Oligonucleotides (ONs) were synthesized using a Biosset ASM-800 DNA synthesizer (Biosset Ltd., Russia) and standard reagents (Glen Research, USA) following standard phosphoramidite protocols. Modified ONs were synthesized using standard reagents and 5′-O-dimethoxytrityl-1′,2′-dideoxyribose-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (GlenResearch, USA). ONs were purified by a preparative-scale reverse-phase high-performance liquid chromatography (HPLC) on a 250 mm × 4.0 mm Hypersil C18 column (Thermo Fisher Scientific, USA) with detection at λ = 260 nm and a 2.5–15% gradient of acetonitrile in 0.1 M ammonium acetate buffer at 55°C. Dimethoxytrityl protection groups were removed via treatment with 80% acetic acid for 30 min, and the detritylated ONs were further purified in a 2.5–15% gradient of acetonitrile in 0.1 M ammonium acetate buffer. The purity of all ONs was approximately 95% by HPLC. The ON concentrations were calculated from the absorbance measured above 90°C and extinction coefficients, which were approximated using the nearest-neighbor model.
Hypersil c18 column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Hypersil C18 column is a reverse-phase liquid chromatography column designed for the separation and analysis of a wide range of organic compounds. It features a silica-based stationary phase with chemically bonded C18 alkyl chains, providing high-resolution separation capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Microtof q mass spectrometer, by Bruker (2 mentions)
Uhplc system, by Thermo Fisher Scientific (2 mentions)
1525 binary hplc pump, by Waters Corporation (2 mentions)
Speedvac, by Thermo Fisher Scientific (2 mentions)
Precellys 24, by Bertin Technologies (2 mentions)
Standard reagents, by Glen Research (1 mentions)
Model g1379b, by Agilent Technologies (1 mentions)
Waters 2707 autosampler, by Waters Corporation (1 mentions)
Microplate reader, by Tecan (1 mentions)
Rotary evaporator, by Eyela (1 mentions)
Uhplc esi tofms, by Bruker (1 mentions)
Avance 3 hd 600 mhz, by Bruker (1 mentions)
Avance 400 nmr spectrometer, by Bruker (1 mentions)
Ltq orbitrap xl instrument, by Thermo Fisher Scientific (1 mentions)
Prominence lc 20ad, by Shimadzu (1 mentions)
Nicolet nexus 470 ft ir spectrometer, by Thermo Fisher Scientific (1 mentions)
Ods gel, by YMC (1 mentions)
Ultimate 3000 uhplc system, by Thermo Fisher Scientific (1 mentions)
Waters alliance 2695, by Waters Corporation (1 mentions)
Lc 20ad pump, by Shimadzu (1 mentions)
19 protocols using hypersil c18 column
1
Oligonucleotide Synthesis and Purification
2
Amino Acid Derivatization and HPLC Analysis
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Compound 1 (0.5 mg) was placed in a 5 mL conical vial containing HCl (6 M, 1 mL), and the sealed vial was heated at 110 °C for 20 h. After evaporation of the solvent, H2O (100 μL) was added. Then NaHCO3 (1 M, 50 μL) and 1-fluoro-2,4-dinitrophenyl-5-l -alanine amide (l -FDAA, 1%, 100 μL) in acetone were added to the hydrolysis solution, and the sealed vial was heated at 40 °C for 2 h. Then HCl (2 M, 20 μL) was added to the reaction mixture to stop the reaction. After evaporation, the reaction products were dissolved in MeOH for HPLC analysis on a Thermo BDS Hypersil C18 column (150 mm × 4.6 mm, 5 μm). MeOH–H2O (0.5% H3PO4) gradient: 0 min: 30% MeOH–H2O; 40 min: 70% MeOH–H2O, with a flow rate of 1 mL min−1. UV detection was performed at a wavelength of 340 nm. Compounds 2–4 were performed by the same protocol as 1.
3
Identification of Rue Essential Oil Metabolites
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The identification of the secondary metabolites of rue essential oil was carried out following the methodology established by Díaz-Montes et al. [40 (link)]. An ultra-high-performance liquid chromatography (UHPLC) system (Thermo Scientific, Waltham, MA, USA) equipped with a photo diode array detector (PDA) was used for the analysis. The chromatographic separation was performed on a Hypersil C18 column (50 mm × 2.1 mm and 1.8 μm particle size) (Thermo Scientific, Waltham, MA, USA). The samples (0.3 μL) were injected into the system. The mobile phases were A: acetonitrile (20%) and B: water–trifluoracetic acid pH 2 (80%) in isocratic conditions, with a flow rate of 104 μL/min for a running time of 9.87 min. The UHPLC system was coupled with a micro TOF-Q mass spectrometer (Bruker Daltonics, Karlsruhe, Germany). The conditions of the micro TOF-Q were as follows: 2.7 kV capillary voltage; ionization source in negative electrospray mode (ESI− and ESI+); scan range, m/z 50–3000; set collision cell RF, 200.0 Vpp; set nebulizer, 0.4 bar; desolvation gas flow set at 4.0 L/min; and source temperature, 180 °C. The mass data were processed using Bruker Compass Data Analysis version 4.1 software (Bruker Datonics, Karlsruhe, Germany).
4
HPLC Assay and Drug Release Quantification
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High performance liquid chromatography (HPLC) was used for assay and drug release studies of the prepared layers. HPLC analyses were performed using Agilent HPLC 1200 Seriesconsisting of a degasser (Model G1379B), a binary pump (Model G1312A), auto sampler ALS (Model G1329A), auto sampler thermostat FC/ALS Therm (Model G1330B), thermostat column compartment (Model G1316A) and a variable wavelength detector (Model G1314B). Separation was performed on Thermo Scientific Hypersil C18 column (150 * 4.6 mm, 5 µm BDS). The mobile phase consisted of buffer (1.2 g KH2PO4/1 liter water, adjusted to pH 6.9 with KOH)/Acetonitrile (90:10, v/v). The HPLC system was operated at a flow rate of 1.0 ml/min at 40 °C. The UV detector was set at 200 nm. Method used in HPLC analysis was according to the USP with some modifications. The analytical method was validated (results are not shown).
5
Quantification of Artemisinin in Artemisia annua
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Dried Artemisia annua L. (15 g) was extracted with 80% methanol, 80% ethanol, and 80% acetone under reflux for 24 hr, and the supernatants were filtered using Whatman filter paper (Macherey-Nagel, Germany). The filtered extracts were evaporated under reduced atmospheric pressure using a rotary evaporator (Eyela, Japan), resulting in a viscous solution. Equal volumes of Artemisia annua L. were extracted with hot water and filtered using Whatman filter paper.
The artemisinin-containing Artemisia annua L. extracts were measured by the Folin-Denis method [20 ]. Folin-Ciocalteu phenol reagent (750 µl) was added to 150 µl of sample and the reaction mixture was incubated for 5 min. After incubation, 600 µl of 10% Na2CO3 was added and the reaction mixture was incubated at room temperature for 1 hr. Absorbance of the reaction mixture was measured at 760 nm using a microplate reader (Tecan, Männedorf, Switzerland). Artemisinin was quantified by HPLC with a Waters® 1525 binary HPLC pump, Waters® 2707 autosampler, Waters® 2489 UV/visible detector, and ODS (250×4.6 mm, 5 µm) Hypersil C-18 column (Thermo Fisher Scientific, Waltham, MA, USA) [21 ]. The mobile phase consisted of a 3:7 water:acetonitrile ratio with a flow rate of 1 ml/min. The injection volume for each sample was 10 µl, and the retention time of artemisinin was approximately 15 min at 220 nm.
The artemisinin-containing Artemisia annua L. extracts were measured by the Folin-Denis method [20 ]. Folin-Ciocalteu phenol reagent (750 µl) was added to 150 µl of sample and the reaction mixture was incubated for 5 min. After incubation, 600 µl of 10% Na2CO3 was added and the reaction mixture was incubated at room temperature for 1 hr. Absorbance of the reaction mixture was measured at 760 nm using a microplate reader (Tecan, Männedorf, Switzerland). Artemisinin was quantified by HPLC with a Waters® 1525 binary HPLC pump, Waters® 2707 autosampler, Waters® 2489 UV/visible detector, and ODS (250×4.6 mm, 5 µm) Hypersil C-18 column (Thermo Fisher Scientific, Waltham, MA, USA) [21 ]. The mobile phase consisted of a 3:7 water:acetonitrile ratio with a flow rate of 1 ml/min. The injection volume for each sample was 10 µl, and the retention time of artemisinin was approximately 15 min at 220 nm.
6
L-threonine and 2,5-DMP Analysis
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UNIVERSAL Hood gel imaging system was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA USA) for gel imaging. UV1000 spectrophotometer was purchased from Lairui Scientific Instrument Co, Ltd. (Shanghai, China) for photometric measurements. Protein electrophoresis system PowerPac™/ Mini-PROTEAN® and HC/Mini-PROTEAN® were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA USA) for SDS-PAGE analysis of expression products. A Hypersil C18 column was purchased from Thermo Fisher Scientific Inc. (Waltham, MA USA) for the quantitative analysis of L-threonine and 2,5-DMP. Avance III HD 600 MHz and UHPLC-ESI-TOF-MS were purchased from Bruker Corporation (Karlsruhe, Germany) for the qualitative analysis of L-threonine and 2,5-DMP.
7
Nanoparticle Encapsulation and Release Study
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The DL of RPTN, RCPTN, and RPN was analyzed using RP-HPLC (1200 Series; Agilent Technologies, Santa Clara, CA, USA) in a Hypersil C18 column (250×4.6 mm, pore size 5 μm; Thermo, Waltham, MA, USA). The full methods are described in the “Supplementary materials” section. DL was defined as the ratio between the quantity of RBG loaded into the NPs to the quantity of the NPs, and EE was defined as the ratio of the quantity of RBG loaded into the NPs to the total RBG quantity added during the preparation procedure. All preparation procedures were performed in sextuplicate.
The in vitro release of RBG in RPTN, RCPTN, and RPN was also evaluated using the dialysis method,24 (link) as described in the “Supplementary materials” section.
The in vitro release of RBG in RPTN, RCPTN, and RPN was also evaluated using the dialysis method,24 (link) as described in the “Supplementary materials” section.
8
HPLC Analysis of Tea Polyphenols
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HPLC analysis of the extracted EGCG and the supernatant from ZnO preparation process was carried out using the Waters 1525 binary HPLC pump coupled to an UV-visible detector (set at 270 nm, Waters 2489) and Hypersil C18 column (100 × 4.6 mm i.d., Thermo Fisher Scientific Inc, Waltham, MA, USA). The flow rate was 0.7 mL min−1, injection volume was 22 μL, and the sample was dissolved in acetonitrile at 100 mg mL−1. The gradient elution started with 95% of 0.1% aqueous formic acid and 5% acetonitrile. Then, the content of acetonitrile was increased linearly to 15% within 14 min and maintained for 11 min. After that, the percentage of acetonitrile was increased to 35% within 28 min, and to 85% within 12 min. Analysis was carried out in triplicate. Series of solutions of each standard were analyzed under the same HPLC conditions to establish the calibration curves. The standard solutions include 30–70 μg mL−1 gallic acid, 150–800 μg mL−1 caffeine, 120–500 μg mL−1 epigallocatechin-3-gallate (EGCG) and 40–120 μg mL−1 epicatechin-3-gallate (all polyphenols were at least 90% pure and were purchased from Chemieliva Pharmaceutical Co., Ltd., Chongqing, China. Each standard was analyzed by 1H NMR).
9
Spectroscopic Analysis of Organic Compounds
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Optical rotations were measured on a Rudolph IV Autopol automatic polarimeter. IR spectra were determined on a Thermo Nicolet Nexus 470 FT-IR spectrometer. 1D and 2D NMR spectra were recorded on a Bruker Avance 400 NMR spectrometer (400 MHz for 1H and 100 MHz for 13C, respectively). Chemical shifts were referenced to the solvent peaks at δH 2.50 and δC 39.8 for dimethyl sulfoxide (DMSO-d6), respectively, and coupling constants were in Hz. ESIMS and HRESIMS spectra were obtained from a Thermo Scientific LTQ Orbitrap XL instrument. Column chromatography was carried out with silica gel (160–200 mesh, 200–300 mesh), and HF254 silica gel for TLC was obtained from Qingdao Marine Chemistry Co. Ltd. (Qingdao, China), ODS gel (50 μm), and Sephadex LH-20 (18–110 μm) were obtained from YMC (Kyoto, Japan) and Amersham Pharmacia Biotech AB, Uppsala, Sweden. HPLC chromatography was performed on an Alltech instrument (426-HPLC pump), equipped with an Alltech UVIS-200 detector and semipreparative reversed-phase columns (Grace Prevail C18, 5 μm, 250 mm × 10 mm). Analytical HPLC chromatography was performed on Prominence LC-20AD (Shimadzu, Kyoto, Japan) equipped with pump LC-20AD, detector Prominence SPD-M20A PDA and Shimadzu VP-ODS column (150 mm × 4.6 mm), Thermo BDS Hypersil C18 column (150 mm × 4.6 mm, 5 μm).
10
Identification of Rue Essential Oil Metabolites
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The identification of the secondary metabolites of rue essential oil was carried out following the methodology established by Díaz-Montes et al. [40 (link)]. An ultra-high-performance liquid chromatography (UHPLC) system (Thermo Scientific, Waltham, MA, USA) equipped with a photo diode array detector (PDA) was used for the analysis. The chromatographic separation was performed on a Hypersil C18 column (50 mm × 2.1 mm and 1.8 μm particle size) (Thermo Scientific, Waltham, MA, USA). The samples (0.3 μL) were injected into the system. The mobile phases were A: acetonitrile (20%) and B: water–trifluoracetic acid pH 2 (80%) in isocratic conditions, with a flow rate of 104 μL/min for a running time of 9.87 min. The UHPLC system was coupled with a micro TOF-Q mass spectrometer (Bruker Daltonics, Karlsruhe, Germany). The conditions of the micro TOF-Q were as follows: 2.7 kV capillary voltage; ionization source in negative electrospray mode (ESI− and ESI+); scan range, m/z 50–3000; set collision cell RF, 200.0 Vpp; set nebulizer, 0.4 bar; desolvation gas flow set at 4.0 L/min; and source temperature, 180 °C. The mass data were processed using Bruker Compass Data Analysis version 4.1 software (Bruker Datonics, Karlsruhe, Germany).
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