L 2130 pump

HPLC analysis was conducted using a VWR^TM-Hitachi (VWR International GmbH, Darmstadt, Germany) consisting of an L-2455 DAD detector, an L-2130 pump, and an L-2200 autosampler. System management and data acquisition were performed by EZChrom Elite^® 3.3.2 SP2 Software (VWR International GmBH, Darmstadt, Germany) involving integration parameters of peak area, retention time, and resolution. A Chromolith^® Widepore 300 Epoxy 100-4.6 mm HPLC column was kindly provided by Merck KGaA, Germany. Enantiomer separations were performed at ambient column temperature and flow rate of 0.8 mL/min with a mobile phase 50 mM Na₂HPO₄ pH 3.5 (adjusted by addition of about 4 mL H₃PO₄ 85% in 1 L buffer solution). An injection volume of 20 µL was set up at detection wavelengths of 230 nm and 240 nm.

Methanolic extracts were quantitatively dissolved in methanol (1.5 mL) and filtered through a Millipore Millex–GP, 0.22 µm. The resultant extracts were analyzed for their contents of phenolic acids by the RP-HPLC method. These analyses were carried out according to the procedure developed by Muszyńska et al. 2016, with some modifications [64 (link)]. HPLC analyses were conducted using an HPLC VWR Hitachi-Merck apparatus: L-2200 autosampler, L-2130 pump, RP-18e LiChrospher (4 mm × 250 mm, 5µm) column thermostated at 25 °C, L-2350 column oven, L-2455 diode array detector at the UV range 200–400 nm. The mobile phase consisted of solvent A: methanol/0.5% acetic acid 1:4 (v/v), and solvent B: methanol. The gradient was as follows: 100:0 for 0–25 min; 70:30 for 35 min; 50:50 for 45 min; 0:100 for 50–55 min; 100:0 for 57–67 min. The comparison of UV spectra and retention times with standard compounds enabled the identification of phenolic acids present in analysis samples. The quantitative analysis of free phenolic acids was performed with the use of a calibration curve with the assumption of the linear size of the area under the peak and the concentration of the reference standard (Sigma–Aldrich, St. Louis, MO, USA).