HPLC analyses were performed as described before, using an 1100 series HPLC system (Agilent) with a Luna 5μ C18 reversed-phase column (Phenomenex) and 50% acetone and 50% 20 mm formic acid as the mobile phase (6 (link)).
1100 series hplc system
Manufactured by Agilent Technologies
Sourced in United States, Germany, France
The 1100 series HPLC system is a high-performance liquid chromatography instrument manufactured by Agilent Technologies. It is designed to separate, identify, and quantify components in a liquid sample.
Automatically generated - may contain errors
Lab products found in correlation
Xbridge c18 column, by Waters Corporation (4 mentions)
Eclipse xdb c18 column, by Agilent Technologies (3 mentions)
Tensor 27 ft ir instrument, by Bruker (3 mentions)
P4 2080 plus hplc system, by Agilent Technologies (3 mentions)
P 1010 polarimeter, by Jasco (3 mentions)
Eclipse xdb c18, by Agilent Technologies (3 mentions)
Spherisorb 5 μm ods2 4.6 150 mm analytical column, by Waters Corporation (2 mentions)
Hpx 87h column, by Bio-Rad (2 mentions)
Zorbax sb c18 column, by Agilent Technologies (2 mentions)
Heleos 2 multi angle light scattering detector, by Wyatt Technology (2 mentions)
Wtc 050n5 size exclusion column, by Wyatt Technology (2 mentions)
Astra 7, by Wyatt Technology (2 mentions)
T rex refractive index detector, by Wyatt Technology (2 mentions)
R114 rotavapor, by Büchi (2 mentions)
Sevencompact s210 ph meter, by Mettler Toledo (2 mentions)
Chemstation software, by Agilent Technologies (2 mentions)
Vortex genie 2 vortex mixer, by Scientific Industries (2 mentions)
4000 qtrap lc ms ms system, by AB Sciex (2 mentions)
Chemstation version b 04, by Agilent Technologies (2 mentions)
Maldisynapt instrument, by Waters Corporation (2 mentions)
173 protocols using 1100 series hplc system
1
Reversed-Phase HPLC for Compound Analysis
2
LC/MS Analysis of ADC Deglycosylation
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Prior to LC/MS analysis, conjugates and metabolites were deglycosylated with PNGase F (NEB, cat#P0704L) under non-denaturing conditions at 37°C overnight. ADCs (500 ng) were loaded into a reverse phase column packed with a polymeric material (Michrom-Bruker, cat# CM8/00920/00). LC/MS analysis was performed using Agilent 1100 series HPLC system, comprising binary HPLC pump, degasser, thermostatted auto sampler, column heater and diode-array detector (DAD), coupled to an Orbitrap Velos Pro (Thermo Scientific) mass spectrometer with electrospray ion source. The resulting mass spectra were deconvoluted using ProMass software (Thermo Fisher Scientific).
3
HPLC-MS Analysis of Glycerophosphoethanolamides
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HPLC-MS and HPLC-MS/MS was performed using an Agilent 1100 Series HPLC system equipped with an Agilent Eclipse XDB-C18 column (4.6 mm x 250 mm, 5 μm particle diameter) connected to a Quattro Ultima mass spectrometer (Micromass/Waters) using a 1:1 split. For HPLC, a 0.1% aqueous acetic acid-acetonitrile solvent gradient was used at a flow rate of 1 ml/min, starting with an acetonitrile content of 5% for 5 min, which was increased to 100% over a period of 40 min. The gradient was followed by propanol-acetonitrile (3:1) wash and reconditioning of the column. We noticed that retention times and especially peak shapes for glycerophosphoethanolamides are very sensitive to cleanliness of the column. Even with the propanol wash after every run performance of the column gradually declined and it was necessary to periodically back-flash it with chloroform (every 10 to 40 runs, depending on the samples). This procedure returned the original performance and dramatically improved the peak shapes. Samples were analyzed by HPLC-ESI-MS in both positive and negative ionization modes using a capillary voltage of 3.5 kV and a cone voltage of 60 V. HPLC-MS/MS screening was performed using argon as collision gas at 2.1 mtorr and 30 eV. CM was either analyzed directly without further processing or extracted with methanol as outlined above.
4
Hepatic Lipid Analysis by HPLC and FPLC
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High‐performance liquid chromatography (HPLC) and fast‐performance liquid chromatography analyses were performed at the Lipid and Lipid Metabolite Analysis Core Facility, part of the Women and Children's Health Research Institute and Faculty of Medicine and Dentistry at the University of Alberta. Hepatic lipids were extracted in the presence of 50 μg dipalmitoyl‐phosphatidyl dimethylethanolamine as an internal standard and separated by HPLC, as described previously.21 Lipid species were separated in an Onyx monolithic silica normal‐phase column (Phenomenex) using a 3‐solvent system in an 1100 series HPLC system (Agilent Technologies) and quantified using in‐line detection with an Alltech ELSD2000 evaporative light‐scattering detector (W. R. Grace). Plasma lipoprotein particles were resolved in a Superose 6 10/300 gel‐filtration fast‐performance liquid chromatography column isocratically with 50 mmol/L NaCl buffer using a 1200 series HPLC system (Agilent Technologies). Cholesterol or triglycerides in lipoproteins were detected enzymatically by in‐line reaction at 37°C.
5
Quantitative HPLC Analysis of Sugars and Organic Acids
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Sugars and organic acid content were determined using an Agilent 1100 series HPLC system, equipped with a diode array detector (DAD) and a refractometer index (RI) detector (Model 132; Gilson, Villiers-le-Bel, France), as previously described by Woznicki et al. [28 (link)]. The methanolic extracts (20 μL) were injected in a randomized order, and separation was performed on a Rezex ROA-Organic acid H+ (8%) column (300 × 7.8 mm; Phenomenex, Torrance, CA, USA) at 45 °C with mobile phase 7.2 mmol/L H2SO4 run at a flow rate of 0.5 mL/min. The detection of the sugars was performed with a RI detector and the organic acid detection was performed with DAD at 210 nm. Identification and quantification was performed according to the method described by Amundsen, et al. [24 (link)], and results were presented on a equivalent g/100 g fresh weight (fw) basis.
6
HPLC Quantification of Salbutamol Sulfate
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To quantify salbutamol sulfate in the homogeneity test or deposition study, a mobile phase containing 95% (v/v) of 25 mM potassium phosphate (monobasic) pH 3.0 and 5% (v/v) of methanol was used. The flow rate of the mobile phase through the HPLC column was 1.5 mL/min with a total run time of 25 min per injection set at a wavelength of 225 nm yielding a retention time of 12 min. To adjust the pH to 3.0, a 1 M HCl solution was used while stirring at 180 rpm, after which the mobile phase was filtered and degassed using a Fisher Scientific (Leicestershire, England) 0.22-μm filter before its use.
HPLC was executed via the Agilent 1100 series HPLC system (Santa Clara, California, USA) where a degasser (G1322A), binary pump (G1312A), variable wavelength detector (VWD G1314A), column thermostat (G1316A), and thermostatic autosampler (ALS G1329A) coupled with the Waters Spherisorb 5 μm ODS2 4.6 × 150 mm analytical column (Milford, Massachusetts, USA). Likewise, internal standards of varying salbutamol sulfate concentrations (0.00. 0.50, 2.50, and 5.00 μg/mL, respectively) were used to calibrate and normalize the results.
HPLC was executed via the Agilent 1100 series HPLC system (Santa Clara, California, USA) where a degasser (G1322A), binary pump (G1312A), variable wavelength detector (VWD G1314A), column thermostat (G1316A), and thermostatic autosampler (ALS G1329A) coupled with the Waters Spherisorb 5 μm ODS2 4.6 × 150 mm analytical column (Milford, Massachusetts, USA). Likewise, internal standards of varying salbutamol sulfate concentrations (0.00. 0.50, 2.50, and 5.00 μg/mL, respectively) were used to calibrate and normalize the results.
7
HPLC Method Development and Validation
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The method development and validation experiments were performed using an Agilent Technologies 1100 Series HPLC system equipped with a quaternary pump, auto sampler, and a diode array detector. The chromatographic data were obtained using Agilent Technologies ChemStation for LC Rev.A10.02[1757] software. The column used was a YMC-Pack ODS-AQ with a column length of 150 mm, an inside diameter of 4.6 mm and a particle size of 3 microns.
8
Profiling Adzuki Bean Phytochemicals
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The chemical constituents of adzuki bean total extract (ABTE), adzuki bean flavonoids (ABF) and adzuki bean saponins (ABS) were identified by liquid chromatography-ion trap mass spectrometry. HPLC analysis was performed on an Agilent 1100 series HPLC system equipped with degasser, binary pump, diode array detector and auto-sampler (San Francisco, USA). The separation was performed on a Phenomenex C8 column (150 × 2.0 mm, 5 μm). Gradient elution was performed using water containing 10 mM ammonium acetate (A) and acetonitrile (B). Initial conditions were 10% B for 10 min, changed to 15% B at 30 min and 25% B at 45 min, and then 35% B at 55 min, 45% B at 60 min and 55% B at 70 min. Flow rate was set at 0.2 mL/min, and UV absorption was measured at wavelength of 205 nm and 262 nm for saponins and flavonoids, respectively. The sample injection volume was 10 μL.
9
Quantitative Analysis of Metabolites by HPLC-ESI-TOF MS
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Metabolites were analyzed using HPLC-ESI-TOF MS as previously described [28 (link)]. Briefly, their separation was conducted on a HPX-87H column with 8% cross-linkage (150-mm length, 7.8-mm inside diameter, and 9-μm particle size; Bio-Rad, Richmond, CA) using an Agilent Technologies 1100 Series HPLC system. Metabolites were eluted isocratically with a mobile-phase composition of 0.1% formic acid in water at a flow rate of 0.5 ml/min. Drying and nebulizing gases were set to 13 liters/min and 30 lb/in2, respectively, and a drying-gas temperature of 330°C was used throughout. ESI was conducted in the negative ion mode and using a capillary voltage of −3,500 V. Luteolin, chrysoeriol (ChromaDex, Inc., Irvine, CA), tricin (ChromaDex, Inc., Irvine, CA), and selgin were quantified via 8-point calibration curves of authentic standard compounds for which the R2 coefficients were ≥ 0.99. Stock solutions of metabolites used for enzymatic assays and standard curves were quantified spectrophometrically using published molar absorption coefficients: S-adenosylmethionine (ε = 15,400 L.mol-1.cm-1 at 254 nm) [29 (link)], Luteolin (ε = 14,790 L.mol-1.cm-1 at 350 nm) [30 (link)], chrysoeriol (ε = 15,400 L.mol-1.cm-1 at 347 nm) [30 (link)], and tricin (ε = 41,000 L.mol-1.cm-1 at 349 nm) [31 (link)].
10
Size-Exclusion Chromatography for Protein Characterization
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SEC-purified main peak fractions of co-expressed protein samples (0.5 mg) were loaded on a Superdex 200 10/300 GL (GE Healthcare) column connected to a 1100 series HPLC system (Agilent Technologies), with its variable UV absorbance detector set to 280 nm and coupled in line to a mini DAWN TREOS MALS detector followed by an Optilab rEX refractive-index detector (Wyatt Technology, 690 nm laser) (Some et al., 2019 (link)). Runs were performed at 23°C and 0.75mLmin−1 flow rate in buffer B supplemented with 1 mM tris(2-carboxyethyl)-phosphine (TRC Canada). Absolute molecular mass was calculated by using bovine serum albumin (BSA) (ThermoFisher) as calibration standard and the ASTRA 6 software (Wyatt Technology) with the dn/dc value set to 0.185mLg−1.
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