I would like to know the separation methods for the terpenes groups which was identified by GC-MS. what would be the suitable method to separate the identified terpenes from the n-hexane and ethanol crude extract and how to develop the purification process for the further terpene quatification ?
These are non-polar compounds, as shown by extraction into hexane and ethanol.
Try running a number of different TLC plates. Silica and alumina, using hexane and solvents such as ethyl acetate, toluene, dichloromethane, an ether, and ethanol. Make sure your developing chamber is well saturated with vapor. The desired compounds should elute with an Rf of 0.2 to 0.5. Lowering the hexane concentration relative to the other solvent (called the "strong solvent"- hexane is the weak solvent) will cause the compound(s) to run faster up the plate, making a higher Rf. You can run the same solvents in a flash system, I recommend solid loading. I also recommend a gradient starting below the concentration of the strong solvent used for the TLC plates, and running a gradient to a strong solvent concentration above that for the TLC plate. Also, try a diol TLC plate, use a water spray to visualize the spots. Non-aqueous reverse phase can work as well.
Nice advice from Jack- agreed. On TLC (silica gel F254 only) analysis of the fractions the following applies-
Unsure if your ethanol extract will have good amount of terpenoids and if any at all! Expect flavonoids and phenolics there. For TLC of these use mobile phase: chloroform: methanol: formic acid (85:15:1)
For true terpenoid analysis, use cyclohexane: ethylacetate (9:1) mobile phase for good separations.
Alternatively, you can use, LH20 (solid loading) for serial fractionation using hexane, toluene, chloroform, ethylacetate, methanol etc. to get the terpenoids and other eluted in different fractions- instead of silica LH20 is expensive but worth it (from experience!).
Another way to find "bioactive fractions" and then narrow down to "bioactive compounds" is TL-Cbioautography (search on Google) and from publications from Professors Kurt Hosttetman and Jean Luc Wolfender. Depends what kind of bioactive fractions/ compounds you are looking for- antimicrobial, anti-amylase, anti-oxidant, anticholinesterase, anti-tyrosinase etc. all can be done on TLC plates and compounds identified and eluted in parallel. Such approaches can be done using HPLC and LC-MS to if you folow their publications. I have some papers on those TLC-bioautography approaches for terpenoids too. Please look up.
GC-MS is a guide only to know what is in there (but do not perform GC-MS for your ethanol fraction!- do LC-MS rather for phenylpropanoids/ flavonoids)! but purification is much much more challenging but feasible.
Thanks,
Biswa
silica column using hexane:ethyl acetate for the hexane extract
phase separation by aqueous ethanol: ethyl acetate then butanol for the alcoholic extract
Classical chromatographic methods are good procedures for terpenes purification. I suggest you to try column chromatography using silica gel as stationary fase (dichloromethane, chloroform, ethyl acetate as solvents) or preparative TLC plates (this used to work out for me)... If it helps, take a look on this paper: Article Antibacterial activity of terpenoids isolated from Cnidoscol...
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