All reagents and solvents were reagent grade and used without further purification, unless otherwise stated. Molecular sieves were activated at 200 °C and cooled down to rt prior to use. Reactions were monitored by analytical TLC on 0.25 mm Merck Millipore silica gel 60 F254 using p-anisaldehyde, ninhydrin, and ceric ammonium molybdate as visualizing agents. Flash column chromatography was performed employing 230–400 mesh silica gel. NMR spectra were acquired by using Bruker-AV-400 (400 MHz). Chemical shifts (δ) are given in ppm relative to 1H: 7.26 ppm, 13C: 77.16 ppm for CDCl3; 1H: 3.31 ppm, 13C: 49.0 ppm for CD3OD; 1H: 2.50 ppm, 13C: 39.52 ppm for DMSO‑d6. Splitting patterns are reported as s (singlet), br s (broad singlet) d (doublet), br d (broad doublet), t (triplet), q (quartet) and m (multiplet). Coupling constants (J) are given in Hertz (Hz). Purities of biologically tested compounds were carried out on a HITACHI D-2000 Elite HPLC system with pump L-2130. Exact mass measurements were performed on VG platform electrospray ESI/MS or BioTOF II. Melting points were measured on a FARGO melting point apparatus MP-1D (Mandarin Scientific Co., Ltd.).
D 2000 elite hplc system
Manufactured by Hitachi
Sourced in Japan
The D-2000 Elite HPLC system is a high-performance liquid chromatography instrument designed and manufactured by Hitachi. The system is capable of performing various analytical techniques, including reversed-phase, normal-phase, and ion-exchange chromatography. The D-2000 Elite HPLC system is equipped with a variety of components, such as a pump, an autosampler, a column oven, and a detector, which work together to separate, identify, and quantify the components of a given sample.
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4 protocols using d 2000 elite hplc system
1
Organic Synthesis: Purification and Characterization
2
Quantification of Compounds in Fig Tree Latex
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Fruits, petioles and trunks of fig trees were cut, and extruded latex was collected. These latexes were immediately frozen in liquid nitrogen and stored at –80°C. Following thawing, 30 mg of latex was added to 300 µl of methanol, and the samples were vortexed at 2500 rpm at room temperature for 10 min. After centrifugation at 20 400 g at room temperature for 5 min, the supernatant fraction was collected. Next, the pellet was subjected again to this extraction procedure. The two supernatant fractions were combined and dried with nitrogen gas. The extract was dissolved in 500 µl of methanol and filtered through Minisart® RC4 (0.2 mm pore; Sartorius Stedim Biotech, Göttingen, Germany). FCs in latex extracts were quantified with a D‐2000 Elite HPLC System (Hitachi, Tokyo, Japan) as previously described (Munakata et al., 2014 ).
3
Detailed Characterization of Synthesized Compounds
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Melting points were recorded on an X-4 microscopic melting point apparatus (Shanghai Jingke Instrument Co., Ltd, Shanghai, China) and are uncorrected. All reactions were routinely checked by TLC on silica gel Merck 60F254. The NMR spectra were recorded on a Bruker Ascend 400 M spectrometer (400 MHz for 1H NMR, 100 MHz for 13C NMR, Bruker Corporation, Billerica, MA, USA) and chemical shifts were given in δ units (ppm) relative to internal standard TMS and refer to DMSO-d6 solutions. HRMS (ESI) was performed using an AGILENT LC/MS (Agilent Technologies Inc., Palo Alto, CA, USA). Analysis of sample purity was performed on a Hitachi D-2000 Elite HPLC system (Hitachi, Ltd., Tokyo, Japan). HPLC conditions were the following: Inertsil ODS-2 250 mm × 10 mm, 5 mm column; mobile phase: CH3CN (0.1% TFA) /CH3OH = 1/1, for 10 min; room temperature; flow rate: 1 mL min−1; detection at λ 254 nm. All final compounds in biological assays have a purity of ≥95%.
4
Chromatographic Separation and Identification of AkPT1 Reaction Products
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Extracts of AkPT1 reaction mixtures were chromatographically separated on a LiChrosphereRP-18 column (4.0 mm × 250 mm, Merck) at a flow rate of 1.0 ml min -1 and at 40 °C under the control of a D-2000 Elite HPLC system (Hitachi, Tokyo, Japan). An isocratic separation program of 20% (v/v) solvent A (water with 0.3% (v/v) acetic acid) and 80% (v/v) solvent B (methanol with 0.3% (v/v) acetic acid) was used except for screening of xanthotoxol O-DT activity.
Extracts of xanthotoxol O-DT assay were analyzed with a linear gradient program, consisting of 20% to 90% (v/v) of solvent B (methanol with 0.3% (v/v) acetic acid) in solvent A (water with 0.3% (v/v) acetic acid) over 45 min. Enzymatic products were scanned at a range of 200-370 nm with a L2445 Diode Array Detector (Hitachi).
Reaction products of AkPT1 were identified using LC-IT-TOF-MS (Shimadzu), a TSK gel ODS-80Ts column (2 mm × 250 mm, Tosoh) and a linear gradient program composed of 20% to 80% (v/v) solvent B (acetonitrile with 0.1% (v/v) formic acid) in solvent A (water with 0.1% (v/v) formic acid) at a flow rate of 0.2 ml min -1 and at 40 °C. Precursor ions for MS 2 analysis were selected in a range of m/z = 50-500.
Extracts of xanthotoxol O-DT assay were analyzed with a linear gradient program, consisting of 20% to 90% (v/v) of solvent B (methanol with 0.3% (v/v) acetic acid) in solvent A (water with 0.3% (v/v) acetic acid) over 45 min. Enzymatic products were scanned at a range of 200-370 nm with a L2445 Diode Array Detector (Hitachi).
Reaction products of AkPT1 were identified using LC-IT-TOF-MS (Shimadzu), a TSK gel ODS-80Ts column (2 mm × 250 mm, Tosoh) and a linear gradient program composed of 20% to 80% (v/v) solvent B (acetonitrile with 0.1% (v/v) formic acid) in solvent A (water with 0.1% (v/v) formic acid) at a flow rate of 0.2 ml min -1 and at 40 °C. Precursor ions for MS 2 analysis were selected in a range of m/z = 50-500.
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