All experiments were performed on a Thermo Scientific Accela UHPLC system with an Accela 600 Pump, Accela Autosampler and Accela PDA Detector coupled to a Thermo Fisher Scientific LTQ Orbitrap Velos Pro mass spectrometer (Thermo Scientific, Waltham, MA, USA). All samples were centrifuged using a 5424R Eppendorf centrifuge (Eppendorf, Hamburg, Germany). All ACQUITY UPLC columns (BEH C18 (2.1 mm × 100 mm, 1.7 μm, 130 Å), BEH Phenyl (2.1 mm × 100 mm, 1.7 μm, 130 Å) and CSH Phenyl-Hexyl (2.1 mm × 100 mm, 1.7 μm, 130 Å)) and all ACQUITY UPLC VanGuard pre-columns (BEH C18 (2.1 mm × 5 mm, 1.7 μm, 130 Å), BEH Phenyl (2.1 mm × 5 mm, 1.7 μm, 130 Å) and CSH Phenyl-Hexyl (2.1 mm × 5 mm, 1.7 μm, 130 Å)) were purchased from Waters (Milford, MA, USA).
Beh phenyl
Manufactured by Waters Corporation
Sourced in United States
The BEH Phenyl is a high-performance liquid chromatography (HPLC) column developed by Waters Corporation. It is designed to provide efficient and selective separations of a wide range of analytes, including polar and non-polar compounds. The column features a proprietary hybrid bead technology that combines the stability of bridged ethylene hybrid (BEH) particles with the selectivity of a phenyl stationary phase.
Automatically generated - may contain errors
Lab products found in correlation
Beh c18, by Waters Corporation (2 mentions)
Hss t3, by Waters Corporation (1 mentions)
Unifi software, by Waters Corporation (1 mentions)
Waters h class uplc, by Waters Corporation (1 mentions)
Beh c8, by Waters Corporation (1 mentions)
Waters 1 class uplc, by Waters Corporation (1 mentions)
Empower software, by Waters Corporation (1 mentions)
Accela 600 pump, by Thermo Fisher Scientific (1 mentions)
Accela uhplc system, by Thermo Fisher Scientific (1 mentions)
Acquity uplc columns, by Waters Corporation (1 mentions)
Accela pda detector, by Thermo Fisher Scientific (1 mentions)
Centrifuge 5424r, by Eppendorf (1 mentions)
Agilent 6490 triple quadrupole, by Agilent Technologies (1 mentions)
1260 hplc system, by Agilent Technologies (1 mentions)
Sb c18 column, by Agilent Technologies (1 mentions)
5 protocols using beh phenyl
1
UHPLC-Orbitrap Velos Pro Mass Spectrometry
2
UPLC-MS Method Development and Validation
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The Waters H-Class UPLC was utilized for the Method development and validation study (Waters Corporation, Milford, MA). The entire study was conducted using empower software to acquire, process, and report chromatographic data (Waters Corporation, Milford, MA). A statistical tool, Design-Expert-13, was employed to screen and optimize the CMPs (Stat-Ease Inc, Minneapolis, USA). Various Acquity UPLC columns (100 mm Length × 2.1 mm ID), such as BEH C8, BEH C18, BEH Phenyl, HSS T3, and Protein BEH C4 were assessed for the separation of components (Waters Corporation, Milford, MA). The Waters Xevo G2-XS Quadrupole time-of-flight (Q-ToF) MS instrument with step wave ion optics and XS collision cell coupled with Waters I-Class UPLC was used to separate and identify unknown impurities (Waters Corporation, Milford, MA). UNIFI software was used to identify molecular and fragment ions and their molecular structures (Waters Corporation, Milford, MA).
3
Quantitative Analysis of Biomolecules by LC-MS/MS
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The liquid chromatography–tandem
mass spectrometry (LC–MS/MS) system consisted of a quaternary
pump, an autosampler, and a column oven (Waters ACQUITY UHPLC, Milford,
MA). An electrospray ionization (ESI) source in the positive-ionization
mode was used in the experiments. The separation was performed on
an ACQUITY UHPLC BEH Phenyl analytical column (100 mm × 2.1 mm,
1.7 μm, Waters, Dublin, Ireland) and maintained at 40 °C
with a column oven. The mobile phase consisted of methanol/0.1% aqueous
formic acid (85:15, v/v) at a flow rate of 0.2 mL/min, and the injection
volume was 10 μL. The apparatus conditions were optimized to
the following: source temperature, 150 °C; desolvation gas, nitrogen;
cone voltage, 24 V; capillary voltage, 3.2 kV; desolvation gas flow
rate, 800 L/h; desolvation temperature 400 °C; and collision
energy, 14 V.
mass spectrometry (LC–MS/MS) system consisted of a quaternary
pump, an autosampler, and a column oven (Waters ACQUITY UHPLC, Milford,
MA). An electrospray ionization (ESI) source in the positive-ionization
mode was used in the experiments. The separation was performed on
an ACQUITY UHPLC BEH Phenyl analytical column (100 mm × 2.1 mm,
1.7 μm, Waters, Dublin, Ireland) and maintained at 40 °C
with a column oven. The mobile phase consisted of methanol/0.1% aqueous
formic acid (85:15, v/v) at a flow rate of 0.2 mL/min, and the injection
volume was 10 μL. The apparatus conditions were optimized to
the following: source temperature, 150 °C; desolvation gas, nitrogen;
cone voltage, 24 V; capillary voltage, 3.2 kV; desolvation gas flow
rate, 800 L/h; desolvation temperature 400 °C; and collision
energy, 14 V.
4
UPLC-MS Identification of Compounds
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The present compounds were identified using an Acquity ultra-performance liquid chromatography (UPLC) system attached to an auto-sampler and a binary pump with a 10 µL loop. Chromatographic separations were conducted following the methodology that was proposed by Kumari, Elancheran, Kotoky, and Devi [58 (link)], with slight modifications using a BEH PHENYL (2.1 mm × 100 mm, 1.7 µm; WATERS, Elstree, Herts UK) analytical column at 40 °C. The compounds in the various extracts were identified by using their full mass spectra and their unique mass fragmentation patterns. Comparison of the observed MS spectra with those found in the literature and databases, such as MassBank (http://www.massbank.jp/ ), ChemSpider (http://www.chemspider.com ), and PubChem (https://pubchem.ncbi.nlm.nih.gov ), was the main tool for the identification of the compounds.
5
Quantifying Plant Metabolites via LC-MS
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25 mg of dry N. benthamiana leaves power were dissolved in 0.75 mL of cooled methanol water extract solution (4 : 1, v/v). Samples were ultrasonic for 30 min at room temperature and kept at 4 °C overnight. The supernatant was collected, and ten times concentrated. Hormones and flavonoids analyses were performed using an LC/MS system (LC, Agilent 1260 HPLC system; MS, Agilent6490 triple quadrupole).
SB C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent) was chosen for the detection of hormones. The flow rate was 0.3 mL·min−1. Multireaction monitoring (MRM) mode was adopted. Buffer A (0.1% formic acid in water) and buffer B (0.1% formic acid methanol solution) were as the mobile phases, and the gradient was set as: buffer B increased from 5% to 48% in 0–0.2 min; 57% in 7.0 min; 96% in 7.5 min; 100% in 10.0 min.
BEH Phenyl (2.1 mm × 150 mm, 1.7 μm, Waters) was chosen for the detection of flavonoids. The flow rate was 0.3 mL·min−1. There were 10 compounds monitored by MRM mode. The flow gradient was as follows (buffer A: 0.1% formic acid in water; buffer B: 0.1% formic acid ACN): buffer B from 5% to 15% in 0–2 min; 15% in 10 min; 100% in 15 min.
SB C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent) was chosen for the detection of hormones. The flow rate was 0.3 mL·min−1. Multireaction monitoring (MRM) mode was adopted. Buffer A (0.1% formic acid in water) and buffer B (0.1% formic acid methanol solution) were as the mobile phases, and the gradient was set as: buffer B increased from 5% to 48% in 0–0.2 min; 57% in 7.0 min; 96% in 7.5 min; 100% in 10.0 min.
BEH Phenyl (2.1 mm × 150 mm, 1.7 μm, Waters) was chosen for the detection of flavonoids. The flow rate was 0.3 mL·min−1. There were 10 compounds monitored by MRM mode. The flow gradient was as follows (buffer A: 0.1% formic acid in water; buffer B: 0.1% formic acid ACN): buffer B from 5% to 15% in 0–2 min; 15% in 10 min; 100% in 15 min.
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