I have confirmed a single spotted compound (having brown colour, so we suggest it may be flavonoid) on TLC, then used column chromatography to separate it, and it was further confirmed by TLC as single spot using acetone:ethyl acetate(1:2) as solvent system. Now the 1H-NMR is confirming some impurities, as the doublets, triplets etc are not of good standard (in terms of their ratio).
You did not mention how much is the difference in the ratio of integration. It is only very small then ignore it. Secondly, it is better to use the reverse phase TLC which will give you the better results. Lastly, you may run a HPLC for your compound which will give you the better separation or LC-MS or LC-NMR which one is available. But do think you will always get two peak in the NMR in the ratio of 1:2 (standard) but you may have 1:2.1 then ignore such a difference.
Yes I agree with previous comments.
One TLC's spot does not means that the compound is pure. For checking the purity of the spot you can run 2D TLC or use at least three different TLC conditions (different stationary and mobile phase).
If you have LC MS or LCqTOFHRMS, (nicer) you can first direct analyze your isolate by LCMS before NMR. By using QtOFHR MS you can have exact MW, molecule formula, MSMS; and using some online data base (Metlin, Metfrag, Massbank or others, the chemical structure can be predicted. For confirmation you need to analyze using NMR (1,2D-NMR), or authentic standard.
Previously mentioned processes are very much supportive. I just add some points.
Could you please use Cholester column for analytical HPLC purpose? It gives sharp difference in retention time if the sample contains impurities specially flavonoids and lignans.
Secondly, in case of flavonoids, Ring C which possess two dublets or double doublet in the ring, shows irregular integration. In that case you can minimize the integration ration. Could you please do C-NMR and check the ration of impurities?
Residual solvents? You did not mentioned in which region are the found impurity signals.
Try different solvent systems; you should download a copy of "TLC ATLAS". The book has good solvent systems for flavonoids. You should also look at residual solvents; solvents you used for running the column and possibly plastics (like Dioctylphthalate). You could also try gel filtration for better results.
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