Uv vis 156

Semi-preparative fractionation of the Olive-tree leaves extract was achieved using a Gilson preparative HPLC system (Gilson, Middleton, WI, USA) equipped with a binary pump (model 331/332), an automated liquid-handling solution (model GX-271), and a UV-Vis detector (model UV-Vis 156). The extract was fractionated at room temperature. A 250 mm x 10 mm i.d., 5 μm Phenomenex RP-C18 column was used to separate the compounds. The mobile phases consisted of acetic acid 0.5% (A) and acetonitrile (B). The following multi-step linear gradient was applied: 0 min, 5% B; 5 min, 15% B; 53 min, 27% B; 54 min, 28% B; 60 min, 100% B; 65 min, 100% B; 70 min, 5% B; 75 min, 5% B. The injection volume was 500 μL and the flow rate used was set at 10 mL/min. The compounds separated were monitored with UV-Vis (240 and 280 nm) and MS, using the time-of-flight mass spectrometer detector microTOF (Bruker Daltonik, Bremen, Germany), as reported in the previous section.

Different fractions of the L. citriodora extract were recovered using a Gilson preparative HPLC system (Gilson Inc., Middleton, WI, USA) which equipped with automated liquid handling solutions (model GX-271), a binary pump (model 331/332) and UV-vis detector (model UV-Vis 156). The solvent of the collected fractions was evaporated in a Savant SpeedVac Concentrator SC250 EXP (Thermo Scientific, Waltham, MA, USA).
The composition of the whole extract and the fractions was analyzed through a HPLC-ESI-TOF-MS system. The chromatographic separation was carried out using an Agilent 1200 series rapid-solution LC equipment (Agilent Technologies, Palo Alto, CA, USA) with an autosampler, a binary pump and a diode-array detector (DAD).
Both HPLC systems were coupled to a time-of-flight (TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) with an electrospray ionization (ESI) interface (model G1607A, Agilent Technologies, Palo Alto, CA, USA). Calibration solution was injected at the beginning of each analysis by a 74900-00-05 Cole-Palmer syringe pump (Cole-Palmer, Vernon Hills, IL, USA) directly connected to the interface.
Finally, absorbance and fluorescence measurements for the antioxidant assays were performed on a Synergy Mx Monochromator-Based Multi-Mode Micro plate reader (Bio-Tek instruments Inc., Winooski, VT, USA) in 96-well microplates.