As 2059

The HPLC–DAD system consisted of a quaternary pump (JASCO-2089 Plus pump), a thermoregulated autosampler set at 4 °C (Intelligent autosampler JASCO AS-2059) and a photodiode array detector (PDA) detector (UV/VIS detector JASCO MD-2018 Plus). The column temperature was 30 °C. Separation of compounds was achieved on a Luna C18 column (5 μm, 4.6 mm × 25 cm) (Phenomenex, USA). The flow rate was set to 0.5 mL/min and the mobile phase consisted of water with o-phosphoric acid (0.01%) as solvent A and acetonitrile with o-phosphoric acid (0.01%) as solvent B. The binary gradient elution was: 80–100% A (0–15 min., linear gradient), 100% A (15–25 min.) and 100–80% A (25–27.5 min., linear gradient).

An LC-4000 Series Integrated HPLC System (JASCO, Tokyo, Japan) equipped with a liquid chromatography pump (model PU-2829 plus), an autosampler (AS-2059 plus), a column oven (model CO-2060 plus), a UV/Vis Photodiode Array Detector (model MD-2818 plus), and a ChromNAV 2.0 software program (JAsco, Tokyo, Japan) was used to analyze the TSP, as reported by Peng et al. [52 (link)]. Samples were loaded onto a C18 Luna column with a 5 μm particle size, 25 cm × 3.00 mm I.D. (Phenomenex, Torrance, CA, USA), with a guard cartridge of the same material. Briefly, the mobile phase was made of water containing 0,2% (v/v) phosphoric acid (solvent A) and 82% (v/v) acetonitrile containing 0,04% (v/v) phosphoric acid (solvent B). The following gradient program was used to run the system: from 0 to 15% B in 15 min, from 15% to 16% B from 15 to 40 min, from 16% to 17% B from 40 to 45 min, from 17% to 43% B from 45 to 48 min, from 43% to 52% B from 48 to 49 min, held isocratic at 52% from 49 to 56 min, reduced from 52% to 43% B from 56 to 57 min, from 43% to 17% B from 57 to 58 min and from 17% to 0% B from 58 to 60 min. The flowrate was 1 mL/min. The injection volume was 20 μL. Peaks were detected at 280 nm and identified by comparison to the retention times of hydrolyzable and condensed tannin standards.