I am specifically referring to butanol fraction but I am also interested in chloroform fraction.
Hi, do you want to isolate compound(s) using preparative HPLC directly by using conventional column (analytical column)?Let us share our experiences at our Lab, we sometimes used conventional column (analytical column) for isolation some compounds, and it worked, we can get a few mg pure compounds (by do the separations several times); before we did prep HPLC we did CC to separated the crude extracts into some fractions, and we injected the fractions into HPLC. In my opinion, it is nice to do first CC for raw extracts forseparating it into fractions than use prep HPLC/TLC to isolate the compound(s) from the fractions
A friend of mine has suggested that butanol fraction cannot be subfractionated. Henceforth, the only option is to run a diluted butanol fraction directly on recycle HPLC for compound isolation. If i have to use conventional column, How many times should I run conventional column before I run final subfraction on recycle HPLC?
How did you get to the butanol sub-fraction? Was there an extract that was washed with several solvents?
Depending on the procedure you used, butanol carries a lot of water from extraction and so contains some fairly polar compounds; C18 would work well. I suggest a further gradient using dichloromethane (assuming a silica-based C18) to wash non-polar material off the column. I would also run the dichloromethane wash if running the dichloromethane extract as well.
The dichloromethane gradient, if run alone is non-aqueous reverse phase. Chaining a water->methanol (or acetetonitrile)->dichloromethane gradient is something I call "wide polarity range chromatography". I use the technique for column screening.
See: http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN77_Wide_Polarity_Range_Chromatography.pdf
yes methanol extract was washed with n-hexane, chloroform & butanol. Jack SIlver
@Irfan- That is very helpful information.
- Methanol extract suggests it is fairly polar
- Washing with the hexane and DCM suggests that non-polar and moderately polar compounds were removed.
- What is left should run on C18, although it still may be a complex mixture.
Hi Irfan,
if you have bad separation for C18 column, try C8 stationary phase. Since preparative columns are very expensive, you can try analytical column (C18 and C8 if available) first to check the general polarity of your sample, before buying a new preparative column.
Good luck,
Marcus
never hurts to check with reversed phase tlc plates to find a solvent system that gives good separation. A tlc plate is cheaper than hplc column. Explore acid, neutral and basic mobile phase.
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