Kinetex 2.6 μm xb c18 100a reverse phase column

Root exudation of vanillin by B. humidicola cv. CIAT 679 (commercial name “Tully”) was assessed as described by Egenolf et al. (2021) (link). In brief, exudation patterns were investigated in a two-factorial hydroponic experiment. For factor “pH,” the pH of the trap solution was adjusted to target values of 4.2 and 6.8 by addition of HCl and Na₂CO₃, respectively. Factor “Trap solution” consisted of a NH₄⁺ and NO₃⁻ treatment [see Experiment 1 in Egenolf et al. (2021) (link)]. Root exudates were collected into fresh trap solution for 4 h and secondary metabolites were extracted via liquid-phase extraction as described above. Samples were then analyzed for vanillin via HPLC-PDA at 280 nm (Accela HPLC/PDA, Thermo Scientific, Waltham, United States) using a Kinetex 2.6 μm XB-C18 100A reverse phase column (Phenomenex, Torrance, United States) with formate buffer (10 mM, pH 3.7) as polar and acetonitrile as nonpolar eluent (flow rate 0.5 ml·min⁻¹), and a commercial standard (Aldrich Chem. Co., Inc., Milwaukee, United States) in the range from 0.25–2.0 mg·L⁻¹. The applied eluent gradients are provided in the Supplementary Table S5 (Supplementary Material).

For HPLC-PDA analysis and semi-preparative HPLC fractionation, ethyl acetate extracts were dried under a N₂ flow (30°C) and resuspended in H₂O/acetonitrile (1/1, v/v) or H₂O/isopropanol/acetonitrile (1/1/1, v/v/v), respectively. Root exudates were screened for major secondary metabolites based on PDA chromatograms obtained via HPLC-PDA analysis at a wavelength range of 200–600 nm (Accela HPLC/LTQ Velos MS, Thermo Scientific, Waltham, United States) using a Kinetex 2.6 μm XB-C18 100A reverse phase column (Phenomenex, Torrance, United States) with formate buffer (10 mM, pH 3.7) as polar and acetonitrile as nonpolar eluent (flow rate 0.5 ml·min⁻¹).
Subsequently, selected major secondary compounds were isolated via semi-preparative HPLC (Knauer Smartline, Berlin, Germany), using an EC 250/10 Nucleodur PolarTec 5 µm reverse phase column (Macherey-Nagel, Düren, Germany) in a first, and a xSelect HSS Prep T3 5 µm 10 mm × 150 mm reverse phase column (Waters, Milford, United States) in a second step. Both fractionation steps were conducted with 0.01% trifluoroacetic acid as polar and acetonitrile as nonpolar eluent (flow rate 5 ml·min⁻¹). The applied eluent gradients are provided in the Supplementary Tables S1–S3 (Supplementary Material).