1H and 13C nuclear magnetic resonance (1H NMR) spectra were measured on a Varian 300 MHz and Bruker 400 MHz AC NMR spectrometers. Based on their solubility, samples were dissolved in different deuterated solvents Deutero® (Kastellaun, Germany). Electron ionization-mass spectrometry (EI-MS) spectra were recorded on Thermo Scientific, Trace gas chromatograph Ultra coupled with ISQ Single Quadruple MS Capillary column (National Research Center, Egypt). Two-dimensional (2D) NMR experiments (double quantum filter correlated spectroscopy [COSY], heteronuclear single-quantum correlation spectroscopy [HSQC]) were carried out with all isolated compounds using the pulse sequences from the Varian and Bruker user library. On the basis of 2D-NMR analyses, assignments of 1H and 13C signals were established. Column chromatography (CC) was carried out on silica gel 60 (0.063–0.2 mm) (Sigma-Aldrich Co., USA) and Sephadex LH-20 (25–100 mm) (Sigma-Aldrich Co., USA) using mixtures of dichloromethane and methanol with different ratios for silica gel 60 and 100% methanol for Sephadex LH-20. Fractions were monitored by thin layer chromatography on precoated silica gel 60 F254 (0.25 mm) (Merck®, Darmstadt, Germany) using either p-anisaldehyde/sulfuric acid spray reagent and heating at 100°C for 7 min, NH3 or 5% AlCl3 reagent.[18 ]
Sephadex lh 20
Manufactured by Merck Group
Sourced in United States, Germany, Japan, China, Chile, Sweden, United Kingdom, Ireland, India, Switzerland
Sephadex LH-20 is a gel filtration medium used for the separation and purification of molecules based on their size and molecular weight. It is a cross-linked dextran polymer that can be used in a variety of applications, including the separation of proteins, peptides, nucleic acids, and other biomolecules. The porous structure of the Sephadex LH-20 beads allows for the efficient separation of molecules, making it a versatile tool for researchers and laboratory professionals.
Automatically generated - may contain errors
Lab products found in correlation
Silica gel, by Merck Group (25 mentions)
Silica gel 60, by Merck Group (24 mentions)
Silica gel 60 f254, by Merck Group (15 mentions)
Diaion hp 20, by Merck Group (13 mentions)
Silica gel, by Qingdao Marine Chemical (13 mentions)
Lobar lichroprep rp 18, by Merck Group (11 mentions)
Lichroprep rp 18, by Merck Group (10 mentions)
Silica gel f254, by Merck Group (10 mentions)
Rp 18 f254s plates, by Merck Group (9 mentions)
Gallic acid, by Merck Group (9 mentions)
Methanol, by Merck Group (9 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (9 mentions)
Cdcl3, by Merck Group (9 mentions)
Kieselgel 60, by Merck Group (8 mentions)
Silica gel 60 pf254, by Merck Group (8 mentions)
Ethyl acetate, by Merck Group (7 mentions)
Rp 18 f254, by Merck Group (7 mentions)
Nicolet 380 ft ir instrument, by Thermo Fisher Scientific (7 mentions)
Dmso d6, by Merck Group (6 mentions)
Acetonitrile, by Merck Group (6 mentions)
309 protocols using sephadex lh 20
1
Spectroscopic Characterization of Compounds
2
Antibacterial Compound Purification by Sephadex LH-20
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Sephadex® LH-20 (Merk, Darmstadt, Germany) was used as stationary phase. The Sephadex was suspended in chloroform to pack the column. The solvent-resistant column was 40 cm long, with a diameter of 2.5 cm and had a glass stopper at the bottom. The Sephadex suspension was introduced gradually, and the final column size was 30 × 2.5 cm. A 243.3 mg ethyl acetate extract was dissolved in 5 mL of chloroform and was passed through the column with flow rate 0.2 mL/min. A gradient solvent system of cyclohexane–chloroform–methanol (3:1:1, 2:1:1, 1:1:1) was used. Fractions of 5 mL were collected.
Each fraction was dissolved in 200 µL ethanol and 10 µL were added to each well and tested against K. pneumoniae using the agar well diffusion method to detect which fraction expressed the most antibacterial activity
Each fraction was dissolved in 200 µL ethanol and 10 µL were added to each well and tested against K. pneumoniae using the agar well diffusion method to detect which fraction expressed the most antibacterial activity
3
Metabolite Extraction and Purification
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Hexane, methanol, dichloromethane, and ethyl acetate (Alfa chemicals, Cairo, Egypt) were employed for the extraction and isolation of metabolites. TLC plates (Merk, Darmstadt, Germany), RP-TLC plates (Merk, Darmstadt, Germany), normal column silica gel plates (Merk, Darmstadt, Germany), reversed phase C18 silica gel plates (Merk, Darmstadt, Germany), and Sephadex LH20 (Merk, Darmstadt, Germany) were utilized for the compound isolation process.
4
Physicochemical Characterization of Compounds
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Optical rotations were measured on a J-715 spectropolarimeter (Jasco, Japan) in a 10.0 cm tube, using the suitable solvent for each compound (MeOH or CHCl 3 ). IR spectra were recorded on an Alpha FTIR spectrometer (Bruker), and ECD spectra on a J-1500 spectropolarimeter (JASCO, Japan). NMR experiments were carried out on an Avance II (600 MHz, Bruker, USA) using tetramethylsilane (TMS) as internal standard (Euriso-Top, France). HRESIMS spectra were recorded on a micrOTOF-Q II (Bruker) operated by Hystar software. For fractionation a Reveleris® system (Buchi, Switzerland) with detection wavelengths always set to 205, 254, and 280 nm, or open columns filled with silicagel, RP-18 material or Sephadex LH-20 (Sigma-Aldrich, USA) were used. Final purifications were sometimes conducted on a semi-preparative Dionex HPLC system, equipped with a P580 pump, ASI 100 automated sample injector, UVD 170 U detector, and Gilson Ambimed 206 fraction collector, using a Phenomenex Synergi 4u MAX-RP 80A column (250 × 10 mm, 4 μm particle size). The flow rate was always set at 2.00 mL/min. All solvents required for extraction and isolation were purchased from VWR (Austria). Ultrapure water was produced by a Sartorius arium UV system (Germany).
5
Solvent Preparation and Filtration
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All solvents were purchased from Sigma-Aldrich (Taufkirchen, Germany). Water was purified by a Milli-Qplus system from Millipore (Milford, MA, USA). Sephadex LH-20 was purchased from Sigma-Aldrich. Nylon filters (0.45 µm pore size) were from Agilent (Agilent Technologies, Palo Alto, CA, USA).
6
Extraction and Purification of Oleocanthal
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Approximately 2 L of n-hexane and 1 kg of EVOO (Sigma-Aldrich, MO) were mixed, CH3CN-MeOH (1 L, 20:80) was added, and the mixture was shaken twice. The dried organic layer (24 g) was subjected to repeated medium pressure liquid chromatography (MPLC) in a 50 × 3 cm column with lipophilic Sephadex LH20 (bead size 25−100 μm, Sigma-Aldrich, MO) using n-hexane-CH2Cl2 (1:9) and isocratic elution followed by MPLC (10 g, 25×1 cm column) with C-18 reversed-phase silica gel and Bakerbond octadecyl (40 μm; Mallinckrodt Baker, Inc.), yielding 13.3 mg of > 97% pure (−)-oleocanthal (measured using HLPC) and several other impure fractions. Identification and determination of purity were also based on comparison of 1H and 13C NMR data to the literature [42 (link)]. Generally, 1:100 ratios of mixtures to be chromatographed to stationary phase were used in all liquid chromatographic purifications.
7
Purification and Isolation of Compounds from Ulmus pumila L.
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Ulmus pumila L. extract (14.5 g) was subjected to liquid-liquid partitioning between water and ethyl acetate. The ethyl acetate fraction (4 g) was further partitioned between hexane and 90% aqueous methanol. The latter fraction was chromatographed over silica bed (silica gel 60) using mixture of methanol in dichloromethane as mobile phase starting from 0% to 100% methanol to afford ten fractions (A-J). Fraction B (150 mg) eluted with 5% MeOH/CH2Cl2 was chromatographed on sephadex LH-20 (Sigma) using 50% MeOH/CH2Cl2 as mobile phase to yield nine fractions of which fraction B-7 (50 mg) was further purified by reversed phase C-18 flash chromatography (Buchi-Reverelis Prep., Reverelis® phase C18 cartridge) with gradient elution from 0% to 100% MeOH in H2O to yield1 (8 mg) and 2 (5.5 mg). Fraction C (100 mg) eluted with 10% MeOH/CH2Cl2 subjected to further purification on sephadex LH-20 with 50% MeOH/CH2Cl2 as mobile phase to yield 3 (10 mg) and 4 (3 mg). Fraction D (120 mg) eluted with 15% MeOH/CH2Cl2 yielded 5 (9.5 mg) and 6 (8 mg) after purification on sephadex LH-20. Finally, fraction E eluted with 20% was purified on sephadex LH-20 and eluted with 100%MeOH to afford 7 (4 mg) and 8 (2.5 mg).
8
Analytical Methods for Chemical Compound Characterization
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Melting points were determined with a Reichert Thermovar apparatus and are uncorrected (Reichert Technologies, Buffalo, NY, USA). Optical rotations were determined at room temperature on a Perkin Elmer 343 polarimeter (Perkin Elmer, Waltham, MA, USA). IR spectra were taken in a Bruker IFS 66/S spectrometer. NMR spectra were run on a Bruker AMX-500 spectrometer with pulsed field gradient using the solvent (CDCl3) as internal standard (Bruker Corporation, Billerica, MA, USA). EIMS and exact mass measurements were recorded on a Micromass Autospec instrument at 70 eV. Preparative and semipreparative HPLC was carried out with a Beckman Coulter 125P equipped with a diode-array detector Beckman Coulter 168 (Beckman Coulter Life Sciences, Brea, CA, USA) and preparative Interstil Prep-sil 20 mm × 250 mm, 10 µm particle size (Gasukuro Kogio, Shinjuku-ku, Tokyo, Japan) and semipreparative Ultrasphere silica 10 mm × 250 mm, 5 µm particle (Beckman Coulter Life Sciences, Brea, CA, USA) size columns. Silica gel 60 F254 (Merck 105715, Darmstadt, Germany) and Sephadex LH-20 (Sigma-Aldrich, St. Louis, MO, USA) were used for column chromatography. Computational analysis was carried out with the Hyperchem 7.0 program applying the Polak-Ribiere minimization algorithm.
9
Chromatographic Purification of Phenolic Compounds
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TLC (Kieselgel 60 GF254, Merck) were developed in n-hexane/EtOAc mixtures and spots were revealed by spraying plates with H2SO4-MeOH (5:95, v/v) and heating at 120 °C. Silica gel (Kieselgel 60, Merck, Santiago, Chile 0.063–0.200 mm) and Sephadex (LH-20, Sigma Aldrich, Santiago, Chile) were used in column chromatography (CC). Technical solvents used in chromatography processes were previously distilled and dried according to standard procedures. Caffeic acid, p-coumaric acid, 1,5-Dicaffeoylquinic acid, isorhamnetin, and quercetin (purity 95%, by HPLC) were acquired in Extrasynthese (Genay, France) or Sigma Aldrich (Santiago, Chile).
10
Spectroscopic Analysis of Green Propolis
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The NMR spectra were run on a Bruker DPX 400 spectrometer (400 MHz for 1 H and 100 MHz for 13 C). The samples were dissolved in DMSO, and the spectra were calibrated with the solvent signals at 2.50 ( 1 H) and 39.5 ( 13 C) respectively. Classic and Vacuum liquid chromatography, respectively CC and VLC (glass columns of 450 X 25 mm and 50-100 mm i.d.) were used for chromatographic fractionation of green propolis extract by using silica gel 60H (Merck, art. 7736). Thin Layer Chromatography (TLC) was undertaken by using plates of 1 mm thickness PF254 and art. 9385 from Merck. Sephadex LH-20 and λ-carrageenan were acquired from Sigma-Aldrich, St. Louis, MO, USA. Commercial hexanes, methanol and ethyl acetate (EtOAc) were purified by distillation in our facilities.
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