600 pump

The HPLC consisted of a Waters apparatus equipped with a 600 pump and pump controller, a Waters autosampler mod. 717, and a UV-Visible Photodiode array detector, mod 2996. For resveratrol determination, an isocratic chromatographic method was used. Mobile phase was composed by 80% of solvent A (10% acetic acid) and 20% of solvent B (acetonitrile). The HPLC column was a Symmetry C18 reverse phase (3.9 mm × 150 mm, 5 μm particle size, with a 10 mm guard column of the same material) and flow rate was 1 mL/min. Resveratrol quantitation was performed by peak area integration at 306 nm. Before the analysis, 20 μL of each sample was diluted 1:10 in mobile phase, filtered onto 0.2 µm filters, and then 50 µL were injected onto the column.
Calibration curve from 75 picomoles to 12 nanomoles was prepared by diluting in appropriate solvents a 20 mM stock solution of resveratrol in dimethylsulfoxide (DMSO). The curve (six data points, in duplicate) is linear with an R² value of 0.999 (Figure 8). Peak detection (retention time: 6 min) and purity were checked with the photodiode array detector to verify the characteristic absorption spectrum of the polyphenol (Figure 8, inset).

HPLC system (Waters, USA), consisting of a 600 pump, an autoinjector, 2998 photodiode array detector 200 to 500 nm, was set up and used to determine the presence of bioactive compound (vitexin and isovitexin) in the crude extract. Separation was carried out using 250 × 4.6 mm ODS 3.3 mm column (Inertsil, Japan) thermostated at 40°C. A gradient method with methanol and deionized water was used for the separation. At 0 min, the mobile phase was set at 10% methanol in deionized water and increased to 90% methanol in deionized water for duration of 45 min. The 90% methanol in deionized water was maintained for further 15 min. The peaks were integrated at the wavelength of 337 nm [23 (link)].

An HPLC-PDA system (Waters Corporation, Milford, MA, USA) consisting of a Waters 600 pump and a 996 PDA detector was used. Analyses were performed using a Waters Sun-Fire C18 column (4.6 × 150 mm, 5 μm). Chromatography conditions were as follows: MeOH: H₂O, 90: 10 to 40: 60 for 40 min; MeOH: H₂O, 40: 60 to 100% MeOH for 1 min; and 100% MeOH for 9 min. The flow rate was 0.1 mL/min; injection volume was 20 μL kudingcha extract (20 mg/mL in petroleum ether); and detection wavelengths were 220, 254, and 280 nm.