Rp 18 column

The production of commercially viable phytochemicals in in-vitro-derived callus cultures of M. oleifera was quantified by HPLC according to Khurshid et al. [59 (link)], equipped with a photodiode array detector, and a pump from Varians-Prostar 230, Metachim Degaset, autosampler Varians- Prostar 410, for separation and quantification, and a Purospher (Merck) RP-18 column was used for separations. Two solvents (A: acetonitrite and B: formic acid acidified (0.1 percent v/v) ultra-pure water) were used in the HPLC system’s mobile phase. During the 60-min run, the mobile phase composition ranged from 5:96–100:0 (solvent A:B, v/v), with linear gradients with 0.80 mL/min flow rates. After every single run, a 10-min re-equilibration period was used. At 204 nm, M. oleifera compounds were detected and quantified based on retention times compared with commercially available standards, and all the results were expressed as µg/g DW.

The feed wastewater characterization and reactor performance were monitored by wet analyses. For the suspended solid (TSS) parameter, standard methods (APHA, 1998) were applied. COD is one of the most important parameters in determining the degree of organic pollution in wastewaters. COD analyses were carried out using the microdigestion and titration method specified in the standard methods (SM 5220B) [20 ].
An HPLC (Shimadzu) equipped with a Diode-Array Detection (DAD) detector and LC-QTOF-MS/MS (Agilent 6530 Accurate Mass–ESI Interface, Santa Clara, CA, USA) was used for liquid chromatography analysis. To achieve the chromatographic separation, the Purospher Star RP-18 column (125 mm × 2.0 mm, particle size 5 µm) was supplied with a C18 guard column by Merck (Darmstadt, Germany). The analyses were performed in Positive Ionization mode with eluent A (acetonitrile-methanol (2:1)) and eluent B (ammonium acetate 5 mM at pH 4.7 (acetic acid)). The flow rate was selected as 0.3 mL/min, and the injection volume was determined as 10 µL. According to the selected method, the eluent gradient started from 5% and rose to 95% of eluent A in 5 min (was held for 4 min) and turned to the initial condition in 5 min.

Serum Trp and Kyn concentrations were measured by reversed-phase high-pressure liquid chromatography on a ProStar Varian system, as described earlier [32 (link), 33 (link)]. In brief, chromatographic separation was performed using a RP-18 column (Merck, Germany) and 15 mmol/l acid-sodium acetate buffer (pH 4.0) as eluent (flow rate: 0.9 ml/min). Serum specimens were deproteinized with trichloroacetic acid (2 mol/l) before analysis. 3-Nitro-L-tyrosine was used as an internal standard. All chemicals were purchased from Sigma-Aldrich (Austria). Trp was monitored by its native fluorescence at 286 nm excitation and 366 nm emission wavelengths (ProStar 360 detector, Varian, USA); Kyn and 3-nitro-L-tyrosine were detected by UV absorbance at the 360 nm wavelength (Shimadzu SPD-6A UV detector, Austria) in the same chromatographic run. Finally, the Kyn to Trp ratio (Kyn/Trp; kynurenine (μmol/l) divided by Trp (mmol/L)) was calculated [6 (link)].

The methods of Vasconcellos et al. [43 (link)] determined the glycine betaine quantification by incubating the isolates in NB with varying concentrations of PEG (0 and 30%) at 27ºC for 24 h. The bacterial cells were pelletized by centrifugation (5000 x g), weighted, and resuspended in 1 ml of ethanol by vigorous shaking (30 min). The extract was then filtered through a 0.45 μm filter and evaluated for glycine betaine production by injecting in HPLC on an RP-18 column (Merck, Germany) and detected at 200 nm.