Methylated RNA was purified using the RNA Clean & Concentrator Kit (Zymo Research). The purified RNA was subsequently enzymatically digested to individual mononucleosides using nuclease P1 (Sigma Aldrich), snake venom phosphodiesterase (Sigma Aldrich), and Calf Intestinal Phosphatase (NEB) [2 (link)–4 (link),20 (link),51 (link)]. The digested samples were separated by analytical HPLC using a Luna C18 reverse-phase column (10 μm, 4.6 mm x 250 mm) (Phenomenex) and as in a previously described protocol [20 (link)]. Synthetic 2-methyladenosine and the unmodified mononucleosides were detected by UV absorption at 256 nm, whereas the 14C-labeled methyladenosines were detected by radiomatic flow scintillation analyzer (Packard 515TR flow scintillation analyzer; Perkin-Elmer).
Luna c18 reverse phase column
Manufactured by Phenomenex
Sourced in United States
The Luna C18 reverse-phase column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. The column utilizes a C18 stationary phase, which provides excellent retention and selectivity for both polar and non-polar analytes. The column is available in various dimensions and particle sizes to suit different application needs.
Automatically generated - may contain errors
Lab products found in correlation
Nuclease p1, by Merck Group (1 mentions)
Rna clean concentrator kit, by Zymo Research (1 mentions)
Calf intestinal phosphatase, by New England Biolabs (1 mentions)
Snake venom phosphodiesterase, by Merck Group (1 mentions)
Data analysis software version 4, by Bruker (1 mentions)
Maxis 3g mass spectrometer, by Bruker (1 mentions)
Hplc system, by Shimadzu (1 mentions)
1260 infinity hplc system, by Agilent Technologies (1 mentions)
Lc 20at prominence, by Shimadzu (1 mentions)
Spd 20a, by Shimadzu (1 mentions)
Nylon membrane filter, by Merck Group (1 mentions)
Lc solution software version 1.24 sp1, by Shimadzu (1 mentions)
Waters 2489 uv detector, by Waters Corporation (1 mentions)
Empower 2, by Waters Corporation (1 mentions)
Waters 717 plus autosampler, by Waters Corporation (1 mentions)
Waters 600 binary pump, by Waters Corporation (1 mentions)
Sil 10af, by Phenomenex (1 mentions)
Milli q water, by Merck Group (1 mentions)
1200 hplc, by Agilent Technologies (1 mentions)
6330 series ion trap lc ms system, by Agilent Technologies (1 mentions)
11 protocols using luna c18 reverse phase column
1
Quantification of Methylated RNA
2
UHPLC-HRMS Analysis of EAS^xO^xO Endoperoxides
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High resolution mass spectrometry analysis of EASxOxO endoperoxides were performed in an UHPLC Agilent coupled to an UHR-ESI-Q-TOF Bruker Daltonics MaXis 3G mass spectrometer with CaptiveSpray source in the negative mode. The UHPLC mobile phase consisted of ammonium formate (solvent A) and acetonitrile:methanol 7:3, v/v (solvent B) with the following linear gradient: 25% B during 15 min, 25 to 70% B for 1 min, 70% B until 25 min, 70 to 25% B during 1 min and 25% B until 30 min. Endoperoxides was separated on a Luna C18 reverse phase column, 250 × 4.6 mm, 5 µM particle size (Phenomenex, Torance, CA) and monitored at 210 nm. The flow rate was 0.8 mL.min−1. Reverse phase column was kept at 30°C. The ESI conditions were: capillary, 4.0 kV; dry heater, 180 °C; dry gas, 8.0 l/min; end plate, −450 V. Nitrogen was used as collision gas and the CID (collision-induced dissociation) energy was 10 eV. The instrument was externally calibrated using an ESI low concentration tuning mix over the m/z range of 100 to 2000. The Bruker Data Analysis software (version 4.0) was employed for data acquisition and processing.
3
Quantitative Analysis of Endoperoxide EASO2
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HPLC-ESI-MS/MS analyses of the anthracene endoperoxide EASO2 were conducted by injecting 25 µL of the sample in a Shimadzu HPLC system (Tokyo, Japan) coupled to a mass spectrometer Quattro II triple quadrupole (Micromass, Manchester, UK). Endoperoxide EASO2 was separated using a Luna C18 reverse phase column, 250 × 4.6 mm, 5 µM particle size (Phenomenex, Torance, CA,) that was kept at 25°C. The liquid phase consisted of 25 mM ammonium formate (solvent A) and acetonitrile:methanol 7:3, v/v (solvent B) with linear gradient of 25% B during 15 min, 25 to 70% B for 1 min, 70% B until 25 min, 70 to 25% B during 1 min and 25% B until 30 min. The eluent was monitored at 210 nm with a flow rate of 0.8 mL.min−1. First 5 min of run gradient was discarded and 10% of flow rate was directed to the mass spectrometer. Ionization of the sample was obtained by electrospray ion source (ESI) in the negative ion mode using the following parameters: source temperature, 120°C; desolvation temperature, 200°C; cone voltage, 15 V; collision energy, 10 eV. The endoperoxides EASxOxO were detected by the loss of the oxygen molecule, in the Selected Reaction Monitoring mode (SRM). The transitions recorded were m/z 228→212 for EAS16O16O, m/z 229→212 for EAS18O16O and m/z 230→212 for EAS18O18O.
4
Phenolic Profile of S. dendroideum Extract
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The phenolic compound profile of S. dendroideum extract was evaluated on a 1260 Infinity HPLC system coupled to a diode array detector (DAD) (Agilent Technologies, CA, USA). The HPLC method followed has been reported previously [10 (link)]. In brief, the analysis was developed using a 5 μm Luna C18 reverse-phase column (Phenomenex, CA, USA), maintained at 25°C. The mobile phase consisted of water (eluent A) and methanol-water (60 : 40% v/v, eluent B). The pH of both eluents was adjusted to 2.4 using phosphoric acid (D. E. Q., Monterrey, NL, Mexico). The gradient used was as follows: 0 min, 100/0; 3 min, 70/30; 8 min, 50/50; 35 min, 30/70; 40 min, 20/80; 45 min, 0/100; 50 min, 0/100; and 60 min, 100/0 (time (%) of eluent A/% of eluent B). The method was run for 60 min at 0.8 mL/min with a pressure of no more than 400 psi. The samples were prepared in DMSO (Research Organics, Cleveland, OH, USA) and further diluted with water to a final concentration of 25 mg/mL. Chromatograms were recorded at 360 nm.
5
Carbamazepine Extrudate Analysis via HPLC
6
Quantitative Analysis of NPX, DCF, and MFN
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A Shimadzu® liquid chromatograph (LC-20AT Prominence) equipped with a UV/Vis detector SPD-20A with a slit of 8nm was used. In the first 7 minutes of analysis, the 256 nm wavelength was used in the UV/Vis (for NPX) and in the last 8 minutes (for DCF and MFN), the 234 nm wavelength. The separation of the compounds was performed on a Luna C-18 reverse phase column (Phenomenex®) (250 mm x 4.6 mm, 5 µm), maintained at 25 °C, using a mobile phase of ACN: acidified water with H3PO4 (pH 2.24) 60/40 (v/v), with a flow of 1.2 mL min -1 . The mobile phase was filtered through 0.45 μm nylon membrane filters (Millipore). To control the equipment and obtain the data, a microcomputer and LC solution® software version 1.24 SP1 of Shimadzu were used.
An MX-S mini vortex and a USC-1400A ultrasound were used for the extraction process.
An MX-S mini vortex and a USC-1400A ultrasound were used for the extraction process.
7
HPLC Analysis of Carbamazepine Extrudates
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A Waters HPLC, consisting of a Waters 600 binary pump, Waters 2489 UV/detector, and Waters 717 plus autosampler (Waters Technologies Corporation, 34 Maple St., Milford, MA, USA), determined the drug content. A Phenomenex Luna ® C18 reverse phase column (5 µm 100 Å, 250 × 4.6 mm) was used as the stationary phase. Empower 2 software (Version 32, Waters, Milford, MA, USA, 2008) was used to analyze the data. The mobile phase was water:methanol:acetic acid (34:65:1 % v/v), and the UV detector was set at 285 nm wavelength. The flow rate was maintained at 1.0 mL/min, and 20 µL was injected from each sample. Drug-content uniformity was assessed by dissolving accurately weighed CBZ extrudates in methanol and subsequently quantified by using the HPLC procedure outlined above.
8
Chromatographic Profiling of Plant Extracts
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Chromatographic profiles of extracts of different species obtained by the diode array alarm coupled with high performance liquid chromatography. (HPLC-PDA) and UFLC-ESI-TOF. First, the extracts were analyzed in a Shimadzu © high performance liquid chromatograph (HPLC) composed of two LC6-AD pumps, automatic injection system SIL-10AF coupled to a detector with a PDA photodiode array), at the 'using a Phenomenex Luna C-18 reverse phase column (4.6 x 250mm, 5µM),
9
HPLC-MS Analysis of Bioactive Compounds
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Samples were analyzed by liquid chromatography (1200 HPLC, Agilent Technologies, USA) equipped with a Luna reverse phase C-18 column (3.00 m, 150 mm × 3.0 mm, Phenomenex, USA).
The instrument was equipped with a binary solvent pump with the following solvents: (A) MilliQ water (Millipore, U.S.A.) with 10% v/v of formic acid and (B), methanol/water/formic acid 50/40/10 v/v. The chromatographic separation was carried out at a constant flow rate (200 l min -1 ) and to a stepwise gradient: from 15% to 45% of B in 15 min, to 70% of B at 35 min, to 90% of B at 45 min, then 99% of B at 55 min, hold for 4 min. The initial mobile phase was re-established for 11 min before the next injection. DAD detector was set at 520 nm. The mass spectrometry analyses were performed with a 6330 Series Ion Trap LC-MS System (Agilent Technologies, U.S.A.) equipped with an electrospray ionization source (ESI) operating in positive mode. Qualitative analyses were performed in scan mode (100-850 m/z) and N2 dry gas temperature was set at 325°C. Mass spectra were processed and analyzed by the DataAnalysis for 6330 Series Ion Trap LC/MS 4.0 software (Bruker Daltonik, Bremen, Germany).
Identification of spectra was done by analysis of fragmentation pattern and by comparison with literature data.
The instrument was equipped with a binary solvent pump with the following solvents: (A) MilliQ water (Millipore, U.S.A.) with 10% v/v of formic acid and (B), methanol/water/formic acid 50/40/10 v/v. The chromatographic separation was carried out at a constant flow rate (200 l min -1 ) and to a stepwise gradient: from 15% to 45% of B in 15 min, to 70% of B at 35 min, to 90% of B at 45 min, then 99% of B at 55 min, hold for 4 min. The initial mobile phase was re-established for 11 min before the next injection. DAD detector was set at 520 nm. The mass spectrometry analyses were performed with a 6330 Series Ion Trap LC-MS System (Agilent Technologies, U.S.A.) equipped with an electrospray ionization source (ESI) operating in positive mode. Qualitative analyses were performed in scan mode (100-850 m/z) and N2 dry gas temperature was set at 325°C. Mass spectra were processed and analyzed by the DataAnalysis for 6330 Series Ion Trap LC/MS 4.0 software (Bruker Daltonik, Bremen, Germany).
Identification of spectra was done by analysis of fragmentation pattern and by comparison with literature data.
10
HPLC Analysis of Oil Samples
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The HPLC system (1100 series, Agilent Technologies) was equipped with a quaternary pump, auto-injector system and a thermo stated column compartment. Two types of detectors were linked to the same system which was Agilent Technologies refractive index detector and an Alltech ELSD 800. The oil samples (10 μL) were analyzed using a Phenomenex Luna reverse phase C18 column (250 ×4.6 mm, 5 um particle size) operating at 35℃. Both methods used isocratic analyses with mobile phase mixtures of acetonitrile:methylene chloride (2:3 v/v) . For ELSD, analysis was conducted with an evaporator temperature of 40℃ and an air pressure of 2.3 bars. For the RID, the detector temperature was set at 35℃. The flow rates for the mobile phase were 0.8 mL/min (HPLC-ELSD) and 1.0 mL/min (HPLC-RID) .
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