Crude organic (EtOAc) extracts of F53 (665 mg) were fractionated using a silica solid phase extraction (SPE) cartridge (Grace Davison, Columbia, MD, USA). Fractions were eluted stepwise with DCM, EtOAc, EtOAc:MeOH (1:1), and MeOH and each fraction was tested for cytotoxicity against RPMI‐8226 cells. The bioactive EtOAc fraction was separated by HPLC with a Discovery BIO Wide Pore C18, (10 × 250 mm, 5 μm; Supelco, Bellefonte, PA, USA) reversed phase column using a Shimadzu Class VP HPLC system equipped with a Shimadzu SPD‐M20A Diode Array Detector. HPLC was performed using a H2O/MeCN (with 0.5% trifluoroacetic acid) gradient at a flow rate of 3.5 ml min−1. Purification was achieved using a solvent gradient from 50% to 100% MeCN over 20 min after an initial 5 min isocratic elution with 50% MeCN. The cytotoxic fraction containing 1 (6 mg) eluted at 15.2 min under these conditions.
Discovery bio wide pore c18
Manufactured by Merck Group
Sourced in United States
The Discovery BIO Wide Pore C18 is a reversed-phase chromatography column designed for the separation and purification of biological macromolecules. It features a large pore size to accommodate the analysis of larger molecules like proteins and peptides.
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2 protocols using discovery bio wide pore c18
1
Bioactive Compound Purification from Crude Extract
2
HPLC Analysis of Radioactive Compounds
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HPLC analysis of the described compounds (cold and radioactive) was performed using the same chromatographic equipment, a Perkin-Elmer LC 200 analytical HPLC coupled to an LC 290 UV/Vis detector and a Berthold LB-507 A radiometric detector. The column used was a Supelco Discovery BIO WidePore C18 (250 × 4.6 mm, 5 μm particle size). Mobile Phases A: H2O (TFA 0.1%) B: ACN (TFA 0.1%). Gradient program: 5 min A-95%, 20 min going from A-95% to B-100%, 1 min going back to A-95% and 4 min A-95%. Mass spectra were acquired in an electrospray ionization/quadrupole ion trap (ESI/QITMS) Bruker HCT mass spectrometer. Samples were injected in mixtures of H2O:ACN at a flow rate of 150 µL h−1.
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