It is very simple always run TLC in a solvent mixtures showing fine polarity adjustment. Run 2 D TLC on a glass plate 12X 20 in size from one side. Try to collect the spot dissolve it and again apply the same sample to coat over the plate in single spot now change the solvent admixture  slightly, run the sample again, you will get a single fine spot and  a single molecule. Take its absorption spectra on UV and IR you will find a single no overlapping  spectra of the compound. Second method is go for affinity chromatography and change the terminal group and generate  linker to add at the terminal position, coat the same material on column substrate, by changing pH elute your desirable compound more precisely a single one from collection tube. 

Dear Muthamil Iniyan,

TLC is older technique, takes more time and some prediction problem, solvent system confusion is increases. so that you can use HPLC-LC-MS from that you can able to separate 100%clear expected target from mixture.

Very simple to isolate peaks from HPLC and detect a compound by LC-MS. 

Whatever sample of mixture you have, just use low concentration in Analytical HPLC and see how much peaks appear and each peak in LC-MS it shows the M+ from that you can avoid unwanted peaks and collect exactly whatever you want. it's very simple.

Good Luck & Regards

R.Selva........

Dear Selva,

Thanks for your suggestion. In HPLC, the problem is the peak carry over of our natural products. To set up a good mobile phase in analytical HPLC itself taking more than 20-30 runs to separate each peak. So that we are purifying it by TLC and CC before HPLC step. So I think prefractionation is needed to isolate a mixture before HPLC and LC-MS. Even in LC-MS, there is a confusion that which is the M+ ion peak.

your problem is very common who have done research with  natural products.

for isolation, i have several suggestions that might be helpful to you.

1) you have to 1st developed TLC from the fraction, and identified the targeted compound,  isolate the minute quantity of compound from TLC plates and identifies their Mass pattern in LC-MS/MS.

2) if you can go with semiprepartive LC also to purify your compounds.... now a days  semipreps. are available which is directly convert your analytical method to prep. method which was injected for mass.

best luck...