I have defatted my extract and then prepare ethanolic extract....after that I dissolved my extract in water and then fractionate firstly with chloroform, ethyl acetate and then with n-butanol (AR) grade, after fractionation the left aqueous extract after separation of butanol fraction, I check for the presence of phenolic and flavonoids.Chloroform fraction lacks both.but Aqueous fraction show more phenolic content as compared to ethyl acetate and butanol.also more antibacterial activity also.I did not get reason behind more phenolic content in aqueous as it should not contain anything as I supposed.I did it twice and get same output..what should I do now?
Should i change my fractionation procedure. Suggest me what to do if I have to target phenolics and flavonoids which are responsible for antimicrobial activity of plant.
while doing fractionation, did you do it until get colourless solution, if not, pls do so...... good luck
Dear Vikas Kumar
You should be sure that your fractionation process has been well performed i.e
1- re-suspension of your extract in water
2-washing your suspension with chloroform then separate in a funnel; and repeat until the chloroform added remain clear before passing to ethyl acetate... and so on
If so, then I can just said that phenolic compounds represent a large family of compounds, and depending of their origin, class and structure, some can have more affinity with water than others.
Regards!
NOBOSSE
Thanks Pierre Nobosse sir. I am again trying for the fractionation process with more time and more solvent until it becomes colourless
Good!
Also note that the chloroform fraction will be in the lower layer in your separating funnel in contrast to other solvents.
Regards!
NOBOSSE
Still my leftout aqueous has more phenolic content as tested by Fecl3 test. It shows more greenish black colour as compared to ethylacetate and utanol fraction
Dear Vikas
Please check your phenolic content by the Folin-Ciocalteu method and use a spectrophotometer. Visual observation is sometime measleading. If you have the same trend by this way, then just consider what I said in a previous answer.
Regards!
NOBOSSE
i have spotted those fractions on TLC and then dipped in 5% FeCl3 solution.Aqueous fraction showed more blackish spot as compared to other fraction and absent in Chloroform fraction. What should i do now?
I would suggest you test the presence of phenolics and flavonoids by using TLC technique instead of FeCl3 test for each fractions. It would give you more information as you can observe both types of compounds under UV (254 and 366 nm) after development and derivatisation. Attachment may help for particular method. You also may access via http://www.hptlc-association.org/
Can u tell me how to preceed for TLC of both fractions as i am not getting proper separation ..its just smear or like that.I am working with ethyacetate: glacial acetic acid:water (8:3:1)....but not proper band are there.....in case of butanol,i am not able too get proper band.how to do that fraction for tlc....?
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