I run a sample (containing lots of compounds) on hplc column to see my required product. Now I want to extract my pure compound peak from Column. I want to know the methodology. If some one has any idea, please let me know. I want to send it for NMR analysis afterwards.
Well, if you know the elution time of your specific peak (related to your required fraction) then just simply collect it from the HPLC line. If you don't know then you have to run a standard of your specific fraction and note the elution time and then collect the fraction by running your actual sample.
Hope it will help
Forgive me but this may be more questions than answers.
1. How do you know which peak on the chromatograph is your "required compound"? Does it have a particular absorbance?
2. Can you collect samples with your HPLC system? If so, simply collect the material from multiple injections always collecting the same fraction of the sample. You can do this with a preparative column but you may not have one. When I was in grad school I had to collect the active fraction of more than 100 separate injections on an analytical column.
3. You may be able to run large amounts of sample with thick TLC or an open chromatographic tube (gravity feed). This works well if your compound is colored or fluoresces. You can load a significant amount of material with either method.
4. Remember, that by either technique you WILL NOT HAVE a clean sample. Other compounds have probably coeluted but you do not see them because they do not absorb the appropriate wavelength or fluoresce. That means that you need to purify the compound before sending it for NMR analysis. Depending upon how much material you isolate and its tendency to crystallize, you may be able to recrystalize the material. Usually, the other material,being at lower concentrations, will stay in teh liquid when your sample crystalizes. (NOTE: Precipitation is NOT crystallization. Precipitation can collect many impurities during formation of the precipitate.). You can also consider using a different solvent system/packing material to generate an orthogonal separation. This will almost always get rid of the impurities but takes longer.
I hope that this helps.
Well, if you know the elution time of your specific peak (related to your required fraction) then just simply collect it from the HPLC line. If you don't know then you have to run a standard of your specific fraction and note the elution time and then collect the fraction by running your actual sample.
Hope it will help
You can calculate the time between when you peak elutes and when you can collect it by pi x tubing radius squared x tubing length/ volumetric flow rate (e.g. 1 ml/min).
I think you can play with the detector wavelength in such a way you get a specific discrete peak, provided you have a reference standard.
what type of detection are you using? If you are using a vw detector then you can monitor multiple wavelengths to ensure you have a pure compound. To collcte the sample when you see your compound come out of the detector collect it in a test tube and if the sample is stable dry it on a rotovap. Reconstitute the dried sample in mobile phase and reject. Repeat until you have enough for the NMR analysis.
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