Diaphorin was extracted and purified as previously described [13 (link)], with some modifications. Briefly, adult D. citri specimens were treated twice with methanol, and the extracts were combined and concentrated in vacuo. The residue was resuspended in methanol and purified in a Shimadzu (Kyoto, Japan) LC10 high-performance liquid chromatography (HPLC) system with an Inertsil ODS-3 C18 reversed-phase preparative column [5 μm, 7.6 × 150 mm, GL Science (Tokyo, Japan)] heated to 35°C. The mobile phase was isocratic 20% acetonitrile in water, with a flow rate of 1.5 mL/min. Diaphorin was detected at a wavelength of 200 nm. The purified samples were combined and dried in vacuo. Diaphorin was re-dissolved in methanol and quantified in an HPLC system as described above, except the mobile phase was 15% acetonitrile in water, with a flow rate of 1.0 mL/min, and an Inertsil ODS-3 analytical column (5 μm, 4.0 × 250 mm, GL Science) was used. Known amounts of synthesized pederin (see below) were used as standards. The purified diaphorin was stored at −20°C until used.
Inertsil ods 3 c18 reversed phase preparative column
Manufactured by GL Sciences
Sourced in Japan
The Inertsil ODS-3 C18 reversed-phase preparative column is a high-performance liquid chromatography (HPLC) column designed for preparative-scale separations. It features a silica-based stationary phase with octadecylsilane (ODS-3) bonding, providing a reversed-phase chromatographic mode. The column is intended for the purification and isolation of a wide range of organic compounds.
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2 protocols using inertsil ods 3 c18 reversed phase preparative column
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Extraction and Purification of Diaphorin
2
Diaphorin Extraction and Quantification from D. citri
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Diaphorin was extracted from D. citri, purified, and quantified as previously described [30 (link)]. Briefly, adult D. citri were treated twice with methanol, and the extracts were combined and concentrated in vacuo. The residue was resuspended in methanol and purified in a Shimadzu (Kyoto, Japan) LC10 high-performance liquid chromatography (HPLC) system with an Inertsil ODS-3 C18 reversed-phase preparative column [5 μm, 7.6 × 150 mm, GL Science (Tokyo, Japan)] heated to 35°C. The mobile phase was isocratic 20% acetonitrile in water, with a flow rate of 1.5 mL/min. Diaphorin was detected at a wavelength of 200 nm. The purified samples were combined and dried in vacuo. Diaphorin was re-dissolved in methanol and quantified in an HPLC system as described above, except the mobile phase was 15% acetonitrile in water, with a flow rate of 1.0 mL/min, and an Inertsil ODS-3 analytical column (5 μm, 4.0 × 250 mm, GL Science) was used.
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