Poroshell 120 ec c18 reverse phase column

Commercial grade reagents and solvents were used without further purification. Thin layer chromatography (TLC) was performed using precoated silica gel 60 F²⁵⁴ plates and visualized using anisaldehyde solution, heat, and UV light (254 nm). Flash column chromatography was undertaken on silica gel (400–630 mesh). ¹H NMR was recorded at 400 MHz and chemical shifts are quoted in parts per million (ppm) versus an appropriate solvent peak or 2.50 ppm for DMSO-d₆. The following abbreviations were used to describe peak splitting patterns: br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, td = triplet of doublets, ddd = doublet of doublets of doublets. Coupling constants, J, are reported in hertz (Hz). HPLC was conducted using an Agilent HPLC unit equipped with an Agilent Poroshell 120 EC-C18 reverse phase column (4.6 × 50 mm, 2.7 Micron) and mass spectroscopy was performed using a quadrupole LC/MS unit.

Identification analysis was performed on a DIONEX UltiMate 3000 UPLC system (Thermo Fisher Scientific, San Jose, CA, USA) coupled to an LTQ-Orbitrap mass spectrometer via an ESI interface. Samples were separated on an Agilent poroshell 120 EC-C18 reverse-phase column (4.6 mm × 150 mm, 2.7 μm). Mobile phase A was water (containing 0.1% formic acid), and mobile phase B was acetonitrile. The gradient elution program was as follows: 0→5 min, 5% B; 5→10 min, 5→30% B; 10→25 min, 30→60% B; 25→40 min, 60→80% B; 40→45 min, 80→95% B. The flow rate was 0.3 ml/min, and the column temperature was 25°C. The injection volume was 10 μl.
The mass spectrometer was operated in negative mode. The spray voltage was 5.0 kV, and the S-lens was set at 60%. The ESI source temperature and capillary temperature were 300°C and 350°C, respectively. The sheath gas and auxiliary gas were high-purity nitrogen, and the flow rates were 50 arb and 5 arb, respectively. The scan range was from 110 to 2,000 Da, and the resolution was 30,000.
The compounds identified were forsythoside E, echinacoside, rehmaionoside B, 2,3,5,4′-tetrahydroxyl diphenylethylene-2-O-glucoside, and forsythin, and they are shown in Figures 1 and 2 and Table 1.

Cell biomass was measured in terms of optical density at 600 nm (OD₆₀₀). The residual phenol and 4-chlorophenol contents were determined using high-performance liquid chromatography (HPLC) performed with an Agilent 1260 infinity instrument equipped with a reverse-phase poroshell 120 EC-C18 column (4.6 × 150 mm, 2.7 μm, Agilent). The mobile phase was a mixture of methanol and MillQ water (60:40 [vol/vol]) at a flow rate of 0.8 mL/min. The injection volumes for all samples were 5 µl and monitored at 210 nm with a variable-wavelength detector. LC-MS analysis was performed with a SHIMADZU LCMS-2020 instrument. Mass spectrometry was performed in ESI positive- and negative-ion modes with a scan range 50–500 m/z. The ESI-MS interface parameters were set as follows: drying gas (nitrogen) flow rate, 1.5 L/min; capillary column temperature, 350℃; spray voltage, 4.5 kV.