I want to know the easiest and least expensive compound fractionation or separation methods and its most commonly used techniques to ascertain the in vitro activity of the separated compounds
You didn't say which is your raw material, I suppose is a plant. As you know plants have hundreds of compounds and they are classified as primary and secondary metabolites. For the last one we use liquid- liquid partition and majorly chromatographic techniques ( TLC, CC, GC, HPLC, electrophoresis and so on). Do you want to separate or isolate a special kind of secondary metabolites? Some times you can get a particular methods. Please, be more explicitly.
I assume your compounds that you want to isolate are from plant extract. I can suggest two ways for your isolation process from raw plant to get crude extract.
1. If you have targeted compounds:
Process: Double Maceration (soak your plant in solvent for a period of time)
Methodology: First, find out from the literature review or monograph the polarity index of your targeted compounds (are they from polar, semi-polar or non-polar group) and macerate your plant (usually grounded to powder if possible) with lower polar solvent to eliminate compounds with lower polarity. Then, macerate them again with solvent that is close to your targeted compound. You will have crude extract that contain mostly your targeted compound in the second maceration. Then, you need to run your crude extract with TLC for screening using appropriate solvent system. Then you may continue your fractionation using column chromatography (CC) or vacuum liquid chromatography (VLC).
2. If you want to isolate all compounds individually from crude extract:
Process: Multiple maceration
Methodology: Macerate your plant using low polarity to high polarity solvent separately. From this method, you will obtain crude extracts of different polarities and simultaneously categorized your compounds according to their polarity. From here, you may continue with fractionation using CC or VLC. However, this can be done if you have a large amount of plant sample.
Another alternatives are by using liquid-liquid extraction (LLE) which might seem the easiest but you have to know the polarity and miscibility of the solvents that you're going to use for LLE. Another one is supercritical fluid extraction method. You may need to develop your method before run.
Here's an article (as attached) that you may use for your study. Hope this help.
Thanks Lock and Azmar by the way if i want to separate secondary metabolites from the plants extracts based on their polarity which chromatographic techniques is feasible to get higher yield of bioactive compounds for example xy is my compound how can i obtain the particular with hyphenated instruments and which method will be convenient for further purifications?
You can try to purify using preparative TLC (prep TLC) or chromatotron. Prep TLC is faster compare to chromatotron but you can only use small amount of sample (1 mg or less). You can collect your targeted compound by scraping the prep TLC according to its band. Make sure to screen your fraction first before running the method to confirm your compound position. You may adjust your solvent system so that your compound can be separated well. It is advisable to run prep TLC using fraction that has major amount of your targeted compound.
Very much depends on the nature of your compound(s). I like to start with a crude fractionation-most biologically interesting compounds are nonpolar so use a normal phase separation. If polar compound(s), use reverse phase. Collect a reasonable amount of fractions from this phase separation (5-10 fractions) and test these fractions against your in vitro assay. Your results from this should help guide rather inexpensive fractionation of compounds from the active fraction(s)...it's a good idea to switch the phase separation at this point (i.e. if you used normal phase for the crude fractionation, switch to reverse phase for HPLC fractionation of the active fractions).
As an addition to my previous question, whether doing normal or reverse phase for the crude fractionation, one should run the a wide range of polarity with the mobile phase (i.e. hexane to EtOH for normal phase). I used a handpacked large column with silica gel that I added water to and baked at low temperature in an oven.
Also, thinking more about compound characteristics, most biologically-active compound tend towards nonpolar but also have some polar characteristics on the same molecule as well.