column chromatography / TLC

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Arvindekar
Arvindekar's picture
column chromatography / TLC

We are tring to separate biological active plant component with anti diabetic activity.  Our expertise is more in biological activity determination but we are stuck when it comes to isolation of active components.
For eg. we have attempted solvent extraction and obtained our desired component in chloroform extract. On running a silica colum with increasing concentration of ethyl acetate with toluene ethyl acetate the 70:30 fraction contained the biological activity.  However an HPLC analysis revealed further five peaks and we are unable to separate them further.  I want suggestions on how we can separate these using column or TLC (we do not have preparatory HPLC facility)

Dr. Analytical
Dr. Analytical's picture
Column chromtography is a

Column chromtography is a "low resolution" technique, while HPLC is "high resolution."  Your results are therefore not surprising.  TLC can be better than column chromatography, but it is also slow, and has a limited sample capacity.
If you don't have equipment for doing a high resolution separation on a large scale, then your only other choice is to separate your active fraction using a different chromatographic system.  By that I mean something with different selectivity (stationary phase and mobile phase).  What you are using now is a "normal phase" system.  You could try a different support (say, alumina vs. silica), or go with a reverse phase system (non-polar support, more polar solvent).
Either way you will have to spend some time learning a little more about chromatography.  Or, maybe you could find a contract lab to do the purification for you.
 

Arvindekar
Arvindekar's picture
Thanks for the suggestion but

Thanks for the suggestion but I guess we have to learn it any way as I do not have the kind of funds to get my analysis from a contract lab.  How much does it cost anyway?
If we decide to work with TLC what solvent system will be best for separation