717 automatic injector

Cell concentration was measured as optical density at 600 nm (OD_600nm) with Spectrophotometer Beckman DU-70. Metabolite concentrations were determined with an HPLC system (600E quaternary bomb, 717 automatic injector, 2410 refraction index, Waters, Milford, MA). For determination of glucose (Glu), xylose (Xyl), arabinose (Ara) and acetic acid, an Aminex HPX-87H column (Bio-Rad, Hercules, CA) was used. Running conditions were: mobile phase, 5 mM H₂SO₄; flow, 0.5 ml/min at 50°C. Under these conditions sugars and acetic acid were detected by refraction index. For analysis of L-Phe, L-Tyr, CA and pHCA, HPLC was performed using an Agilent 1100 System (Agilent technologies, Palo Alto, CA) with photodiode array detection. The column used was a Phenomenex Synergi Hydro RP C18, 150 × 4.6 mm, 4 μm (Phenomenex, CA, USA). Running conditions were: mobile phase B, 0.1% TFA in methanol; mobile phase A, 0.1% TFA in water. Flow was set to 1 mL/min and column temperature of 45°C. Mobile phase gradient started with 5% solvent B increasing at 80% in 8 min; this ratio was maintained for 2 min and returned to 5% solvent B in 2 min. From 10 to 12 min reduce solvent B at 5%. Samples were centrifuged and supernatants were filtered with nylon syringe filters

The determination of amino acids was performed using high-performance liquid chromatography, HPLC (Agilent Technologies, mod. 1260 Infinity, Santa Clara, CA, USA), in the reverse phase by precolumn derivatization reaction with orthophthaldehyde (OPA) in the presence of mercaptoethanol. The liquid chromatograph used consisted of a 1525 pump system, a 2475 fluorescence detector, 356 nm excitation and 445 nm emission wavelength (λ), and a 717 automatic injector (Waters, Milford, Massachusetts, USA). Equipment monitoring and data acquisition and processing were performed through Waters’ Breeze program. Separations were carried out using a Nova-Pack C18 column (3.9 × 150 mm) from Waters. OPA was used as a derivatizing reagent; 750 mg of OPA was dissolved in 5 mL of methanol and 0.5 mL of 2-mercaptoethanol was added. The protocol was followed, and the chromatographic conditions used are detailed in Pripis-Nicolau et al. (2001) [26 (link)].