1525 separation module

Optical rotations were measured using a P-1020 polarimeter (JASCO). UV spectrua were obtained with a DU 640 spectrophotometer (Beckman). ECD spectra were acquired on a JASCO J-815-150S CD spectrometer. IR spectra were obtained via a Nicolet-Nexus-470 spectrometer. NMR spectra were recorded on an Agilent DD2 NMR spectrometer (500 MHz). ESIMS and HRESIMS spectra were obtained by a Q-TOF (Micromass) and a LTQ Orbitrap XL (Thermo Scientific) spectrometer, respectively. Single-crystal analysis were performed on a Gemini A Ultra system using Cu Kα radiation (Aglient Technologies). A 1525 separation module (Waters) equipped with a C₁₈ (Kromasil, 5 μm, 10 × 250 mm) column was used for semi-preparative HPLC. ODS (Unicorn; 45–60 μm), Sephadex LH-20 (Amersham Biosciences), and silica gel (200–300 mesh; Qing Dao Hai Yang Chemical Group Co.) were applied for column chromatography. TLC (G60, F-254; Yan Tai Zi Fu Chemical Group Co.) was used in the compounds detection.

The loaded hydrogel samples were subjected to HPLC-PDA analysis using a Waters 1525 Separation Module linked to a Waters 2996 photodiode array detector (Waters Corporation, Milford, MA, USA), in accordance with a modified method reported by Carpentieri [77 (link)]. The detected compounds were separated analytically in a Waters Spherisorb C18 reverse-phase column (5 μm ODS2, 4.6 250 mm, Milford, MA, USA).
The mobile phase consisted of phosphoric acid (0.1%, eluent A) and methanol (100%, eluent B). Analytical separation was carried out by the following gradient elution: 0–30 min from 5% B to 80% B, 30–33 min 80% B, and 33–35 min from 80% B to 5% B. The injection volume and the flow rate of the mobile phase were 5 µL and 0.8 mL/min, respectively. The signal was provided for the quantification of each compound at the wavelength of maximum absorbance. All commercial standards were dissolved into distilled water to generate 6-point standard calibration curves (R² = 0.999). The results were represented in terms of mg of the target components (gallic acid, catechin, epicatechin, and rutin) per L of the withdrawn sample.