Dg 2080 53 3 line degasser

Reversed-phase liquid chromatography was performed using a Jasco system containing an UV-2075Plus Intelligent UV/Vis Detector, an AS-2050Plus Intelligent Sampler, an LG-2080-02 Ternary Gradient Unit, a PU-2080Plus Intelligent HPLC Pump, a DG-2080-53 3-Line Degasser, and an LC-NetII/ADC (Jasco, Gross-Umstadt, Germany). The mobile phase acetonitrile/water 10:90 was degassed prior to use and the flow rate was 1 mL min⁻¹ with the following gradient: a linear gradient for 2 min from 89% solvent A (water containing 10% acetonitrile) to 33% solvent B (100% acetonitrile) holding it for 4 min, a linear gradient for 4 min to 100% solvent B holding it for 2 min, a linear gradient for 0.1 min to 89% solvent A holding it for 3 min. A reversed-phase ec MN Nucleodor C18 (5 µm, 4.0 × 125 mm²) column (Macherey-Nagel, Düren, Germany) was adjusted to 40 °C by a column thermostat (W.O. Electronics, Austria). For each measurement, 10 µL of a sample was injected and the absorbance was monitored at 240 nm. The relative product level was evaluated using the relative peak area of the product compared to the combined peak areas of educt and product. The values of the three samples were averaged out (mean average), and standard deviations were calculated.

HPLC analyses were performed on a Jasco system (Gross-Umstadt, Germany) equipped with a PU-2080 Plus Intelligent HPLC Pump, a DG-2080-53 3-Line Degasser, a LG-2080-02 Ternary Gradient Unit, a AS-2057 Plus Intelligent Sampler, a Column-Thermostat Jetstream Plus, and a MD-2010 Plus Multiwavelength Detector. Proanthocyanidins were separated on a reversed phase Aqua 5 µ C-18, 125 Å, 250 × 4.6 mm column (Phenomenex, Aschaffenburg, Germany) at 280 nm protected by a guard column of the same material. The same eluent and gradient were used as in the polyphenol LC-MS/MS analysis, with a flow rate of 0.5 mL/min at 25 °C. Seed samples were diluted in acetonitrile/water (1:9, v/v; approximately 5.5 mg/mL). Briefly, 20 µl from all samples was injected three times, and (-)-epicatechin standard solutions in the range of 0.5 to 1000 mg/L were injected twice.