I Never packed column with alumina, I only used silica gel. I am not able to separate plant compounds with silica gel as stationary phase. So I thought of packing column with Alumina. Can you please suggest how to pack column bed with Alumina? My solvent system was Chloroform:Methanol (For separating Flavonoids).
Is there any other solvent system? Can you help me in this regard. Thank you.
I think it is not good idea to use alumina for polyphenol separation. It absorbs polyphenols too much. You can use silica gel but change the solvent system or you can try different mobile phases like polyamide (which is also efficent for isolation of flavonoids) or Sephadex LH-20. My suggestion is use silica gel column with different solvent mixtures.
You can use chloroform and a drop of methanol or you can use ethyl acetate and a drop of acetic acid to elute flavanoids and polyphenols. It 's better to use silica column.
For polyhydroxy compounds like flavonoid s alumina is not at all a suitable solid phase in column (due to high affinity and adsorption.). Polyphenols can be conveniently separated by partition chromatography rather adsorption columns.
Silica gel columns with suitable solvent system ( hexane with different proportions of EtOAc, Me2CO,EtOH are normally suitable). Try first on small plates of(40x20 mm cut plates a Silicagel 254 TLC plates and identify suitable solvents, if not clearly separated try double phase chromatography of same once developed plate.............you will get interesting results)
@ Dr.B.K.Chakravarthy... Sir, I had run the TLC with different solvent systems started from ethyl acetate and ended with methanol. In 100% ethyl acetate it is showing one brick red spot in Long UV and not showing any spot under Iodine vapors. In chloroform 100% it is showing nice separation with 4 brick red spots and same happens in iodine. I increased the ratio to 1:1 Chl : Methanol, the top spots moved up and 3 more spots come up...So my guess was totally around 7 compounds are there in my compound. But I am confusing to select the mobile phase...
So please suggest the best mobile phase.
Thank you @Ramesh Kumar, @ Didem... Sir thank you for u r valuable suggestions. I will follow u r suggestions and will pack with silica gel 200-400 Mesh (will it be good). I do not have Sephadex.
May be all 4 spots are free poly phenol in nature . Just spray with mild FeCl3 soln.in MeOH. Those which take blue color you can focus on.
Another way of separating combined polyphenols in a close by Rf value mixture is by converting all in to methyl esters ( with dry Di methyl sulphate and try to separate)
May the readers know which plant extract you are working......is it known one?
If it is new and unknown one just let us know the family..some useful references can be provided.
@Dr. B.K., what did you meant by double phase chromatography? it it the 2D TLC?
I didnt get good separation with silica gel so had to give a try with Alumina neutral. It was my first experience with alumina. I got a compound pure from this column but the compound which present in sufficient quantity was not at all eluted or shown up in the TLC. If this compound is more polar and bound more affinity with alumina how can i elute it out? 30% methanol in Chloroform didnt help. Please give me some suggestions.
Use silica gel but change the solvent system and try different mobile phases on try and error bases
It is like preparing a silica column. Two methods:
1- dry: add alumina to your column then run the solvent through it
2- wet: make a sludge of alumina an your solvent an fill the column. Remove the solvent and start running your ctg.
I think you should not loose hope and continue with the silica column only. Important thing is that you should try different combinations of the solvents rather than using alumina. Flavonoids are readily separated in silica columns. Try using ethyl acetate, chloroform and methanol in different ratios.
I understand that your problem in not the column pacing material but the solvent system you used. You should give another try with other solvents and I am sure you can get the separation in chloroform:methanol. Some hit and trials with EtOAc and toluene as well will help u further.
Change of column will complicate the process further.
Gud Luck
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