1200 hplc system

UQ₁₀ was quantified on the basis of the method in (52 (link)). Briefly, reverse-phase HPLC (RP-HPLC) was performed using an Agilent 1200 HPLC system. Approximately 0.02 nmol of RC-LH1₁₆ or RC-LH1₁₄-W was dissolved in 50 μl of 50:50 methanol:chloroform containing 0.02% (w/v) ferric chloride and injected onto a Beckman Coulter Ultrasphere ODS 4.6 mm × 25 cm column preequilibrated in HPLC solvent (80:20 methanol:2-propanol) at 40°C at 1 ml⁻¹ min⁻¹. Isocratic elution was performed in HPLC solvent for 1-hour monitoring absorbance at 275 nm (UQ₁₀), 450 nm (carotenoid), and 780 nm (BChl). The peak in the 275-nm chromatogram at 25.5 min, which did not contain any other detectable compounds, was integrated. The integrated area was used to calculate the molar quantity of extracted UQ₁₀ by reference to a calibration curve calculated from injection of 0 to 5.8 nmol of pure standard (fig. S14). Each sample was analyzed in triplicate with reported errors corresponding to SD of the mean.

The following parameters were measured daily on influent and effluent of both full-scale reactors: TOC was analyzed using a TOC analyzer (TOC-L CPN Basic System, Shimadzu, Japan). TSS was measured following the procedure of APHA (1998) [19 ]. For U1: Methanol and methyl acetate were detected using gas chromatography with FID on an HP5890 with a RTX-1 nonpolar column. Acetate is detected on a hp 5890 Series II with a DB-Wax polar column; and TA, BZ, IA, OA, PT, and TMA were detected using an Agilent 1200 HPLC System with multiple wavelength detector, or equivalent; the HPLC column was an Agilent SB-C18, 4.6 mm i.d. x 50 mm, 1.8 μm particle diameter (p/n 822975–902). For reactors E and F: TA, BZ, IA, OA, PT, and TMA were aromatic compounds, fatty acids and methyl compounds were detected using a high-performance liquid chromatography (Agilent ZORBAX Eclipse XDB-C18, Rapid Resolution HT 4.6 mm i.d. x50 mm, 1.8 μm particle diameter, operated at 600 Bar).

The metabolite extraction was dissolved in 5 mL methanol following ultrasonic treatment and purification by filtering through a 0.22 µm membrane. Chromatography was performed by an Agilent 1200 HPLC system. qTOF-MS was performed using an Agilent 6510 Q-TOF system equipped with atmospheric pressure chemical ionization (APCI) in positive ion model. An HHS T3 column with a 3.5 μm, 4.6 × 150 mm column was used (Waters, USA), with the temperature maintained at 35 °C. The mobile phase consisted of 0.1% formic acid aqueous solution (v/v, solvent A) and methanol/0.1% formic acid (v/v, solvent B). The flow rate was set at 1 mL/min, and the injection volume was 20 μL. The linear gradient with the following proportion of phase B (tmin, B%) was used: (1, 90), (3, 100), (10, 100), (12, 90), and (15, 90). The optimum MS conditions were documented as the following: corona current = 4 uA; capillary voltage = 5.0 kV; skimmer voltage = 65 V; segmented fragmentor = 135 V; gas temperature = 350 °C; vaporizer temperature = 400 °C; drying gas flow rate = 8 L/min; and nebulizer pressure = 60 psi. Nitrogen was used as the collision, drying, and nebulizer gas. MS spectra were acquired at 150–1000 m/z using an extended dynamic range and a scan rate of 1.5 spectra/s by varying collision energy with mass.

Lyophilized peptides were resuspended at 1 mg/mL in a 1:1 solution of H₂O/CH₃CN with 0.1% TFA, then analyzed on an Agilent 1200 HPLC system using an Agilent Zorbax 5 µm 300SB-C3 column (2.1 mm × 150 mm) with a 5–65% gradient of CH₃CN with 0.08% TFA in H₂O with 0.1% TFA and a flow rate of 0.8 mL/min. Representative IMPs were also characterized using two additional columns: Phenomenex Aeris 3.6 µm WIDEPORE C4 column (4.6 mm × 150 mm) with a 5–65% gradient of CH₃CN with 0.08% TFA in H₂O with 0.1% TFA and a flow rate of 0.8 mL/min; and Phenomenex Luna 5 µm C18(2) column (4.6 mm × 250 mm) with a 0–70% gradient of CH₃CN in H₂O with 0.1% TFA and a flow rate of 1.5 mL/min.
A 1:100 dilution of each 1 mg/mL peptide solution was prepared and analyzed by LC/MS on an Agilent 6550 ESI-Q-TOF mass spectrometer equipped with an Agilent Poroshell 5 µm 300SB-C3 column (1 mm × 75 mm) with a 1–91% gradient of CH₃CN in H₂O with 0.1% formic acid and a flow rate of 0.4 mL/min.

Individual gel regions corresponding to major protein bands from different polyacrylamide gels were manually excised, destained, reduced, carbamidomethylated, and trypsin digested as described in Addis et al.41 (link). Tryptic peptides were supplied to the proteomic facility of Porto Conte Ricerche Srl (Tramariglio, Alghero, Italy) for LC MS/MS analysis using a XCT Ultra 6340 ion trap equipped with a 1200 HPLC system and a chip cube (Agilent Technologies, Palo Alto, CA) as described in Biosa et al.42 (link). Mass spectrometry output data were analysed on the software provided by the manufacturer (6300 Series Ion Trap LCMS) employing Mascot Daemon MS/MS ion search software (Version 2.3, Matrix Science, Boston, MA) for protein identification. Data were then processed against the NCBI database (http://www.ncbi.nlm.nih.gov).

Fructose, glucose, maltose, and saccharose values in the fermented radish roots were determined according to the GB 5009.8-2016 national food safety standard. This method was previously reported by our group, with slight modifications (24 (link)). Radish sample (2.0 g) extraction was conducted by ultrasonic extraction for 30 min by adding 25 mL of water, and then the mixture was passed through 0.22 μm polytetrafluoroethylene filters. The mixture was then analyzed with an Agilent 1,200 HPLC system (Agilent, Palo Alto, CA, USA) with an NH2 column (250 × 4.6 mm, i.d. × 5 μm; Agilent). The available sugars were separated at 30°C using a mobile phase acetonitrile aqueous solution (70%, v/v) with a flow rate of 1.0 mL/min. The compounds were then quantified using external standards and expressed as g/100 g of sample.