Crude organic extracts were fractionated using a Shimadzu preparative HPLC system consisting of a CBM-20A controller, an LC-20AP pump, an SPD-20A detector, and an FRC-10A fraction collector, equipped with a C18 reversed-phase Reprosil column (10 μm, 120 Å, 250 by 22 mm). The system was controlled with Shimadzu LabSolutions software. A 12.5-mL flow was used with a linear gradient of buffer B (0 to 95%), 5 min of 95% buffer B, and then 100% buffer A for 5 min.
Preparative hplc system
Manufactured by Shimadzu
Sourced in Japan
The Preparative HPLC system is a high-performance liquid chromatography instrument designed for the purification and isolation of compounds on a larger scale. It is used to separate and purify target molecules from complex mixtures, such as those found in chemical, pharmaceutical, or biotechnology applications.
Automatically generated - may contain errors
Lab products found in correlation
Labsolutions software, by Shimadzu (1 mentions)
Luna c18 2, by Phenomenex (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Ecs 400 mhz nmr spectrometer, by JEOL (1 mentions)
Ion trap time of flight it tof mass spectrometer, by Shimadzu (1 mentions)
C18 column, by Shimadzu (1 mentions)
Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Xbridge c18 column, by Waters Corporation (1 mentions)
Lc 20ap, by Shimadzu (1 mentions)
Sil 10ap, by Shimadzu (1 mentions)
Frc 10a, by Shimadzu (1 mentions)
Spd 20a, by Shimadzu (1 mentions)
Cbm 20a, by Shimadzu (1 mentions)
Kinetex c18, by Phenomenex (1 mentions)
Chromnav software, by Jasco (1 mentions)
Hplc system, by Shimadzu (1 mentions)
8 protocols using preparative hplc system
1
Preparative HPLC Fractionation of Crude Extracts
2
Preparative HPLC Purification of Ammodendrine
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Preparative high-performance LC (HPLC) was carried out on a Shimadzu preparative HPLC system with ultraviolet detection (190 to 350 nm) and automatic fraction collection. The individual components of the system were: DGU-20A5 degasser, SIL-10AP autosampler, LC-20AT pump, CTO-10ASvp column oven, SPD-M20A photodiode array detector, and FRC-10A fraction collector.
Analytes were separated on a Luna C18(2) semi-preparative column (250 mm by 10 mm, 5 μm, 100 Å, Phenomenex) kept at 30°C. Mobile phases A and B consisted of, respectively, 0.05% formic acid in water and 0.05% formic acid in acetonitrile. Analytes were eluted using the following gradient at a constant flow rate of 2 ml/min: 0 to 2 min, 2% B (constant); 2 to 32 min, 2 to 25% B (linear); 32 to 37 min, 25 to 65% B (linear), 37 to 42 min, 65 to 95% B (linear); 42 to 45 min, 95% B (constant); 45 to 50 min, 95 to 5% B (linear); and 50 to 62 min, 2% B (linear). Ammodendrine was collected in the fraction eluting between 21.82 and 22.41 min (λmax = 240 nm).
Analytes were separated on a Luna C18(2) semi-preparative column (250 mm by 10 mm, 5 μm, 100 Å, Phenomenex) kept at 30°C. Mobile phases A and B consisted of, respectively, 0.05% formic acid in water and 0.05% formic acid in acetonitrile. Analytes were eluted using the following gradient at a constant flow rate of 2 ml/min: 0 to 2 min, 2% B (constant); 2 to 32 min, 2 to 25% B (linear); 32 to 37 min, 25 to 65% B (linear), 37 to 42 min, 65 to 95% B (linear); 42 to 45 min, 95% B (constant); 45 to 50 min, 95 to 5% B (linear); and 50 to 62 min, 2% B (linear). Ammodendrine was collected in the fraction eluting between 21.82 and 22.41 min (λmax = 240 nm).
3
Synthetic Peptide Characterization and Purification
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Protected Fmoc-amino acid residues were purchased from Novabiochem or Auspep (Melbourne, AU) with the exception of the neopentyl-protected sulfotyrosine, which was purchased from Bachem. The Fmoc-Rink resin and Fmoc-Wang resin were purchased by Auspep and Peptides International (Louisville, USA), while the 2-chlorotrityl resin was purchased from Novabiochem. Peptide-synthesis grade dimethylformamide (DMF), dichloromethane (DCM), diisopropylethylamine (DIEA), and trifluoroacetic acid (TFA) were supplied by Auspep, while 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate (HBTU), triisopropylsilane (TIPS), HPLC-grade acetonitrile, ammonium bicarbonate, and ammonium acetate were supplied by Sigma-Aldrich (Sydney, AU). Assay reagents at the highest grade were also supplied by Sigma-Aldrich unless otherwise stated.
The Symphony automated peptide synthesizer was supplied by Protein Technologies Inc. Peptides were analysed using a Shimadzu Prominence HPLC system coupled to a PE Sciex API 150EX turbo ionspray mass spectrometer and purified on a Shimadzu Preparative HPLC system. The high-throughput FLIPRTetra fluorescent plate reader was supplied by Molecular Devices (CA, USA).
The Symphony automated peptide synthesizer was supplied by Protein Technologies Inc. Peptides were analysed using a Shimadzu Prominence HPLC system coupled to a PE Sciex API 150EX turbo ionspray mass spectrometer and purified on a Shimadzu Preparative HPLC system. The high-throughput FLIPRTetra fluorescent plate reader was supplied by Molecular Devices (CA, USA).
4
Purification and Characterization Protocol
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Flash vacuum chromatography was performed using Bulk Phenomenex C18 (50 µm). Preparative high-performance liquid chromatography (HPLC) was performed using a Shimadzu C18 column (250 mm × 21 mm) attached to a Shimadzu Preparative HPLC system with a fraction collector. 1D and 2D-NMR spectra were recorded on Jeol ECS 400 MHz NMR spectrometer, with spectra referenced to 1H and 13C resonances in the deuterated solvents. Accurate mass spectrometric data were analyzed by Shimadzu ion-trap-time-of-flight (IT-TOF) mass spectrometer.
5
Shotgun Proteomics Analysis Pipeline
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Tryptic digest was then pre-separated into 20 fractions with a reverse phase C18 Xbridge column (Waters Corp., Milford, MA) by a preparative HPLC system (Shimadzu, Japan) at high pH condition. Each fraction was lyophilized and further analyzed in a nano-HPLC coupled Orbitrap Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA). The raw spectra data was processed by Maxquant software (v1.4.1.2) [14 (link)]. MS/MS spectra data was searched against the Uniprot human database (88,817 sequences) by Andromeda search engine [15 (link)].
6
Microwave-Assisted Automated Peptide Synthesis
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Microwave-assisted automated solid-phase peptide synthesis (Liberty Blue CEM Corporation, Matthews, NC, USA) was used to synthesize PS1-2, PS1-5, and PS1-6 (PS1-2: KWYKKWYKKWYK-CONH2, PS1-5: RWYRRWYRRWYR-CONH2, and PS1-6: KWLKKWLKKWLK-CONH2). Rink Amide resin (Novabiochem) (0.55 mmol/g) was used and Fmoc deprotection was assessed by 20% piperidine in dimethylformamide (DMF). Each coupling step of Fmoc amino acids was achieved using microwave heating in the presence of DIC and Oxyma pure in DMF. Resin-synthesized peptide was transferred to a conical tube, washed with dichloromethane, and allowed to air-dry. Peptide cleavage was performed by treatment with trifluoroacetic acid (TFA)/triisopropylsilane/DiH2O (95:2.5:2.5, v/v/v) for 2 h at room temperature. The cleaved peptide–TFA solution was precipitated with diethyl ether and then dried under a vacuum pump (Edwards RV5, Seongnam-si, Korea) to obtain a powder. The synthesized peptides were purified using a Zorbax C18 column (21.2 × 250 mm, 300 Å, 7 μm) on a Shimadzu Preparative HPLC system (Kyoto, Japan) using 5%–60% acetonitrile gradient in water with 0.05% TFA. Molecular masses were confirmed using a matrix-assisted laser desorption ionization mass spectrometer (MALDI II, Kratos Analytical Ltd., Manchester, UK) [34 (link)].
7
Preparative HPLC for δ13C Isolation
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For the pretreatment for the δ13C measurements (in Section 2.3.1 .), a preparative HPLC system manufactured by Shimadzu Corporation (Kyoto, Japan) was used to isolate glutamic acid from the eluate of a strongly acidic cation exchange column, which consisted of a controller (CBM-20A), a feed pump (LC-20AP), an autosampler (SIL-10AP), a UV detector (SPD-20A), and a fraction collector (FRC-10A). The preparative column and guard column included a Shodex Asahipak NH2P-90 20F (300 mm × 20 mm i.d., 9 μm particle size) and a Shodex Asahipak NH2P-130G 7B (50 mm × 7.5 mm i.d., 13 μm particle size), respectively (Showa Denko, Tokyo, Japan). A mixture of 100 mmol L−1 aqueous ammonium bicarbonate and methanol (35:65, v/v) was used as the mobile phase at a flow rate of 7 mL min−1. The column temperature was set at room temperature, and ultraviolet absorbance at 210 nm was used to detect the signal corresponding to glutamic acid. Since it was difficult to completely remove ammonium salt in the glutamic acid isolated by preparative HPLC, δ15N could not be simultaneously analyzed with δ13C by EA/IRMS.
8
Analytical and Preparative HPLC for Compound Separation
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The analytical HPLC were performed on a Shimadzu HPLC system (Kyoto, Japan) composed of LC-10AD pump, SLC-10A controller and Gastor 154 degasser and equipped with a Kinetex C18, 100 Å, 5 μ, 150 × 4.6 mm column (Phenomenex, Torrance, CA) equilibrated and eluted with a binary solvent system: A (0.05% TFA in H 2 O) and B (0.05%TFA in 9:1 v/v MeCN/H 2 O); λ detector: 280-320 nm; flow: 1 mL/ min; elution program: isocratic elution to 3% B for 5 min and then linear gradient from 3 to 15% B in 45 min. Chromatograms were acquired and processed using the ChromNav software (Jasco, Tokyo, Japan).
The separation was performed on a preparative HPLC system (Shimadzu, Kyoto, Japan) composed of two LC-8A pumps, an SCL-8A controller and an SPD-6A spectrophotometric detector. Solvents were degassed by an ERMA ERC-3562 degasser. The column used for separation was a Kinetex C18, 100 Å, 5 μm, 150 × 21.2 mm (Phenomenex), eluted with the same solvent system used for analytical purposes; λ detector: 280 nm; flow: 12 mL/min; elution program: isocratic elution to 8% B for 5 min and then linear gradient from 8 to 20% B in 45 min.
The separation was performed on a preparative HPLC system (Shimadzu, Kyoto, Japan) composed of two LC-8A pumps, an SCL-8A controller and an SPD-6A spectrophotometric detector. Solvents were degassed by an ERMA ERC-3562 degasser. The column used for separation was a Kinetex C18, 100 Å, 5 μm, 150 × 21.2 mm (Phenomenex), eluted with the same solvent system used for analytical purposes; λ detector: 280 nm; flow: 12 mL/min; elution program: isocratic elution to 8% B for 5 min and then linear gradient from 8 to 20% B in 45 min.
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