Purospher star rp 18 endcapped

Partial purification of extract produced by strain MSt1^T was performed according to Ding et al. (2011 (link)) with modification. Briefly, strain MSt1^T was grown on TSA for 3–4 days at 30°C. Whole agar including the bacteria was placed into a glass jar and extraction was performed three times using 100% acetonitrile (Merck, Germany). The extract was concentrated under reduced pressure using a rotary evaporator and it was then freeze-dried. The dried crude extract was then packed into a C-18 (Merck) column and eluted with 50:50, 70:30, and 100:0% methanol:water. The active fraction (100% methanol) was collected and concentrated under reduced pressure using a rotary evaporator followed by freeze-drying. The active fraction was further fractionated using a preparative HPLC system [Agilent Infinity 1260 Quaternary LC system (Agilent, USA)] with a reverse phase column [Purospher STAR RP-18 endcapped (Merck), 4.6 μm, 250 × 20 mm]. Mobile phase A consisted of 100% Milli-Q water and mobile phase B consisted of 100% methanol. A linear gradient of 60–95% B in 15 min at a flow rate of 18 mL/min were used as elution and monitored at 254 nm. All isolated peaks were collected and the active fraction was evaporated and stored at -20°C for further analysis.

The chromatographic separations were conducted on an Agilent 1220 LC VL system consisting of Agilent 1260 Infinity II photodiode array detector (DAD), a binary pump, and a 20 μL Rheodyne injection loop. OpenLAB CDS Chemstation software was used for data acquisition. The separation was performed on a Purospher^® STAR RP-18 end-capped (150 × 4.6 mm I.D., 5μm) from Merck (Darmstadt, Germany).
A vortex mixer (50 Hz) from Scientific Industries, Inc. (Bohemia, NY, USA), and a centrifuge (H-11n, Kokusan, Tokyo, Japan) were used for extraction and centrifugation in the extraction step, respectively. An ultrasonic water bath (35 kHz and 320 W) with temperature control from Bandelin Sonorex (Berlin, Germany) was also used. A transmission electron microscope (TEM; TECNAI G2 20, FEI, Hillsboro, OR, USA) and zeta potential analyzer (Zetasizer Nano ZS, Malvern, U.K.) were used for the morphology of SUPRAS and charge on the surface of SUPRAS, respectively.

The method described by Soto et al. [20 ], with some modifications, was used. The Knauer Azura analytical UHPLC system (Knauer, Berlin, Germany) was equipped with an Azura DAD 2.1 L diode array detector with high-sensitivity Knauer 3950 autosamplers, and an Azura P 6.1 L pump operated at 25 °C. A reversed-phase column (Purospher^® STAR RP-18 end capped, 150 mm × 4.6 mm, 3.0 µm, Merck, Darmstadt, Germany) was used. The separation was achieved using (A) 1% formic acid in the water, and (B) acetonitrile as mobile phase at 0.7 mL/min with a gradient: at 0 min, the A:B ratio was 95:5; at 6 min, 70:30; at 16 min, 50:50; at 26 min, 30:70; at 36 min, 5:95; and at 46 min, 95:5. The system was allowed to run for another 10 min to equilibrate the column before each injection. The most likely identification of phenolics was achieved by comparing the retention times and HPLC spectra of each peak in the sample with those of the respective phenolic compound standards.

The quantification of BTXs (benzene, toluene and xylenes) and some PAHs (pyrene, benzo(b)fluoranthene and benzo(a)pyrene) was performed in a VWR-Hitachi LaChrom Elite HPLC system (Hitachi, Japan), equipped with a degasser, auto sampler, column oven and diode array detector (DAD), according to the previously optimized method.^{31 (link)}An analytical column (0.25 m × 4.6 mm, 5 μm film) Purospher® Star RP-18 end capped (Merck-Millipore, Germany) was used. The data acquisition and processing were done using EZChrom Elite software (Agilent, USA). Hydrocarbons solutions were daily prepared and calibration curves were obtained for each of the hydrocarbons.
Briefly, the following methodology has been used: aqueous solutions of PAHs and BTX were analyzed in HPLC-DAD system, in gradient mode, using a ternary mixture as the mobile phase (methanol, acetonitrile and ultrapure water, 20 : 50 : 30%), by direct injection of each sample (20 μL) with a flow rate of 1.5 mL min⁻¹. The detection of BTXs and PAHs were analytically determined at the following wavelengths: λ(ben) = 207 nm, λ(tol and xyl) = 211 nm, λ(pyr) = 239 nm, λ(B(b)F and B(a)P) = 256 nm.

The LC–MS/MS analysis was performed using a Waters Acquity UPLC™ system (Waters Co., Manchester, UK) consisting of binary solvent manager, an automatic liquid chromatographic sampler and a Waters Xevo^TM tandem quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source. The chromatography was performed on a Purospher^® STAR RP-18 endcapped (100 mm × 2.1 mm, 2 μm, Merck KGaA, Darmstadt, Germany) analytical column and maintained at 40 °C in a column oven. The mobile phase consisted of 0.1% formic acid-methanol (15:85, v/v), and the flow rate was set at 0.2 mL/min. The injection volume was 10 μL. The positive ion mode with a multiple reaction monitor (MRM) was used for UPLC-MS/MS analysis. The following precursor-to-product ion transitions were used: m/z 433.22→415.19 for schizandrin and m/z 226.26→76.91 for methyl yellow (internal standard, IS).The instrument parameters were optimized as follows: capillary voltage, 3.2 kV; desolvation gas, nitrogen; desolvation temperature, 400 °C; desolvation gas flow rate, 800 L/h; cone voltage, 18 V; collision energy, 12 V and source temperature, 150 °C. MassLynx 4.1 software (Waters Corporation, Milford, MA, USA) was used for data processing.