I would like to isolate and subsequently identify an antimicrobial molecule present in the 70% methanolic extract obtained from a cyanobacterium. What steps would you suggest to purified this molecule?
Thanks in advance
Sephadex LH-20 is designed for relatively non-polar compounds and for repetitive application. If you apply crude plant extracts of aqueous and aqueous alcoholic region, it is not advisable to subject them straight to sephadex LH-20 separation. You would see, your sephadex colourised and contaminated with something not eluted from your column.
You have two options to approach this: fractionate your crude extract by solvent-solvent fractionation. Suspend your sample in water and fractionate in the order of polarity – petroleum ether, then chloroform, ethyl acetate, butanol and you will live with the final water fraction. Your sephadex is ideal for separations from the non-polar fractions. Second, you may fractionate with solid (like silica)-liquid based approach. Vacuum liquid chromatography type if you need a quick fractionation or open column chromatography if you have the patience and also want to try isolate compounds. Fractions collected from the above approaches can be loaded to your sephadex LH-20 column.
I routinely use chloroform-methanol mixture for sephadex LH-20 columns. For very non polar compounds, chloroform; chloroform-methanol (1:1) for vast array of compounds in the middle polarity range; and you can use methanol for relatively polar compounds.
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