How can I set up conditions (eluent, amount) for the separation of an organic compound from reaction crude with Sephadex?
I am trying to purify heavy organic compounds (m.w. 1300-1500) from impurities (m.w. 300-700) with a Sephadex column. How can I choose suitable eluents? I have already tried, as suggested in some publications, some organic solvents (that I'd like than aqueous buffers), such as methanol or dichloromethane, but no fractioning occurred. However, how is the ratio of the amount of crude and that of Sephadex powder?
Sephadex relies on swelling with water to generate the pores needed for size exclusion, so I doubt that you'll have success with organic solvents. Sephadex LH-20 can be used with organic solvents, but it doesn't have the ability to do SEC in the size range you're interested in.
You might want to look into polystyrene or polyacrylate media (e.g. Toyopearl HW-40) for the application you have in mind.
We cannot use Sephadex to isolate organic compounds. The organic compound must be dissolved with a aqueous buffer solvent, then used the suitable number of Sephadex that isolate the heavy organic compounds from impurities.
Thank you for your answers,
However, @Ramling Shivanandayya Mathpati, my compounds are very polar and little impurities were not separated by C18 column purifications. For this reason, I am trying to apply SEC protocols.
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