hi.first step, we should prevent the degradation by adding ...its depends on the molecules.

2nd step, centrifuge the samples at 3500 rpm for 10 min. at temperature 4 in an refrigerated centrifuge with a microtitre vials.

3rd step,acidification:transfer the supernatants in separate tubes and add immediately 100ul of 1N HCL per ml of plasma.Usually HCL can be used to acidify or you can choose.

4th step:Centrifuge at 3500 rpm for 5 min at temperatue 4.

5th step:transfer the supernatants in separate tubes and store at -20 in freezer for later use or use immediately.

Dear, There is two way

You can prepare a Preparative TLC on appropriate mobile phase and than scrap the prominent portion and carry out Spectroscpy study.

Using Coloum chromatography you can isolate the targated molecules in good quantity.

Preparative TLC is best method if you require minor quantity for identification.

As I know, for the isolation of active chemical constituent from Extract, the main thing that is required is Polarity of your compound.

If you knows the polarity of (probable) active chemical constituent, you can directly select the respective solvent for isolation of ACC i.e. if you want to isolate phenol than you can use some polar solvent like alcohol and water.

But if you don't know the structure or polarity of your active chemical constituent, then you have to go for successive solvent treatment, i.e. starting from Liquid paraffin, Benzene or n-Hexane, CHCl3, Acetone, Methanol, Water. Then take TLC of each.

In whatever solvent you got single and desired spot, select that for further process of isolation.

Then to isolate the compound simply treat the extract with selected solvent, and at the end evaporate your solvent to get crystals of your desired constituent.

Hope this will help in your research work...

is it possible to isolate specific constituent from polyherbal extract

Dear Dr Tirgar,

Yes natural products is a little bit tough job on the basis of isolation of active constituents.But that's not a problem. First of all you need to identify your extracts for its phytochemical constituents what kind of compounds are there:

1.glycosides? 2. sterols? 3. alkaloids? 4. terpenes? or any other specialised class?

Depending on this you have to decide on the Fractionation/ purification steps how to go for it. A random fractionation of extracts highlights two simple ways: Liquid-Liquid partition (which we do normally in labs) or chromatographic fractionation (column chromatography what we do in labs). So every action of purification will be again guided by your TLC (Thin Layer Chromatography) what will tell you what is there in your extract and how it is behaving and separating.

Now depending on TLC and fractionation of your extracts (Here fractionation means separating your extracts into fractions from Non-polar-----> Polar) then you decide further purification by chromatographic techniques Column, VLC (Vacuum Liquid Chromatography), Prep-HPLC, Prep-TLC, HSCCC, Ion-exchange chromatography, Radial chromatography and so on. After purification and crystallisation of your active compounds you will go for characterisation studies: UV-Vis, FT-IR, NMR, HRMS and X-ray and then calculate the appropriate structures based on so many different studies like functional group information (IR), chromophore identification (UV-Vis), Carbon and proton skeletal framework (NMR), Molecular weight and fragmentation (MS studies) and systematically you will deduce a structure. For information i will advice you to consult more natural product isolation papers and also suggest one article of mine

:http://www.sciencedirect.com/science/article/pii/S0926669012006632

Dear Sravanthi,

Isolation can be achieved from polyherbal formulation but it will be rather difficult and would involve complex purification steps. Instead of, it would be better advisable first have a draft study on the several herbs involved in preparing polyherbal formulation. So accordingly isolate marker compounds from your extract of single herbs and then do your quantitative studies as we normally do in herbal drugs standardisation.Because you cant identify any new compounds from any polyherbal drug or even if you do you can not form a rationale to suggest which plant does it belong to? So, easy way is to achieve separation and isolation by above steps only.