Hplc pda system

Analysis was performed using a Waters HPLC-PDA system (Waters Co., Milford, MA, USA) consisting of an e2695 separation module and a 2998 photodiode array (PDA) detector. The control of chromatographic processing and data acquisition was performed using Empower^® 3 Software. Aegispak C₁₈-L column (5 µm, 4.6 mm × 250 mm) was used (30 °C). The eluents consisted of 0.1% FA in acetonitrile (A) and 0.1% FA in water (B), as follows: 0.0–5.0 min, 13–20% A; 5–23 min, 20–30% A; 23–25 min, 30–70% A; 25–30 min, 70% A. (flow rate: 1 mL/min). The UV wavelength of the detector was set to 340 nm, and 10 μL of the samples were injected into the HPLC-PDA system for analysis.

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.

Fractionation of triterpenoids from crude cuticular wax extracts was conducted on a semi-preparative HPLC-PDA system (Waters, Milford, MA, USA) using an ACE C18 semi-preparative column (5 µm, 250 × 10 mm; ACT, Aberdeen, UK). The crude cuticular wax extracts were reconstituted with methanol and then filtered through a 0.45 µm membrane before injection into the system using an injection volume of 0.5 mL. Gradient elution at a constant flow rate (4 mL/min) was carried out with a total run time of 85 min as follows: starting from an isocratic run at 100% solvent A for 33 min, then increasing to 100% solvent B over 10 min and maintaining isocratic at 100% solvent B to the end of the run. Equilibration time was at least 15 min between runs. Solvent A was acetonitrile and water (89:11, v/v) and solvent B was methanol and acetonitrile (90:10, v/v). Fraction collection was based on time, monitoring the chromatograms at 205 nm and allowing the collection of known and unknown components to generate many different fractions with no more than two major peaks in each for the screening of anticancer agents. The fractionation scheme used in the present study is presented in Figure 1. Obtained fractions were evaporated to dryness and stored at 4 °C in air-tight containers until required.