I am having an unknown bacterial hit so far, which is inhibiting the growth of multidrug-resistant bacteria. I want to characterize that antimicrobial compound.what should I do to characterize it..i also tries to extract by using hexane, chloroform, ethyl acetate, but in all case, my hit is coming in aqueous phase. It is hard for me to run in the HPLC collum, as we do not have set system for aqueous phase. But having a good setup for the organic phase.
Hello Chankit Giri,
I believe you could look at sequential extraction with solvents (chosen basing on polarity index - less polar to highly polar) and then go with fractionation to understand, which class of molecules are responsible for the said activity. You could then devise protocols for separation and/or partial purification.
Please see these links for a better idea-
https://www.researchgate.net/post/Can_anyone_provide_me_the_protocol_of_sequential_extraction_method_for_plant_extraction
https://www.researchgate.net/post/Is_there_a_difference_between_sequential_extraction_and_fractionation_of_medicinal_plant_extracts
Article Antibacterial activity of sequentially extracted organic sol...
All the best.
Agreed with Prasanth! You can also try a methanol or acetonitrile spin down of your sample for this more hydrophilic compound. Its possible to purchase different types of columns for sample clean up that vary in polarity too i.e. use C8 instead of C18 cartridges for solid phase extraction, or even normal phase silica packed columns. That might help you to isolate your compound a little better.
I cannot imagine what analytical method of separation you can apply without knowing the nature of the antimicrobial compound. If you know at least about its polarity, your job can be easier with the appropriate analytical separation method.
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