G1367e autosampler

NMR spectra including ¹H, ¹³C, HSQC, HMBC, COSY, NOESY, and TOCSY were recorded in a Varian Inova spectrometer at 400 (¹H) and 95 MHz (¹³C) or a Bruker DMX500 spectrometer operated at 500 MHz (¹H) or 125 MHz (¹³C); chemical shifts were recorded as δ values. ESI-MS were recorded on a Thermo Scientific LTQ Orbitrap XL hybrid FTMS (Fourier transform mass spectrometer). Data were collected in both positive and negative ionization modes via a liquid chromatographic/autosampler system that consisted of an Agilent HPLC system. Analytical and preparative HPLC analyses were performed in an Agilent 1260 Infinity system equipped with a G1311B Quaternary Pump, a G1367E Autosampler, and a G1315C DAD VL+ and controlled by Agilent ChemStation software. For analytical and semipreparative HPLC, Phenomenex (Luna Omega Polar C₁₈, 50 × 2.1 mm id., 1.6 μm) and Macherey-Nagel (Nucleosyl C₁₈, 250 × 4.6 mm id., 5 μm and Nucleosyl C₁₈, 250 × 10 mm id., 5 μm) columns, respectively, were used. Column chromatography (CC) was conducted on silica gel (70-230 mesh, Merck) or Sephadex LH-20 (Sigma-Aldrich Chemical). Thin-layer chromatography analyses were carried out on silica gel 60 F₂₅₄ plates (Macherey & Nagel) using ceric sulfate (10%) solution in H₂SO₄ as the color reagent.

To determine the phytochemical profile of the tested extract, we performed high-performance liquid chromatography (HPLC) with an Agilent 1260 HPLC instrument equipped with a G1311B quaternary pump, a G1367E autosampler, and an Agilent G1315C UV diode array detector (DAD). HPLC-grade solvents were purchased from JT Baker. Chromatographic profile elaboration was performed using a Phenomenex reversed-phase column (Luna Omega Polar C18, 50 × 2.1 mm i.d., 1.6 μm). Gradient elution was performed with 0.1% aqueous formic acid as solvent A and acetonitrile as solvent B, and the column temperature was maintained at 35°C. System control, data collection, and data processing were accomplished using OpenLAB LC 1260 chromatography software. The working solution of each sample was prepared by dissolving 10.0 mg of the extract in 1 mL of water, and 2 μL of working solution was injected using an autosampler. For UV detection, the wavelength program was set at acquisition wavelength (λ) values of 240, 254, 280, 320, and 365 nm. Additionally, thin-layer chromatography (TLC) was performed using known standards as controls. Ten milligrams of dry aqueous extract was weighed and, with the help of a sonicator, dissolved in a solution of 1 mL of water-methanol-acetonitrile (30 : 35 : 35). Then, 15 μL was applied to silica gel Merck F₂₅₄ plates.