Frc 10a fraction collector

Semipreparative HPLC was carried out on a Shimadzu system consisting of DGU-20A5R degassing unit, LC-20AT solvent delivery pump, SIL-10AF autosampler, CBM-20A controller, CTO-20AC column oven, SPD-M20A diode array detector and FRC-10A fraction collector (all Shimadzu, Kyoto, Kinki, Japan). Stationary phase was a Luna C-10(2) column with 250 × 10 mm and 10 µm particle size (Phenomenex). Mobile phase consisted of water (A) and acetonitrile (B). Elution at 4 mL/min and 35 °C column temperature started at 40% B, rising to 100% B at 20.0 min. All compounds of interest eluted within this gradient, followed by varying plateaus of 100% B (0–5 min duration) for column cleaning and re-equilibration at 40% B. Injection volume was 200 µL.

Semipreparative HPLC was carried out on a Shimadzu system consisting of DGU-20A5R degassing unit, LC-20AT solvent delivery pump, SIL-10AF autosampler, CBM-20A controller, CTO-20AC column oven, SPD-M20A diode array detector and FRC-10A fraction collector (all Shimadzu, Kyoto, Kinki, Japan). Stationary phase was a Luna C-10(2) column with 250 × 10 mm and 10 µm particle size (Phenomenex). Mobile phase consisted of water (A) and acetonitrile (B). Elution at 4 mL/min and 35 °C column temperature started at 40% B, rising to 100% B at 20.0 min. All compounds of interest eluted within this gradient, followed by varying plateaus of 100% B (0–5 min duration) for column cleaning and re-equilibration at 40% B. Injection volume was 200 µL.

Fractions of crude extracts H and E were obtained through vacuum liquid chromatography (VLC) on silica gel (0.040–0.063 mm, Merck, Darmstadt, Germany) with solvent mixtures of increasing polarity (hexane, ethyl acetate, methanol, all Roth, Germany). Fractions were controlled by thin-layer chromatography (silica gel 60 F₂₅₄, Merck, Darmstadt, Germany) and those of similar substance patterns combined to yield five fractions for H (H1–H5) and 9 fractions for E (E1–E9).
Four compounds were isolated by semi-preparative HPLC using a Shimadzu CBM-20A controller, LC-20AT solvent delivery module, SIL-10AF autosampler, CTO-20AC column oven, SPD-M20A diode array detector, and FRC-10A fraction collector (all Shimadzu, Kyoto, Japan).
All separations were performed on a Luna C18(2) column, 250 × 10 mm, 10 μm (Phenomenex, Torrance, CA, USA) via isocratic elution with a flow rate of 4 mL/min, 25 °C column temperature; 200 μL injection volume.
Compounds 1 and 7 were obtained from E8 (VLC fraction with hexane:ethyl acetate:methanol = 4:80:16) by isocratic elution with a solvent composition of acetonitrile:water = 70:30 (v/v). Compound 4 was isolated with acetonitrile:water = 63:37 (v/v) from H4 (hexane:ethyl acetate:methanol = 36:60:4) and compound 6 with acetonitrile:water = 75:25 (v/v) from E2 (hexane:ethyl acetate = 70:30).

The solvents used for extraction and separation were all from Thermo Fisher Scientific if not explicitly mentioned. The crude extract (1 g) was loaded into a column with the diameter and length of the column of 2.5 cm and 30 cm, respectively. The fine silica used for column chromatography was of particle size 40–63 µm (60 × 100 mesh) (Sigma Aldrich, St. Louis, MO, USA). Then, the column was eluted with 200 mL of hexane, followed by elution sequentially with 400 mL of 50% ethyl acetate in hexane, 500 mL of 5% methanol in dichloromethane and 300 mL of 100% methanol. All four fractions were dried, and their activities in blocking malaria transmission were determined as described above. The fractions containing active compounds against malaria transmission were further fractioned using a Shimadzu HPLC system (an LC-20AD pump, SPD-M20A UV and visible detector, and FRC-10A fraction collector, Shimadzu, Columbia, MD, USA). A semi-purification column (Gemini C18 250 mm × 10 mm, 5 μm, Phenomenex, Torrance, CA, USA) and a gradient solvent of MeOH-H2O (50:50–100:0) was used to obtain the pure bioactive compound.

The Varian (Palo Alto, CA, USA) Mercury Plus 400 MHz and VNMRS 600 MHz FT-NMR spectrometers provided the NMR spectra. HRSIMS was performed by using a Bruker APEX II spectrometer (Bremen, Germany). The V-650 spectrophotometer from Jasco (Tokyo, Japan) was utilized to measure the UV data. For measuring infrared data, the Jasco FT/IR-4X spectrophotometer was selected. Circular dichroism data were recorded using a Jasco J-815 CD spectrometer. A Jasco P-2000 polarimeter was used to quantify specific optical rotation. Merck KGaA (Darmstadt, Germany) silica gel 60 (0.015–0.040 mm) was utilized to pack columns. For high-performance liquid chromatography (HPLC), Phenomenex (Torrance, CA, USA) C18, phenyl-hexyl, and biphenyl columns were utilized. The LC-40D solvent delivery module, DGU-405 de-gassing unit, CBM-40 system controller, CTO-40S column oven, SPD-M40 photo diode array detector, and FRC-10A fraction collector made up the Shimadzu (Kyoto, Japan) HPLC apparatus.

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.).