L 2455

The following apparatuses were used for the stability studies:–

The High-Performance Liquid Chromatography (HPLC) system consisted of an ELITE LaChromVWR/ Hitachi plus autosampler, a VWR photodiode array detector L- 2455, and a VWR L-2130 HPLC pump. Data were acquired and integrated using EZChrom Elite (VWR, Agilent).

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pH meter (Bioblock Scientific model 93313).

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PAMAS particle counter, Rutesheim, Germany.

The experimental setup consisted of a standard VWR-Hitachi LaChrom Elite^® HPLC system with a quaternary gradient pump L-2130, Autosampler L-2200 and diode array detector L-2455 (VWR International, Radnor, PA, USA). The later was not used for the ICM but for comparative measurements. For the ICM measurements, a Smartline DAD 2600 with 10 mm, 10 µL flow cell from Knauer Wissenschaftliche Geräte GmbH, Berlin, Germany was used. Experimental validations were based on SEC analysis after online fractionation with a Foxy Jr.^® from Teledyn Isco, Lincoln, NE, USA.

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).