The dried C. tubulosa stem was ground into powder, and then extracted five times with 75% EtOH. After solvent evaporation under reduced pressure, the crude extract was subjected to Macroporous resin AB-8 column chromatography with H2O/EtOH gradient solvent systems from 20% EtOH up to 100% EtOH. According to the thin layer chromatography, four fractions (Fr.1~Fr.4) were collected for further separation. Fr. 2 was subjected to preparative high performance liquid chromatography (HPLC) on a COSMOSIL® 5C18-AR-II column (250 mm × 20 mm i.d., 5 μm) using 18% acetonitrile as the mobile phase system. The flow rate was 15 mL/min. Three major peaks of interest were selectively collected. The fractions containing the targeted compounds were further condensed to dryness and produced 1 , 2 , and 3 , respectively.
5c18 ar 2 column
Manufactured by Nacalai Tesque
Sourced in Japan
The 5C18-AR-II column is a reversed-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a C18 stationary phase with a specific ligand chemistry that provides improved peak shape and resolution. The column dimensions and particle size are suitable for a variety of HPLC applications.
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12 protocols using 5c18 ar 2 column
1
Isolation and Purification of Bioactive Compounds from Coeloglossum tubulosa
2
HPLC Fingerprint Analysis of N11
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The high performance liquid chromatography (HPLC) fingerprint of N11 was conducted on a Hitachi HPLC system (L-2000 series, Tokyo, Japan). The concentration of N11 was 4 mg/ml. The separation was performed using a Cosmosil 5C18-AR-II column (5 µm, 25 cm×4.6 mm I.D.) at an elution flow rate of 0.8 ml/min and with a mix solvent of A-B (A = H2O, B = CH3CN), which was varied as follows: 0–10 min, 98% A, 2% B; 10–15 min, 98–0% A, 2–100% B; 15–30 min, 0% A, 100% B. The injection volume was 10 µl, and the UV detection wavelength was set at 220 nm. Ultrapure water and acetonitrile elution solvent were used as mobile phases in a series of experiments. Identification of N11 was dependent on retention time and UV spectra in comparison with the standard.
3
HPLC Fingerprint Analysis of Samples
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HPLC was performed on a Shimadzi UPLC Nexera X3 series instrument (Shimadzu, Kyoto, Japan) including an LC-40D X3 (pump), CBM-40 (system controller), DGU-405 (degassing unit), Sil‐40C X3 (autosampler), and CTO-40S (column oven) and equipped with an ODS COSMOSIL 5C18-AR-II column (4.6 mm × 250 mm). In HPLC fingerprint analysis, the mobile phase consisted of double-distilled water with 0.3% phosphoric acid and acetonitrile (ACN) using a gradient condition. The mobile phase flow rate and the injection volume were 1.0 mL/min and 10 μL, respectively. The column oven set at 40 ℃. The concentration of the tested sample was 10 mg/mL in ddH2O.
4
HPLC Analysis of S-allylcysteine
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For the WA and standard (S-allylcysteine, SAC), a high-performance liquid chromatography (HPLC) profile was established. The instruments comprised a Shimadzu LC-10Avp HPLC system and SPD-M10A Diode array detector. Analyses were executed in the experiment by employing a COSMOSIL 5C18-AR II column (4.6 × 250 mm2, 5 µm), with the mobile phase being observed to contain acetonitrile and 10 mM KH2PO4 with an isocratic elution (2:98). All standards and samples in the experiment were passed through a 0.45-µm Minipore filter prior to injection (10 µL) into the column. A 220-nm detection wavelength was employed, and the determined flow rate was 1.0 mL/min. Each analysis required 20 min.
5
SARS-CoV-2 3CL Protease Inhibition Assay
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Enzyme assays were carried out using recombinant SARS 3CL R188I mutant protease at an enzyme concentration of 120 nM. The reaction mixture (40 mM AcONa buffer, pH 4.0, containing 10% grecerol, 10 mM DTT and 4 M NaCl) was analyzed on a Cosmosil 5C18-AR-II column (4.6 × 150 mm), employing a linear gradient of MeCN (10–40%, 30 min) in aq 0.1% TFA. Each IC50 value was obtained from a sigmoidal dose–response curve obtained from the decrease of the substrate in the reaction mixture. Each experiment was repeated three times.
6
Synthesis and Purification of E[c(RGDfK)]{c[RGDf(4-I)K]}2
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Compound 5 (3.0 mg, 1.5 µmol) was treated with a mixture of 95% TFA, 2.5% water, and 2.5% triisopropylsilane (TIS) for 2 h at room temperature. The crude peptide was purified by RP-HPLC on Cosmosil 5C18-AR-II column (10 × 250 mm) at a flow rate of 4 mL/min with a gradient mobile phase of 35% methanol in water with 0.1% TFA to 55% methanol in water with 0.1% TFA for 20 min. The solvent was removed by lyophilization to provide 6 (1.5 mg, 71%) as a white powder.
E[c(RGDfK)]{c[RGDf(4-I)K]}2 (6 ): MS (ESI+) calcd for C59H87IN19O16 [M + H]+: m/z = 1444.56; found, 1444.59.
E[c(RGDfK)]{c[RGDf(4-I)K]}2 (
7
HPLC Analysis of Quercetin Glycosides
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HZW (7.1 mg) was dissolved in 2 ml methanol and responded by ultrasonic wave for 10 minutes at 25°C. After the filtration through a 0.45 μm polyvinylidene fluoride membrane, the filtrate was injected into the Agilent 1100 series HPLC (10 μl) system. The separation was performed on a COSMOSIL 5C18-AR-II column (5 μm, 250 × 4.6 mm, i.d.), which was eluted at a flow rate of 1.0 ml/min with a mobile phase gradient of A–B (A = H2O (0.05% formic acid), B = MeOH) varying as follows: 0–10 min: 90–80% A, 10–20% B; 10–40 min: 80–0% A, 20–100% B; and 40–50 min: 0–0% A, 100–100% B. The UV detection wavelength was set at 254 nm. The two standards are quercetin-3-O-β-D-glucopyranosyl-4′-O-β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranoside (compound 1) and quercetin-4′-O-β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranoside (compound 2) [27 (link)].
8
HPLC Quantification of Melatonin
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The concentration of MEL was determined by an absolute calibration method using a high-performance liquid chromatography (HPLC) system equipped with a diode array detector (Shimadzu, Kyoto, Japan). The HPLC system consisted of an LC-20A solvent delivery unit with highpressure flow-line selection valves, an SIL-20A auto sampler, a CTO-20A column oven, and an SPD-M20A diode array detector connected to the LC solution software. Chromatography was conducted using a COSMOSIL 5C 18 -AR-II column (particle size 5 μm, column dimensions 4.6 mm×150 mm). Column temperature was maintained at 40°C, and the samples were separated using a mobile phase consisting of 20 mM phosphate buffer (pH 7.0) and acetonitrile (3:7, v/v) at a flow rate of 0.5 mL/min. The diode array detector was set at 360 nm.
9
Quantification of Test Compounds by HPLC
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The concentration of the test compounds in the serosal solution and plasma sample were measured using reversed-phase high performance liquid chromatography system (LC-2000Plus; JASCO Company, Tokyo, Japan) equipped with a variable wavelength UV-visible detector (UV-2070plus; JASCO Company) and a fluorescence detector (FP-2025plus; JASCO Company). Griseofulvin was separated on an octadecylsilyl (ODS) column (COSMOSIL 5C18-AR-II column, 150 Â 4.6 mm 2 ) using a mobile phase consisting water and acetonitrile (3:2, v/v) at a flow rate of 1.0 mL/min at 30 C and monitored fluorometrically at excitation/emission wavelengths of 492/515 nm. Diclofenac was separated on the ODS column using a mobile phase consisting methanol and water (3:1, v/v) containing 0.1% phosphoric acid at a flow rate of 1.0 mL/min at 40 C, and monitored spectrophotometrically at a wavelength of 286 nm. Similarly, antipyrine was separated on the ODS column using a mobile phase (methanol/50 mM potassium dihydrogen phosphate ¼ 1:1, v/v) and monitored at 254 nm. Theophylline was separated on the ODS column using a mobile phase (methanol/ water ¼ 1:4, v/v) containing 1% acetic acid and monitored at 270 nm. The concentrations of FD-4 were determined using a fluorescence microplate reader (Varioskan™ Flash 2.4; Thermo Fisher Scientific Inc., Kanagawa, Japan) at excitation/emission wavelengths of 492/515 nm.
10
Detailed Analytical Techniques for Chemical Synthesis
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Unless otherwise noted, all materials used in this study were purchased from commercial suppliers and used without further purification. Unless otherwise noted, work-up and purification procedures were performed with reagent-grade solvents under air. Analytical thin-layer chromatography (TLC) was performed using E. Merk silica gel 60 F254 precoated plates (0.25 mm). The developed chromatogram was analyzed with a UV lamp (254 nm) and ethanolic ninhydrin. Flash column chromatography was performed with an Isolera Spektra instrument equipped with a Biotage® Sfär Silica HC D 10 g or Sfär Silica HC D 50 g cartridge. Preparative high performance liquid chromatography (HPLC) was performed on a Prominence HPLC system (Shimadzu) with a 5C18-AR-II column (#38150-41, Nacalai tesque). High-resolution mass spectrometry (HRMS) was measured using a Bruker micrOTOF II (ESI). Nuclear magnetic resonance (NMR) spectra were recorded on a JEOL ECS400 (1H 400 MHz, 13C 100 MHz) spectrometer. Chemical shifts for 1H NMR are expressed in parts per million (ppm) relative to CHCl3 (δ 7.26 ppm) in CDCl3, HOD (δ 4.79 ppm) in D2O. Chemical shifts for 13C NMR are expressed in ppm relative to CDCl3 (δ 77.19 ppm) in CDCl3. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), coupling constant (Hz), and integration.
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