Hello Shaista, 

This can be a VERY big question. Many laboratories in the botanical field implement what is called "Bioactivity Guided Fractionation".

The synopsis is this: 

• Start with a bioactive extract (Usually a hit in a high throughput screen)
• Fractionate an extract
• Test the resulting fractions in the bioassay 
• Fractionate the active fractions. 
• Repeat until a pure compound is isolated. 

However, this causes issues with supply. If you are looking for a new product, it can be very difficult to assign bioactivity to a peak in a chromatogram. There are multiple technologies that are being developed and improved, but are highly dependent on the facilities that you have to work with. 

As far as extraction, we used the following, as reported in a PLOS ONE article from 2015: 

"Dried plant material was macerated for at least 24 hr, and the methanol extract was subsequently separated from the plant material. This extract was dried in a rotary evaporator to reduce the volume of methanol, and partitioned against an equal volume of hexane. The resulting mixture was stirred for at least one hr and the layers were collected separately using a separatory funnel. The methanol partition was added to water and chloroform in a ratio of 1:5:4, stirred for at least 1 hr, then separated. The chloroform partition from this step was evaporated and used as the starting material for all experiments "

As far as isolation, this may depend on your PI's approach as well. I would say that with my experience with botanical extracts, it might be necessary to run both normal and reverse phase chromatography. The normal phase will help to get rid of the waxes and non-polar molecules, as they tend to not be the bioactive constituents.

If you are only interested in isolating the major constituent of your sample, perhaps a few HPLC gradient experiments will help. However, keep in mind that in Natural Products, what is active is not always what is most abundant, and what is most abundant, is most likely not active.

As far as Characterization: 

In order to be published in most journals, you will need HRMS and NMR profiles of the component. However, keep in mind that it may already be known. The Dictionary of Natural Products is a good database that you can search once you have a molecular formula of your bioactive constituent. It will be able to provide you with citations for the NMR profiles so you can compare against your own. 

What kind of compound are you interested in purifying and the purpose? NB: We have more than 25,000 phytochemicals and some of them have already established standard procedures for extraction and purification. For research purpose, some articles in my profile can help.

https://scholar.google.com/citations?user=hpPJbPcAAAAJ&hl=en

For isolation, you can use different methods like Hot extraction or cold extraction. 

For purification, you can use different chromatographic techniques i.e column, Preparative TLC, reverse phase separation.  

For characterisation of bioactive, you can use Mass, 1H-NMR, 13C-NMR and Elemental analysis.

As Joseph said, it is a multi step research methodology : collection of the plant material, shade drying of the plant material, grinding,  extraction of the bioactive principle, bioassay to test on certain pest. Also rearing the targeted pest. The isolation is another story.

Please see the following link:

http://www.phytojournal.com/archives/2017/vol6issue1/PartA/6-1-23-924.pdf

After obtaining yourvextract, the first question to answer is : What bioactivity are you interested in? You may begin with different assays on your extract and focus eventually on the most promising one. Afterwards, a solvent /solvent partitioning, column chromatography, preparative thin layer chromatography or HPLC, all guided by the chosen bioassay should follow. The article: Sweet potato peels and cancer prevention might be useful. Best wishes!

Please go through the following links

http://www.sciencedirect.com/science/article/pii/S0021967316307531?via%3Dihub

http://www.sciencedirect.com/science/article/pii/S1570023204006476

http://www.sciencedirect.com/science/article/pii/S1570023204007196

http://www.sciencedirect.com/science/article/pii/S0731708513001179?via%3Dihub

Hello Sir,

Kindly check the attached files

Thanks

There are some classes of Bioactive natural products such as flavonoids, alkaloids, etc. and each class has different properties. So it Will need different type of purification techniques.

First of all do thorough literature search to find out what is reported from that particular genus and then do some bio-assay directed isolation work. Mr. Amit has given sufficient literature to follow up. 

Hai mam

Thanks for your valuable information mam. I will surely go the literature of Mr.Smith.

From

Shaista jabeen

Thank you so much to you all for giving me such an elaborate information on this particular question.i will surely go through those links and search my requirements.

By using the HPLC, TLC, column,

Column chromatographic techniques can be used for the isolation and purification of the bioactive compounds. Developed instruments such as High Pressure Liquid Chromatography (HPLC) accelerate the process of purification of the bioactive molecule. Different varieties of spectroscopic techniques like UV-visible, Infrared (IR), Nuclear Magnetic Resonance (NMR), and mass spectroscopy can identify the purified compounds