Dear Aparna

There is no a single standard universal method for this purpose. You have to opt a method most suitable for you. Extract the sample in different solvents (in increasing order of polarity) so that generally a group of compounds are isolated in each solvent based on their polarity. Then, screen each of them for the desired activity. A most active extract has to be then subjected to TLC fractionation followed by bioautography to detect the active band (may be pure or composite). If a well separated active band is available, then you can try separating that compound by preparative TLC. If not then preparative TLC followed by a column chromatography. Preparative HPLC will help in isolating a particular compound more accurately and also you can check the purity by that. Once again confirm the activity of that purified compound. If you get a compound more than 85% pure, then you can go for NMR (both H1 and C13: one dimensional and two dimensional) analsysis and LCMS analysis. Further purified compound can be used for IR analysis. IR spectra gives you the functional groups present in your copound. LC-MS gives either empirical or exact (HRMS) molecular weight of your compound and NMR spectra reveals the C and H environments. Based on these data you can predict the structure of your molecule. Another relative method is there. You can subject your active extract to LC-MS analysis and compare the MS pattern with a MS library. If the compound is known and reported you may get the structure immediately.

I just gave you the outline above. Actual process needs a lot of standardization and technical expertise, where you have to work upon. 

All the best

thank you for your suggestions. I will go through those papers.

Why not try a preparative TLC to obtain more quantities of the spot (including all the constituents indicated by your LC-MS).  This will then give you enough material to try various solvent systems on either TLC (prep) or even Column Chromatography (preferably Lobar CC).

You can use preaparatve chromatography (HPLC) Agilent and varian have this.

Also if you have access try preparatve LC-MS. Where you can separate mass based fractions. Try in chemistry or Biotechnolgy departments in Mysore Univ. probably thet have this.

Dear Aparna

There is no a single standard universal method for this purpose. You have to opt a method most suitable for you. Extract the sample in different solvents (in increasing order of polarity) so that generally a group of compounds are isolated in each solvent based on their polarity. Then, screen each of them for the desired activity. A most active extract has to be then subjected to TLC fractionation followed by bioautography to detect the active band (may be pure or composite). If a well separated active band is available, then you can try separating that compound by preparative TLC. If not then preparative TLC followed by a column chromatography. Preparative HPLC will help in isolating a particular compound more accurately and also you can check the purity by that. Once again confirm the activity of that purified compound. If you get a compound more than 85% pure, then you can go for NMR (both H1 and C13: one dimensional and two dimensional) analsysis and LCMS analysis. Further purified compound can be used for IR analysis. IR spectra gives you the functional groups present in your copound. LC-MS gives either empirical or exact (HRMS) molecular weight of your compound and NMR spectra reveals the C and H environments. Based on these data you can predict the structure of your molecule. Another relative method is there. You can subject your active extract to LC-MS analysis and compare the MS pattern with a MS library. If the compound is known and reported you may get the structure immediately.

I just gave you the outline above. Actual process needs a lot of standardization and technical expertise, where you have to work upon. 

All the best

thank you pradeepa.... the procedure you have given I have followed and standardized till colum chromatography. I have identified the compound now i only have to isolate it in pure form for the NMR and IR analysis and for doing final bioactivity test.

Dear Aprana

There are various chromatographic method for the isolation of  pure compound like VLC, Centrifugal Thin Layer Chromatography (CTLC), PTLC, CC and HPLC, the choice is your. I thing first you have to develop an analytical condition on analytical HPLC of your partially purified fractions and identify that your fractions contain how many compounds  and after that you have to upscale the condition on prep HPLC to isolate the pure compound.

Good Luck...   

Thank you anupam. I will do that

for get  purified compound from the plants extract

 used 

1- Silica gel

2- Sephadex 

3- Thin Layer Chromatography (CTLC)

4- HPLC

5- LC-MS.

6- HNMR

You should use 2 D TLC run second time scratch out single sopt from the TLC, leach it, and dissolve it in solvent put spots on TLC now change the polarity of solvent mixture by taking two low to high polar solvents now run TLC, you will find a single spot. Now use it for any analysis you will get  a single peak if your compound is not photosensitive. 

Dear Apana

You have a lot of advices and I think it would  be  very difficult for you to choose one of them.  I don't know the polarity of your mixture but I suggest you to run a TLC using dichloromethane : methanol  (1:1) and observe  and then change the relation of this mixture  if it necessary to be  less or more polar. After you find the best mixture try a preparative  TLC or run a CC . 

You need to start trying something and then make the necessary correction. Good luck.

The brief steps are:

Conduct preliminary analysis using laboratory chemical test methods. Then run TLC to know the type of fractions. After that Column Chromatography with HPLC, after isolated pure fraction run FT-IR, LC-MS, HNMR, CNMR for structure elucidation. You can read my papers too.

Dear Ms. Aparna

The attached information may be useful to you. You may also follow the thread:

Good luck.

https://www.researchgate.net/post/Can_someone_list_out_the_methods_of_extraction_crude_extract_compounds_isolation_purification_identification_and_structural_elucidation