were thawed on ice, the 2 lyophilized phases were both dissolved in 85% methanol/15% water, according to their previous weight (15–25 μl added solvent) and both phases were added to the filter plate of the kit. Subsequently, 10 µl internal standards were added. The samples were derivatized using phenylisothiocyanate, dried, and metabolites extracted using 40% methanol in water. Acetonitrile, chloroform, formic acid (FA), methanol and water were all HPLC grade and purchased from Sigma-Aldrich (Darmstadt, Germany).
4
Synthesis and Characterization of Chiral Compounds
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Unless otherwise stated, reactions were performed under a nitrogen atmosphere using dried solvents. Commercially available reagents were used without further purification. Thin-layer chromatography (TLC) was performed using Jiangyou TLC silica gel plates HSG F254 and visualized using ultraviolet light or phosphomolybdic acid. Flash column chromatography was performed over silica gel (200 to 300 mesh). 1H and 13C nuclear magnetic resonance spectra were recorded in chloroform-d, unless otherwise noted, on a Bruker AVANCE 600-MHz or a Bruker AVANCE 400-MHz spectrometer. High-resolution electrospray ionization and electronic impact mass spectrometry was performed on a Thermo Scientific Q Exactive mass spectrometer (mass analyzer type: Orbitrap). Analytical chiral high-performance liquid chromatography (HPLC) was performed with an Agilent 1260 Series HPLC using CHIRALPAK AS-H (4.6 mm by 25 cm), CHIRALCEL AD-H (4.6 mm by 25 cm), CHIRALCEL OJ-H (4.6 mm by 25 cm), or CHIRALCEL OD-H columns (4.6 mm by 25 cm) obtained from Daicel Chemical Industries Ltd. with visualization at 254 or 210 nm.
5
Isolation and Characterization of (+)-Usnic Acid from Marine Fungus
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The (+)-usnic acid was isolated from the extracts of marine fungus, Mycosphaerella sp., which was collected from a marine sediment at Donghae-si, Korea. The fungus was cultivated in 6 L of potato dextrose broth (PDB) dissolved in seawater in 27 °C, at 140 rpm shaking incubator for 7 days. The broth was extracted with ethyl acetate and yielded 4.01 g extract. The crude extract was fractionated into 3 fractions with a silica gel column chromatography using CH2Cl2 and MeOH as solvent. Fractions were further purified by C18 HPLC (Phenomenex luna C18 column, 250 mm × 10 mm, 5 μm) using 65% CH3CN in H2O to yield 6.8 mg (+)-usnic acid. NMR spectra were measured using a Bruker Ascend 700 MHz spectrometer. Electrospray ionization source (ESI) mass data was obtained using Agilent Technologies 6120 quadrupole mass spectrometer coupled with Agilent Technologies 1260 series HPLC. The optical rotation of (+)-usnic acid was measured in chloroform using a 10 mm path length cell on a Digital Polarimeter P-2000, Jasco Inc.
6
HPLC Analysis of Chemical Compounds
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The chromatographic system consisted of an Agilent 1260 series HPLC with a binary gradient pump. For the analytical separation, a Phenomenex Luna PFP (2) 4.6 × 150.0 mm column with 5.0 µm particles (Alchrom, São Paulo, Brazil) was used. Extracts (10 µL) were injected into the column, and the analytes eluted at a flow rate of 0.8 mL/minute at 30 °C with solvent A (0.1 % formic acid in H2O) and solvent B (100 % acetonitrile, ACN). The solvent gradient started with 2 % solvent B (98 % solvent A) for 1.3 min, followed by a linear increase to 60 % B for 7 min, then to 100 % B in 1 min and maintained at this concentration for an additional 2 min followed by the return to the initial conditions. The total run time was 15 min.
7
Chiral HPLC Analysis of Benoxacor Enantiomers
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In the normal-phase chiral HPLC analysis, benoxacor enantiomers were separated with a Shimadzu LC-20A HPLC system (Tokyo, Japan), which included two LC-20AD pumps, a DGU-20A degasser, a CTO-20A column oven, a SIL-20AD sample injector, and an SPD-20A photodiode array detector. Signal collection and data analysis were processed by Shimadzu Labsolution (Tokyo, Japan). In the reversed-phase analysis, an Agilent 1260 series HPLC was used to separate benoxacor enantiomers, which included a G1311B pump, a G1315D diode array detector, a G1329B autosampler, a G1316A column compartment, and a G1322A degasser (Santa Clara, CA, USA). Signals were collected and analyzed using an Agilent Chemstation.
8
Photocatalytic Degradation Analysis
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A 250 W Xe lamp was used as the visible light source with a filter (λ ≤ 420 nm). The samples at regular intervals were collected and the concentration of leftover TS was analyzed using Agilent 1260 series HPLC equipped with Eclipse XDBC18 (5 μm) reverse phase column (4.6 × 150 mm). Water and acetonitrile at 30 : 70 v/v ratio was used as the mobile phase at an injection volume of 1 mL min−1 for 10 minutes. Further, the degradation products identification was carried out by liquid chromatography tandem mass spectrometry (Agilent 1290 Infinity Binary LC system, Agilent 6460 Triple Quadrupole LCMS/MS system employing the Zorbax eclipse plus C18 column; rapid resolution, 2.1 × 50 mm, 1.8 μm). The mobile phase of water and acetonitrile at 30 : 70 (v/v) was used for about 60 min. To electro spray ionization (ESI) was used to obtain the mass spectra under the helium gas flow at approximately 1 mL min−1 and 16 V of fragment voltage.
9
Comprehensive Analytical Characterization of Compounds
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Optical rotation was measured on a JASCO P-1020 polarimeter (Jasco, Tokyo, Japan) using a 1 cm cell. The UV spectra were recorded on a Hitachi U-3010 spectrophotometer (Hitachi High-Technologies, Tokyo, Japan), and the IR spectra were recorded on a JASCO 4200 FT-IR spectrometer (Jasco, Tokyo, Japan) using a ZnSe cell. NMR spectra were recorded in CDCl3 containing Me4Si as an internal standard on Bruker Avance 600 spectrometers (Bruker, Karlsruhe, Germany). Proton and carbon NMR spectra were measured at 600 and 150 MHz, respectively. High-resolution ESI-Q-ToF-MS/MS mass spectrometric data were obtained on an Agilent Technologies 6530 Accurate-Mass Q-ToF LC/MS spectrometer (Santa Clara, CA, USA) with an Agilent Technologies 1260 series HPLC. Low-resolution ESIMS data were recorded on an Agilent Technologies 6130 quadrupole mass spectrometer with an Agilent Technologies 1200 series HPLC. HPLC was performed on a Shimadzu LC-6AD equipped with a Shimadzu RID-10A refractive index detector (Shimadzu, Kyoto, Japan). All solvents were spectroscopic grade or distilled in a glass prior to use.
10
Lipidomic Analysis of Cell Cultures
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Supernatant collected from cell culture was stored with 50% MeOH at −80°C. Lipid mediator analysis was performed as described previously by Henkel et al (2019 (link)). Automated solid phase extractions were performed with a Microlab STAR robot (Hamilton). Prior to extraction, all samples were diluted with H2O to a MeOH content of 15% and 10 μl of IS stock solution was added. Samples were extracted using Strata‐X 96‐well plates (30 mg, Phenomenex) and eluted with MeOH. Samples were evaporated to dryness under N2 stream and redissolved in 100 μl MeOH/H2O (1:1).
Chromatographic separation of eicosanoids was achieved with a 1260 Series HPLC (Agilent) using a Kinetex C18 reversed phase column (2.6 μm, 100 × 2.1 mm, Phenomenex) with a SecurityGuard Ultra Cartridge C18 (Phenomenex) precolumn. The QTRAP 5500 mass spectrometer (Sciex), equipped with a Turbo‐VTM ion source, was operated in negative ionization mode. Samples were injected via an HTC PAL autosampler (CTC Analytics), set to 7.5°C. Identification of metabolites was achieved via retention time and scheduled multiple reaction monitoring (sMRM) as previously specified. Acquisition of LC‐MS/MS data was performed using Analyst Software 1.6.3 followed by quantification with MultiQuant Software 3.0.2 (both from Sciex).
Chromatographic separation of eicosanoids was achieved with a 1260 Series HPLC (Agilent) using a Kinetex C18 reversed phase column (2.6 μm, 100 × 2.1 mm, Phenomenex) with a SecurityGuard Ultra Cartridge C18 (Phenomenex) precolumn. The QTRAP 5500 mass spectrometer (Sciex), equipped with a Turbo‐VTM ion source, was operated in negative ionization mode. Samples were injected via an HTC PAL autosampler (CTC Analytics), set to 7.5°C. Identification of metabolites was achieved via retention time and scheduled multiple reaction monitoring (sMRM) as previously specified. Acquisition of LC‐MS/MS data was performed using Analyst Software 1.6.3 followed by quantification with MultiQuant Software 3.0.2 (both from Sciex).
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