I think most of my compounds are non-polar, I tried solvent system 50dcm:50meoh, cannot separate at all, then add more hexane, using 70dcm:20hex:10meoh, better than before, but I don't know why, is that because non-polar solvent can add more affinity with sephadex? and how to adjust the ratio to get a better separation? at first I thought may be it's because of hexane, but when I add more hexane, like 50dcm:40hex:10meoh, still cannot separate more compounds, now I don't know how to do next step...should i add more meoh? and how does the size exclusion work in different solvent system? anyone can share some knowledge of sephadex lh20...I will be very appreciate it.
Try silica column using gradient hex: ethyl acetate solvent for non- polar compounds
Sephadex column rely on the simple principle of size/molecular weight exclusion. Not on the polarity. So, I will suggest you to first solubilize your extract in the appropriate solvent system and do elution with the same solvent. Be sure column is packed properly, no any crack and bubble inside.
However, only Sephadex will not be enough to get the pure compound. you have to do the successive elusion using other column too. like, reverse phase chromatography (ODS) and normal phase silica. For more details visit column chromatography expert Dr. Khem Raj Joshi, Kumamoto University.
In our laboratory, we prefer 20, 40, 60, 80 and 100 % MeOH based on the solubility of extract/fractions in Sephadex
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