Ultimate 3000 hplc system
Manufactured by Phenomenex
Sourced in United States
The Ultimate 3000 HPLC system is a high-performance liquid chromatography (HPLC) instrument designed for analytical and preparative separations. The system features a modular design, allowing for customization to meet specific laboratory requirements. It includes a variety of components such as a pump, autosampler, column compartment, and detector, which work together to provide efficient and reliable separation and analysis of diverse analytes.
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12 protocols using ultimate 3000 hplc system
1
HPLC and MALDI-TOF Analysis of NLY01
2
Quantifying 3-HB Consumption in E. coli and C. necator
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To analyse the consumption of 3-HB by E. coli MG1655 and C. necator H16, single colonies of the wild type strains were used to inoculate 2 ml of minimal medium. From the saturated overnight culture, 6 ml of fresh medium were inoculated 1:100 in 50 ml Falcon tubes and incubated at 30 °C and 200 rpm. At an OD600 of 0.4–0.5, stock solutions of D- (21 ) or L-3-HB (22 ) were added to the cultures to a final concentration of 5 mM. 0.5 ml samples were taken immediately, 1, 2, 3, 6 and 24 hours after supplementation of 3-HB. The OD600 was determined for every time point. Subsequently, samples were centrifuged for 5 min and 13,000 rpm. The supernatant was removed and subjected to HPLC analysis as described previously58 with slight modifications. Briefly, to the cell-free supernatant samples, an equal volume of mobile phase was added which was spiked with 50 mM valerate as internal standard. The mobile phase was composed of 0.005 M H2SO4. Subsequently, the mixture was passed through a 0.22 µm pore size membrane filter. Samples were analysed using a Thermo Scientific Ultimate 3000 HPLC system equipped with a Phenomenex Rezex ROA-organic acid H+ (8%) 150 mm × 7.8 mm × 8 µm column and a diode array detector with the wavelength set at 210 nm. The column was operated at 35 °C with an isocratic flow rate of 0.5 ml/min. Samples were run for 30 min and the injection volume was 20 µl.
3
Glucosinolates Extraction and Quantification
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Glucosinolates were extracted and purified as described by Gigolashvili et al. (2007) (link). Separation of desulfoglucosinolates was performed on a Dionex Ultimate 3000 HPLC system (DGP-3600MB, WPS-3000TB, PDA-3000) equipped with a Phenomenex Luna Security Guard C18 column (4.0 × 3.0 mm) and a Luna C18(2) reverse-phase column (5 µm, 250 × 4.6 mm) at 25 °C column temperature and a flow rate of 1 ml min−1 using the following gradient: 0–5 min, 0% acetonitrile (ACN); 5–30 min, 30% ACN; 30–32 min, 40% ACN; 32–36 min, 40% ACN; 36–40 min, 0% ACN; 40–50 min, 0% ACN. Peaks were quantified at 229 nm relative to an internal benzyl glucosinolate standard using the respective response factors described by Brown et al. (2003) (link).
4
HPLC and MALDI-TOF Analysis of NLY01
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(1) HPLC analysis . NLY01 purified by preparative RP-HPLC was analyzed by using a Dionex Ultimate 3000 HPLC system (Sunnyvale, CA, USA) with a Phenomenex Gemini RP-18 column (250 × 4.6 mm, 5 μm) at ambient temperature and a constant flow-rate of 1.0 mL/min under UV monitoring at 280 nm. The mobile phase consisted of 0.1% (trifluoracetic acid (TFA) in deionized water (eluent A) and acetonitrile (AN) containing 0.1% TFA (eluent B), and these were applied as linear gradients from 30% B to 60% B over 20 min. (2) MALDI-TOF mass spectrometry. Molecular weight of NLY01 was determined by a Bruker Daltonics Microflex MALDI-TOF mass spectrometer (Bremen, Germany) with a 337 nm nitrogen laser. Mass spectrum was obtained in the linear and positive-ion mode with an acceleration voltage of 20 kV. As a matrix solution, a saturated solution of sinapinic acid in acetonitrile:water (50:50, v:v) containing 0.1% TFA was used. Each analyte was mixed with a matrix solution at a ratio of 1:1 (analyte:matrix, v:v) and 1 μl of the analyte-matrix solution was applied and air-dried on the sample plate.
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5
Comprehensive Phospholipid Analysis by HPLC-MS
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MS analysis of phospholipids was performed on an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher). Phospholipids were separated on a normal phase column (Luna 3 μm Silica (2) 100 Å, 150 × 2.0 mm, Phenomenex) at a flow rate of 0.2 ml min−1 on a Dionex Ultimate 3000 HPLC system. The column was maintained at 35 °C. Analysis was performed using gradient solvents (A and B) containing 10 mM ammonium acetate. Solvent A contained propanol:hexane:water (285:215:5, vol/vol/vol) and solvent B contained propanol:hexane:water (285:215:40, vol/vol/vol). All solvents were LC–MS grade. The column was eluted for 0–23 min with a linear gradient of 10–32% B; 23–32 min using a linear gradient of 32–65% B; 32–35 min with a linear gradient of 65–100% B; 35–62 min held at 100% B; 62–64 min with a linear gradient from 100% to 10% B followed by and equilibration from 64 to 80 min at 10% B. The instrument was operated with the electrospray ionization probe in negative polarity mode. Analysis of LC–MS data was performed using software package Compound Discoverer (ThermoFisher) with an in-house generated analysis workflow and oxidized phospholipid database. Lipids were further filtered by retention time and confirmed by fragmentation mass spectrum.
6
HPLC-DAD Analysis of Analytes
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HPLC-DAD analysis was carried out using a Thermo Fisher UltiMate 3000 HPLC system (Germering, Germany) with a Phenomenex Kinetex C18 column (250 × 4.6 mm, 5.0 μm, 100 Å) (Torrance, CA) maintained at 25°C. The temperature of the sampler during analysis was maintained at 10°C to prevent evaporation of the solutions. A step gradient system using an aqueous acetonitrile (MeCN) system (A: H2O containing 0.1% formic acid/B: MeCN) over 30 min (0.0−14.0 min, 40–55% B/14.0−24.0 min, 55–90% B/24.1−30.0 min, 40% B) at a flow rate of 1 mL/min was adopted. Ten microliters of solution were injected for every analysis, and a 250 nm wavelength was applied with consideration of the absorption maximum of the analytes.
7
Chromatographic and Mass Spectrometric Analysis
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Chromatographic analysis was performed with the DIONEX Ultimate 3000 HPLC system coupled to a chromatographic column (Phenomenex Synergi 4 u Hydro-RP 80A 250 × 3.0 mm) set at 40 °C and a flow rate of 0.9 mL/min. Gradients of mobile phases (mobile phase A: 0.1% formic acid in water and mobile phase B: 0.1% formic acid in acetonitrile) were performed over a total of 25 min. MS analysis was carried out on a Thermo Scientific Exactive Plus Benchtop Orbitrap mass spectrometer. The heated electrospray ionization source (HESI II) was used in positive and negative ion modes. The instrument was operated in full scan mode from m/z 67 to m/z 1000. High-resolution accurate mass (HRAM) full-scan MS and top five MS/MS spectra were collected in a data-dependent fashion at a resolving power of 70,000 and 35,000 at FWHM m/z 200, respectively. The post-treatment of data was performed using the MZmine2 version 2.53 (http://mzmine.github.io/ ; Boston, MA, USA) [45 (link)]. Metabolites were identified using the Human Metabolome Database version 4.0 [46 (link)]. LC-MS analyses were performed in a blinded assessment; that is, without revealing the identity of the samples to the mass spectroscopist prior to analyses.
8
Synthesis and Purification of Zinc Hook Peptides
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Zinc hook peptides (Hk peptides) were synthesized by solid‐phase peptide synthesis (SPPS) using an Fmoc‐strategy on a TentaGel R RAM Amide Rink (Rapp Polymere GmbH, Tübingen, Germany) resin (substitution 0.2 mmol g−1) and a Liberty 1 microwave‐assisted synthesizer (CEM) as described previously.33 , 85 Peptides were N‐terminally acetylated with acetic anhydride or fluorescently modified with a dansyl (Dns) moiety or 5(6)‐carboxyfluorescein (FAM )derivatives. Cleaved peptides were precipitated and washed with cold diethyl ether and purified on a C18 column (Phenomenex) with a gradient of acetonitrile and 0.1 % TFA using a Dionex Ultimate 3000 HPLC system. The identity of peptides was confirmed with an API 2000 Applied Biosystems ESI‐MS instrument. Concentration of peptide stocks in 10 mm HCl was determined using a sulfhydryl‐group reactant, DTNB (ϵ=14,150 m −1 cm−1 at 412 nm), prior to each experiment.86
9
Pesticide Analysis in Microcosms
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Two hours and 18 days after application of the treatments, 4 mL-water subsamples of three microcosms per ARO treatment were taken and frozen at −20°C. Later, the samples were filtered (0.2 µm) and analyzed for their concentration of the three pesticides used in the ARO mixture. Measurement of the pesticides was conducted using a UltiMate3000 HPLC System combined with an LTQ-OrbiTrap XL (Thermo Scientific, USA). These samples were then analyzed with an UltiMate3000 HPLC System (column: Phenomenex, Art.-No. 00B-4462-Y0) and an attached LTQ Orbitrap XL (Thermo Scientific) operated in positive ionization mode.
10
Protein Characterization by LC-ESI-MS
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Liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) analyses were carried out using an Advion CMS expressionL mass spectrometer and a Dionex Ultimate 3000 HPLC system equipped with a Phenomenex Aeris 3.6 μm WIDEPORE C4 200 Å column (100 x 2.1 mm). Separations upstream of the mass spectrometer were achieved with an aqueous (95% H2O, 5% MeCN, 0.1% formic acid) / organic (MeCN, 0.1% formic acid) mobile phase at 0.3 mL/min (method: hold 10% organic 0.0–1.0 min, linear gradient of 10–90% organic 1.0–7.0 min, hold 90% organic 7.0–9.0 min, linear gradient of 90–10% organic 9.0–9.1 min, re-equilibrate at 10% organic 9.1–12.0 min). Deconvolution of protein charge ladders was achieved using either Advion Data Express software or the maximum entropy algorithm provided by Analyst 1.4.2 software.
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