Dear Dr! congratulations on purifying phytochemicals but there are some points about your question that appeared in my mind and I think they will help you! At first it is needed to know which phytochemicals group extraction was your purpose of research? second; which extraction process did you keep on? You know every extraction process based on the solvent you use will cover some phytochemical groups and exclude some groups! Third; based on what do you claim that you have purified a compound? At the end when you are sure about purity of a compound using spectroscopic technique can help structure elucidation! IR method can help predicting functional groups there are in a compound but golden method is NMR (1 & 2 dimentional methods) that should be in accordance of mass spectrum. It should be added that High Resolution Mass Spectroscopy (HR-EI-MS or HR-ESI-MS) is really helpful in this step. I would be glad to help you more and more based on on information you send me.

I think that you have check whether you have a single compound. The best way is not to check using TLC, but rather DOSY NMR technique. I hope so that you have heard for this NMR technique and that the pulse sequence is known to you (if not, please check publications of the inventor of this NMR technique and a big NMR expert, Dr Gareth Morris from University of Manchester, United Kingdom. If you are sure that you have a single compound in your sample, then you should monitor UV and IR spectra of your compound. Furthermore, you need to record 1D and 2D NMR spectra without exception. Also, high resolution mass spectra will be necessary.

I wish you good luck in your work. Hopefully, this will be helpful to you.

Thank to All.

I think that you have a mixture of compounds. The TLC technique is effective for separation of compounds that differ in the values of Rf.

1) Begin by analytical TLC plates coated by silica gel 0.25 mm. Assuming that the compounds are polar utilize development system of petroleum ether:ether:acetic acid 80:20:1. At the beginning of the plate will stay the polar compound or other polar compounds. You will visualize the spots in chamber with iodine vapors.

2) Follow preparative TLC. In plate having silica gel 1 mm thick apply 50 mg of the initial mixture from one end of the plate to the other and develop with CHCl3: MeOH: H2O 65:35:6. The compound or compounds would occur at different heights as stripes when placing in iodine chamber. Take the strips with the adsorbent and extract with a polar solvent.

3) You can repeat with many plates to receive large quantities.

4) If the compound or mixture of compounds are non polar they will move at different heights and you will pick them up.

5) Try to measure the specific rotation of all compounds in a polarimeter to determine if there are stereogenic centers.

6) Examine the separated compounds by IR and Raman spectroscopy and MS spectrometry.

7) Also you have performed all available NMR experiments.

Also, if you have experience in computational chemistry you can use the programs Avogadro (free) and Gaussian 09 in order to support the proposed structures. With Avogadro you can design the structures of the compounds you isolated and search all conformers of each compound.

Choose those that have less energy and save to a input file for Gaussian.

In this file, I propose to add in the route section the command:

# T B3LYP/6-311G (d) Opt Freq = Raman Test

After calculation you will receive the file.out which contains all frequencies. The calculated frequencies will be multiplied by the appropriate factor to correct them. The corrected theoretical values will be compared with those received in the experimental spectra. If the corrected frequencies do not differ more than 50 cm-1 the proposed structures will be accepted.

Keep also the file file.chk. This file is necessary for the theoretical calculation of chemical shifts of NMR in another calculation in the Gaussian program.

For Structural confirmation and all you can for First NMR both proton and Carbon, And NOE studies for structural studies and XRD studies like x ray difraction , If you want to know about the elemental composition go for EDX, For Groups IR Spec, Then For Mass and GC, Purification by HPLC, For all this you need the same compound at least 200 mg. But in some cases XRD studies need more compound...... Hopefully it helps you..

MASS by ESI if the molecular wt is more than 200, or if it is less than 200 go for EI