The compound is polyphenol from plant extract. I hv collect the fraction, which hv the same retention time with the standard.when I run nmr, the spectrum shows a mixed of strange compound. What is possible reason for this?
Plant extracts are generally too complicated for one-dimensional HPLC and your problem will be co-elution with other compounds. What you should do is re-inject the collected fraction using another HPLC column that has a different retention mechanism (possibly a cyano if you have used a C18). This is called two-dimensional HPLC and has the capacity to resolve many more peaks than 1D-HPLC. Run your standard on the second column, collect your new fraction, and let us know how it goes.
you need to be more precise !
what strange signal do you see in NMR ?
if it look strange anyway, it should mean that you have coelution of compounds that lead to an enrichment and not a purification of the polyphenol.
Plant extracts are generally too complicated for one-dimensional HPLC and your problem will be co-elution with other compounds. What you should do is re-inject the collected fraction using another HPLC column that has a different retention mechanism (possibly a cyano if you have used a C18). This is called two-dimensional HPLC and has the capacity to resolve many more peaks than 1D-HPLC. Run your standard on the second column, collect your new fraction, and let us know how it goes.
The faction apparently having the same Rt can be mix of more than one compound co-eluted. You have not mentioned in detail about the nmr pattern of the fraction and the strange signal. However, you may attempt hplc separation by changing the operating conditions like column, solvent- system, elution time, etc. Also, you can re-inject the collected fraction using another HPLC column having different retention mechanism. 2D-HPLC will also be helpful in solving your problem as suggested by Stevenson.
If u have enough sample (5-10 gm) u can get the pure compound u can use seperation by column chromatography, u have to set up column by using suitable silica gel, apply the sample and elute with non polar solvent (hexane) follow by semi polar and polar solvent. The fraction u can compare with standard by using TLC and/or HPLC.Thank u.
Please check whether compound have fluorescence property or not. If yes then go for preparative chromatography by TLC.
Can you modify the method? For instance, you can make changes on the ingredient of mobile phase to see whether the peak are splitted to two peaks.
It seems you have the standard already for comparison. You can do HPTLC and compare the Rf with the standard compound. It will also resolve if other compounds are present, they will have a different Rf than your standard. You can measure quantitatively and also confirm the presence of standard in your sample by using UV overlain spectra generated by the instrument instantly. If they are the same they will overlap each other.
Hi
I think the problem is that compounds with the same retention time as standard. The best way use to change the solvent system and flow rate and see whether it helps. For NMR u should have pure compound mixture of compounds gives confusing pattern. U should separate those mixed components and analyse individually in NMR.
If you are doing Preperative HPLC the fraction with same RT as Standard can be collected and analysed in HPTLC using different solvent system to see if the fraction is mixture. I hope this will help for your further plan of work. all the best.
An extract with numerous components with possibly same or nearby same retention time is the cause for the problem. I also encountered the same problem. to solve the case you ned to do further fractionation and seperation may be u can use column chromatography to seperate and then run the sample on HPLC using 1D column. Use C-18 column.
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