I am doing Thin layer chromatography for my active fraction 20% Ethanol (20%Ethanol with water), to get some idea which solvents have to used for column chromatography. i have tried many combinations but unable to get good results. Can any one suggest best combination to get at least 2-3 spots for my fraction, want to mention that this fraction already showed very good results against different bacteria. And now i want to check the active compounds. Feeling very confuse> my ultimate goal is to isolation and purification of active compounds from this fraction. More likely that i have some polar compounds but i am not sure.
Dear Shakeel, if you got a plat extract in 20% EtOH your best option is to run a reverse phase FCC. You can purchase C18 colums at low price and use it as a pre-pached column if you don't have a biotage or companion where to mount it. Eluent will have to be water and organic (acetonitrile or MeOH). Run a C18 TLC to get an idea of how well your components separate.
Column can be re-used many times (up to 100 times in my experience) as long as you wash it well with 100% organic phase and store them wet in the dark once you have finished your separation.
C18 TLC can also be re-used, wash it with MeOH or acetonitrile and dry in air to restore it.
Dear Shakeel, if you got a plat extract in 20% EtOH your best option is to run a reverse phase FCC. You can purchase C18 colums at low price and use it as a pre-pached column if you don't have a biotage or companion where to mount it. Eluent will have to be water and organic (acetonitrile or MeOH). Run a C18 TLC to get an idea of how well your components separate.
Column can be re-used many times (up to 100 times in my experience) as long as you wash it well with 100% organic phase and store them wet in the dark once you have finished your separation.
C18 TLC can also be re-used, wash it with MeOH or acetonitrile and dry in air to restore it.
Agreed with Filippo. Also pH can have a huge effect on your resolution (how tight your bands are). So when you are scouting conditions with the C18 plates, you can try small amounts of different acids - phosphoric, TFA, acetic; buffers if you wish, or a little NH4OH, always keeing your pH between 2 and 8 to maintain integrity of your stationary phase. Good luck!
That is a fairly polar compound. You may need to run a system with water if you haven't done so (it is helpful to explain what you have already tried). This is called aqueous normal phase. C18 is also a possibility as mentioned above.
Please see:
http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Application%20Notes/HILIC%20Purification%20Strategies%20App%20Note.pdf#search=HILIC
http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Application%20Notes/RediSep%20Rf%20Gold%20Silica%20and%20Highly%20Polar%20Solvents%20App%20Note.pdf#search=HILIC
http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Posters/Strategies%20for%20Flash%20Purification%20of%20Highly%20Polar%20Compounds%20Poster.pdf#search=HILIC
The problem with TLC with natural products is that one can often resolve spots, but we don't know if those spots are active.
To work around the problem, I use "wide polarity range chromatography" with several types of columns. You may find another column type may work well, possibly an ion exchange column.
Here are some links for screening different columns and solvents:
http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Posters/New%20Techniques%20in%20Extract%20Column%20Screening%20Poster.pdf#search=column%20screening
http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Posters/New%20Techniques%20in%20Extract%20Column%20Screening%20II%20Poster.pdf#search=column%20screening
The column screen is useful in that it suggests a purification strategy since the compound(s) may well be resolved with different columns from different impurities.
Links updated 14 Jan 17
Dear Researcher
Kindly go through this paper and try the liquid combination used for the Column chromatography. It could be perfect considering the different solubulities of the chemicals present in your desired material.
Good luck!!!
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