Factorfour vf 5ms column

Biotransformations were performed in amber glass vials (40 mL) in a total reaction volume of 1 mL. Whole-cell (WC) and cell-free extract (CFE) biotransformations were performed as previously described [7 (link)]. Reactions with purified BVMO were performed in 100 mM Tris-HCl buffer (pH 8) containing 2 μM BVMO, 0.5 U BmGDH, 100 mM glucose, 0.3 mM NADP⁺ and 10 mM substrate. Reactions were maintained at 20°C with shaking (200 rpm), where after they were extracted using an equal volume (2 x 0.5 mL) ethyl acetate containing either 2 mM 1-undecanol or 3-octanol as internal standard. GC-FID (and GC-MS for product identification) was performed on a Finnigan Trace GC ultra (ThermoScientific) equipped with a FactorFour VF-5ms column (60 m x 0.32 mm x 0.25 μm, Varian). Steady-state kinetics were performed by monitoring the oxidation of NADPH spectrophotometerically at 340 nm (ε₃₄₀ = 6.22 mM^-1.cm^-1) or 370 nm (ε₃₇₀ = 2.70 mM^-1.cm^-1). To investigate optimal pH, temperature, stability and effect of organic solvents, reactions typically contained 2 μM BVMO, 0.3 mM NADPH, 1 mM phenylacetone, 1% (v/v) methanol (100 mM Tris-HCl, pH 8; 25°C).

A Shimadzu GC-MSQP2010 fitted with a AOC-20i injector and FID and MS detectors (Shimadzu, Tokyo, Japan) was used for the GC-MS analysis. The GC was run with a nonpolar Supelco MDN-5S fused silica capillary column (30 m × 0.25 mm ID, 0.25 µm film thickness) commonly used for the analysis of VOCs. The injection volume (EO dissolved in chromatography grade hexane) was 1 μL. Helium was used as the carrier gas at a constant flow of 1 mL/min. The column temperature was set at 50 °C for 30 s, then increased from 50–150 °C at 4 °C/min, from 150–175 °C at 1.5 °C/min and from 175–300 °C at 20 °C/min for a total analysis time of 58.42 min. The injector temperature was set to 250 °C and the injection was accomplished with a split ratio of 1/50 throughout the entire run. The MS was operated in the electron impact mode at 70 eV, with a scan range of 40–400 m/z. The temperatures were set to 200 °C for the ion trap, 50 °C for the manifold and 305 °C for the transfer line.
A Varian 450-GC fitted with an MS240 iontrap MS and a Combipal autosampler (Varian Instruments, Sunnyvale, CA, USA) and run with a nonpolar Varian FactorFour VF-5ms column (30 m × 0.25 mm ID, 0.25 μm film) commonly used for the analysis of VOCs was used under the same conditions to assist in compound identification.