Dad model spd m20a

The HPLC apparatus consisted of an LC-20A Prominence (Shimadzu^®, Kyoto, JPN) equipped with a quaternary pump and a DAD model SPD-M20A (Shimadzu^®). A 15 µm Phenomenex C18 column 250 × 21.2 mm I.D. (Torrance, CA, USA) connected to a fraction collector FRC-10A (Shimadzu^®, Kyoto, Japan). Elution was performed with a mixture of solvents according to Silva al. [6 (link)] with small modifications: solvent A was 1% formic acid, and solvent B, 80% methanol at a linear gradient (0–25 min, 11–55% of solvent B). The sample injection volume was 400 µL at 3.5 mL/min flow rate, and separations were monitored at 536 nm. Red-violet-colored fractions containing betanin were concentrated by rotary evaporation, and pure and concentrated betanin was suspended in deionized water, freeze-dried, and stored at −80 °C for further analysis (Figure S1).

Concentrated beetroot juice was purified by RP-HPLC. The HPLC apparatus consisted in an LC-20A Prominence, (Shimadzu^®, Kyoto, Japan) equipped with a quaternary pump and a DAD model SPD-M20A (Shimadzu^®, Kyoto, Japan). A 15 µm Phenomenex C18 column (250 × 21.2 mm I.D., Torrance, California, USA) connected to an FRC-10A fraction collector (Shimadzu^®) was used in the semi-preparative HPLC. The elution conditions were performed according to Cai et al. [39 (link)] with modifications. Solvent A was 1% formic acid, and solvent B was 80% methanol at a linear gradient (0–25 min, 11–55%). The injection volume was 100 μL and a flow rate of 5.5 mLmin⁻¹ was used. Separations were monitored at 536 nm and, after purification, magenta fractions, containing betanin, were concentrated by a rotary evaporator (Rotavapor^® R-215, Buchi, São Paulo, Brazil) at 24 °C, 150 rpm and a water bath at 40 °C. The extracts were then suspended in 1 mL deionized water and stored at −30 °C under an N₂ atmosphere for further analysis. The purified betanin was analyzed using a Nucleosil 100-C18 column (250 × 4.6 mm I.D., 5 μm) with 30 µL injection volume and a flow rate of 1.0 mL min⁻¹. The mobile phase and gradient conditions were similar to the purification step and betanin concentrations were quantified in comparison to a betanin standard solution (Sigma-Aldrich Co.).