All ''negative'' samples were furthered analyzed by using the same anion-exchange chromatographic method but with Q-Exactive mass spectrometry detection as the final confirmation tool. The detector was a Thermo Q-Exactive Orbitrap™ (Thermo Scientific, San Jose, CA, USA), equipped with heated electrospray ionization (HESI) source. Capillary temperature and vaporizer temperature were set at 330°C and 280°C, while the electrospray voltage was set at 3.50 kV operating in negative mode. Sheath and auxiliary gas were set at 35 and 15 arbitrary units, with S lens RF level of 60. Instrument calibration was performed for every analytical session with a direct infusion of a LTQ Velos ESI Negative Ion Calibration Solution (Pierce Biotechnology Inc., Rockford, IL, USA). Full Scan acquisition (FS) with resolving power set at 70000 Full Width at Half Maximum (FWHM) was used. Detection of NO3 -was based on its retention time and exact mass (61.98834) accompanied with by characteristic isotopic pattern. Chromeleon TM software (Thermo Fisher Scientific, Waltham, MA) was used to control the IC system while Xcalibur TM 3.0 software (Thermo Fisher Scientific, San Jose, CA, USA) was used to control the HRMS system and the exact mass of the compounds and to record and elaborate data.
Chromeleon software
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, France
Chromeleon software is a chromatography data system that provides instrument control, data acquisition, and data processing capabilities for analytical laboratories. It offers a comprehensive solution for managing the entire chromatography workflow.
Automatically generated - may contain errors
Lab products found in correlation
Ultimate 3000, by Thermo Fisher Scientific (9 mentions)
Hypersil gold c18 column, by Thermo Fisher Scientific (5 mentions)
Dionex ultimate 3000 system, by Thermo Fisher Scientific (4 mentions)
Xcalibur software, by Thermo Fisher Scientific (4 mentions)
Dionex ultimate 3000, by Thermo Fisher Scientific (4 mentions)
Ultimate 3000 system, by Thermo Fisher Scientific (4 mentions)
Vanquish hplc system, by Thermo Fisher Scientific (3 mentions)
Ultimate 3000 hplc system, by Thermo Fisher Scientific (3 mentions)
High performance liquid chromatography (hplc), by Thermo Fisher Scientific (3 mentions)
P680 hplc pump, by Thermo Fisher Scientific (3 mentions)
Wps 3000 autosampler, by Thermo Fisher Scientific (2 mentions)
Ics 5000, by Thermo Fisher Scientific (2 mentions)
Erc refractomax 521, by Thermo Fisher Scientific (2 mentions)
Rezex rpm monosaccharide pb2 column, by Phenomenex (2 mentions)
Chromquest, by Thermo Fisher Scientific (2 mentions)
Vanquish uplc system, by Thermo Fisher Scientific (2 mentions)
C18 combiflash redisep column, by Teledyne (2 mentions)
Avance 3 hd, by Bruker (2 mentions)
Orbitrap q exactive high field mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Dionex ics 5000 plus system, by Thermo Fisher Scientific (2 mentions)
120 protocols using chromeleon software
1
Nitrate Confirmation via Anion-Exchange Chromatography
2
Ion Chromatography Analysis of Samples
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The analyses were accomplished by an Ionic Chromatography (IC) Dionex ICS-5000+ system (Sunnyvale, CA, USA) made up of a Dual Pump (DP), a Conductivity Detector (EG), a Detector/Chromatography Module (DC) and an Autosampler (AS-AP). The ion chromatography separation column was a Thermo Scientific Dionex IonPac AS19-4 μm (2 × 250 mm, 4 μm particle size) with a guard column Dionex IonPac AG19-4 μm (2 × 50 mm) maintained at 30 °C. The eluent flow rate was 0.30 mL/min with a gradient from 15 mM KOH (aq), held for 8 min, increased to 55 mM KOH (aq) at 20 min, held in these conditions for 4 min and back to 15 mM KOH (aq) at 24.1 min, with a cycle time of 30 min. The KOH eluent was neutralized using Dionex anion self-regenerating suppressor set to 50 mA (ASRS II, 4 mm). Chromeleon TM software (Thermo Fisher Scientific, Waltham, MA) was used to control the IC system and to elaborate the data obtained.
3
Evaluation of Traditional Chinese Medicine for Burn Wound Healing
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BO (pharmaceutical batch number: 201671HZ) was supplied by the Department of Pharmacy at the Wuhan Integrated Traditional and Western Medicine Hospital (Wuhan, China). The BO used in this study is a hospital preparation which contains Rheum palmatum L., Angelica sinensis (Oliv.) Diels, Codonopsis pilosula (Franch.) Nannf., Asarum sieboldii Miq., Borneolum syntheticum, and Calomelas. Burn moisturizing scald ointment (BMS) was purchased from Meibao Pharmaceutical Co., Ltd. (Shantou, China); ELISA kits were obtained from Neobioscience Technology Co., Ltd. (Shenzhen, China); Collagen I primary antibody; phosphorylated PI3K, AKT, and mTOR; unphosphorylated PI3K, AKT, and mTOR polyclonal antibody; and HRP-labeled secondary antibody were purchased from Servicebio (Wuhan, China); Microscope (XSP-C204) was from Motic China group and microscope camera was purchased from Cognex (Massachusetts, United States); Toe volume measuring instrument (PV-200) was from TECHMEN Co., Ltd. (Chengdu, China); full-wavelength microplate reader 1,510 was obtained from Thermo Fisher Scientific (Massachusetts, United States); Ultimate 3000 series HPLC system consisting of the computer-controlled system with the CHROMELEONTM software and a SQL database, equipped with a WPS-3000 autosampler, was from Dionex (California, United States).
4
Dextran Formation via Dextransucrase Activity
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For identification of short-chain gluco-oligosaccharides produced by dextransucrases in the presence of maltose (“Dextran Formation After Pre-Cultivation in mMRS Broths Media Containing Sucrose”), high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) (ICS5000, Thermo Fisher Scientific, USA) on a CarboPac PA20 column (Thermo Fisher Scientific, USA) was performed. The separation was accomplished at a flow rate of 0.5 mL/min and an isocratic elution with 150 mM NaOH (Merck Millipore, USA) for 80 min and final flushing step with 200 mM NaOH and 1 M sodium acetate (Merck Millipore, USA) for 20 min. For identification and quantification of oligosaccharides, external sugar standards were used (Carbosynth, Switzerland) and the calibration curves were generated with the ChromeleonTM software (version 6.8, Dionex, Germany).
5
Sugar Quantification by HPLC-RI
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Mono- and disaccharides were quantified by high-performance liquid chromatography (HPLC) coupled to refractive index detection (ERC Refractomax 521, Thermo Fisher Scientific, USA). Sugar separations were performed on a Rezex RPM-Monosaccharide Pb2+ column (Phenomenex Ltd., Germany) with water as mobile phase at a constant flow rate of 0.6 mL/min at 85 °C and 20 μL of injection volume. For identification and quantification of the respective sugars, sugar standards were used and the calibration curves were generated with the ChromeleonTM software (version 6.8, Dionex, Germany).
6
Carbohydrate Profiling of S. flexneri
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The sugar composition of the S. flexneri PS was determined by HPAEC-PAD using a Carbo Pac PA1 column (50 mm × 250 mm) coupled to a CarboPac PA1 guard column and connected to Dionex KS5000 system. Samples diluted at 10 µg/ml of saccharide were treated with NaOH at a final concentration of 2 M, heated at 110 °C for 6 h in a closed screwcap test tube and filtered with 0.45 µm filter before the analysis. The separation was performed with a flow rate of 1 ml/min using gradient elution of 100 mM NaOH for 32 min with increased concentration of NaNO 3 ranging from 8 to 50%, followed by a washing step for 22 min. The chromatography was monitored using the pulsed amperometric mode with a gold working electrode and an Ag/AgCl reference electrode. A quadruple-potential waveform for carbohydrates was used. The chromatographic data were processed using Dionex Chromeleon TM software. The calibration curve was set up with glucose (Fluka) in the range of 1.0-25.0 µg/ml which was treated the same way as the samples mentioned above [20] . All the above experiments have been performed in triplicates and the results are the mean of those three readings.
7
HPLC-DAD Method for Compound Quantification
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Analysis was performed by a high-performance liquid chromatograph (HPLC) (U3000 Ultimate, Thermo Fisher Scientific, Courtaboeuf, France) coupled to diode array detection (DAD). The conditions proposed by Mahajan et al. and Pujeri et al. were taken as a starting point for optimization [12 (link),19 (link)]. A Zorbax® C18 column (dimensions, 75 by 4.6 mm; particle size, 3.5 µm) was used for separation. The mobile phase consisted of methanol (A) and formate buffer solution (B: 10 mM, pH adjusted to 3.5) in gradient mode (Tmin/A:B; T0/35:65; T9/35:65; T35/65:35; T40/35:65). The column temperature, the flow rate, and the detection for quantification were set at 25 ± 2 °C, 1.0 mL·min−1, and 246 nm, respectively. Data acquisition (e.g., peak time, area) was carried out using Chromeleon® software (v6.80, Thermo Fisher Scientific).
8
Size-Exclusion Chromatography of Biomolecules
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SEC was performed at ambient temperature using a Dionex UltiMate 3000 UHPLC system with a Phenomenex TSK G3000SWXL, 7.8-mm inner diameter × 300-mm column (Phenomenex). Mobile phase consisted of 0.2 m sodium phosphate and 0.1 m sodium sulfate (pH 6) at a flow rate of 0.5 ml/min. 20 μg of sample reconstituted in mobile phase was injected, and the column effluent was monitored at 280 nm. Data were analyzed by NIBSC for consistency using Chromeleon software (Thermo Fisher Scientific).
9
Analysis of Human Milk Carbohydrates by GPC
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Total HM carbohydrates were analyzed using GPC. Glycans were separated by the GPC stationary phase and eluted according to size and charge. Neutral mono-, di-, and oligosaccharides, and acidic oligosaccharides with different degrees of polymerization (DP), could be detected. HM carbohydrate solution was prepared by dissolving 0.2 g/mL of total HM carbohydrates in ultrapure water (Sartorius Arium Pro) containing 2% (v/v) 2-propanol at 37 °C. Five milliliters of 0.2 µM filter-sterilized HM carbohydrate solution were injected for each GPC run. The sample loop was cleaned by ultrapure water prior to analysis. Two connected Kronlab ECO50 columns (5 × 110 cm) packed with Toyopearl HW 40 (TOSOH BIOSCIENCE) were used. Milli-Q water was maintained at 50 °C using a heating bath (Lauda, RE 206) for column equilibration. Milli-Q water containing 2% (v/v) 2-propanol was used as the eluent. The flow rate of the eluent was set at 1.65 mL/min. Eluting glycans were monitored by refractive index detection (Shodex, RI-101). The resulting chromatograms were analyzed by using Chromeleon® software (Thermo Scientific).
10
Monosaccharide Analysis of Butyrivibrio Cultures
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The monosaccharides released during Butyrivibrio monoculture and coculture growth were determined using a HPIC method (55 ). Culture samples from the eight time intervals (0, 2, 4, 6, 8, 10, 12 and 16 h) were used for analysis, and the HPIC system used was the DIONEX ICS-5000 system (Thermo Fisher Scientific). Data analysis was performed with Chromeleon software (Thermo Fisher Scientific). Using a two-tailed, unpaired Student's t test, differences between sample means were considered statistically significant if the P value was <0.05. Quantitative analyses were carried out using standard solutions of the monosaccharides arabinose (Sigma-Aldrich), fucose (Sigma-Aldrich), galactose (Sigma-Aldrich), glucose (VWR International Ltd., Poole, UK), rhamnose (VWR), and xylose (VWR).
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