I am doing extraction of secondary metabolites with etoac from endophytic fungus Trichoderma, but after the first wash, when I add more etoac, it becomes turbid and watery. When I try to evaporate it on rotary, water remains there. Am I missing something? And am I using commercial grade etoac and distill it on rotary before using?
Dear Hira,
your extraction medium /ethylacetate/ is only partly soluble in water. Adding et.ac. to wet fungi a two layer solvent system is formed: a lower layer rich for H2O and upper one rich for ethylacetate /causing the system turbidity/. That is the whole your problem.
When you used rotary vacuum evaporator ethyl acetate evaporated as first due to its low boiling point /approx. 77oC/, water evaporated only partly /b.p. 100oC under normal conditions/ .
Try to remove water prior to extraction of metabolites. You may get the fungi dry at ambient temperature /you may lose some metabolites due to oxidation and secondary reactions/, or better lyophylize them. Best might be to freeze-dry the fungi and afterward extract the unhydrous material with ethylacetate.
Instead of freeze drying the fungi, you may remve water from the sample with addition of n-hexane and rotavap diestillation. Then the fungi can be submitted to et. ac. extraction.
Regards,
R.S.
Dear Dr. Rastislav
Thankyou so much for your help. I am doing solid state fermentaion on PDA (Potato dextrose media), and when I mix ethyl acetate with the agar plates, then this whole etoac turns into turbid layer. What should I do in that case?
And another thing is that should I lypholize crude extracts?
Derar Hira,
1. I hope that I am not wrong, agar is polysachcharide consisting of beta and alpha D-galactopyranose units, some of the building units are rarely and randomly esterified with H2SO4.
It is soluble in H2O and creates gel like system. It may be partly soluble in et. ac., too, and agar as a natural polysaccharide also forms gel. If I were you I would try maybe propyl-, isopropyl or buthylacetate instead of et. ac. They are weaker solvents of polar substrates..
2. I think, you may try it, it should work. I would prefer freeze dying, if it is accesible for you and when there is no chance to contamine the inside surface of the instrument.
Yes, same agar which we use as gelling agent and thankyou so much again for your valuable suggestions.
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