Asi 100 autosampler

Optical rotations were measured in MeOH using a Perkin-Elmer 341 Polarimeter. 1 H-, 13 C-NMR and 2D-NMR measurements were recorded in CD3OD on a Bruker Avance III 600 spectrometer
( 1 H at 600 MHz and 13 C at 150 MHz) equipped with a 5 mm TCI cryoprobe. 2D-NMR experiments were performed using standard Bruker microprograms (TopSpin 3.5 software). HR-ESI-MS analysis was conducted using a Micromass Q-TOF micro instrument. Flash chromatography was carried out on a Grace Reveleris system equipped with dual UV and ELSD detection using Grace® cartridges (Silica gel or RP-C18). HPLC separations were performed on a Dionex apparatus equipped with an ASI-100 autosampler, an Ultimate 3000 pump, a STH 585 column oven, a diode array detector UVD 340S and a Chromeleon software. A prepacked RP-C18 column (Phenomenex 250 x 10 mm, Luna 5 µ) was used for semi-preparative HPLC. The eluting mobile phase consisted of H2O with TFA (0.0025%) and CH3CN with a flow rate of 5 mL/min and the chromatogram was monitored at 205 and 210 nm. Thin-layer chromatography (TLC) was carried out using silica gel 60 F254 pre-coated aluminium plates (0.2 mm, Merck). After developing with solvent systems, spots were visualized by spraying with 50% H2SO4 followed by heating.

The stock solutions of each of the standard compounds of daidzein, genistein, glycitein, daidzin, genistin, and glycitin were prepared by dissolving 1 mg of each in 10 ml of 80% aqueous methanol and were stored in the refrigerator. Each isoflavone standard solution was injected into the HPLC, and the peak areas were determined. The lyophilized samples were dissolved in methanol, filtered through a 0.45 µm syringe filter (Pall, Ann Arbor, MI, USA), and used for HPLC analysis. HPLC was conducted with a Dionex P680 instrument (Dionex Corporation, Sunnyvale, CA, USA) equipped with an ASI-100 auto sampler (Dionex) and a UVD 170 UV-vis detector (Dionex). A Sunfire C18 column (150 mm × 4.6 mm, 3.5 µm particle size) from Waters (Milford, MA, USA) and TCC-100 thermostatted column compartment (Dionex) were used, and the column was maintained at 30 o C during the separation. The mobile phase consisted of solvent A (0.1% (v/v) trifluoroacetic acid in water, pH 2.5) and solvent B (acetonitrile) with the following gradient: 0-5 min, 15% B; 5-45 min, linear gradient from 15% to 40% B. The injection volume of standards and samples was 20 µl, and the flow rate was 1 ml/min. The retention times of daidzein, genistein, glycitein, daidzin, genistin, and glycitin were 25.7, 35.5, 28.1, 8.6, 15.6, and 10.3 min, respectively.

Optical rotations were measured on a Perkin Elmer model 341 polarimeter (589 nm, 20 °C).
NMR data were performed in CD 3 OD on Bruker Avance 500. HRESIMS data were gained using a Micromass Q-TOF high-resolution mass spectrometer. Mass spectra were recorded in the positive-ion mode in the range m/z 100-2000, with a mass resolution of 20000 and an acceleration voltage of 0.7 kV. CC was carried out on HP-20 resin (Sigma Aldrich). Flash chromatography was conducted on a Grace Reveleris system equipped with dual UV and ELSD detection using Grace® cartridges (Silica gel or RP-C 18 ). HPLC separations were performed on a Dionex apparatus equipped with an ASI-100 autosampler, an Ultimate 3000 pump, a STH 585 column oven, a diode array detector UVD 340S and a Chromeleon software. A prepacked RP-C 18 column (Phenomenex 250 x 15 mm, Luna 5 µ) was used for semi-preparative HPLC. The eluting mobile phase consisted of H 2 O with TFA (0.0025%) and CH 3 CN with a flow rate of 5 mL/min and the chromatogram was monitored at 205 and 210 nm. TLC were carried out using silica gel 60 F 254 pre-coated aluminium plates (0.2 mm, Merck). Spots were visualized through developing agent (CHCl 3 :MeOH:H 2 O, 14:6:1) and chromogenic agent (50% aq. H 2 SO 4 ) subsequent heating.