I am trying to separate my active compound through HPLC. When I injected my samples, I get more than 20 peaks and I was collected all peaks into 10 different fractions. After doing bioassay, I have shown that all fractions was shown activity i.e means all fractions contained active compound. Is it possible?I dont understand. Why all fractions was shown activity--that means my compound not separated or others reasons. Please give me some suggestions.
A compound can not be in all fractions, especially when you have 10 .... would think that many compounds have similar activity, I recommend doing a test with thin layer chromatography under the same conditions.
That would give you an idea if you have the same copuesto in all fractions, which I think is very, very strange.
Again I say,, quite possibly you have many compounds with similar activity.
Dear Dr. Daniel Delgado,
Thank for your nice suggestions. I am also surprised to see my bioassay result. But is it possible about 20 compounds have similar activity? I dont know. Actually it is a growth promoting compound which promote the growth of uncultured bacteria in laboratory conditions and its origin is another bacteria. When I used equivalent concentration, all factions did not show similar actiivity but when I used 10 fold concentrations, all fractions shown similar activity. There is a possibility of concentrations effect but it is true, all factions have shown similar activity.
Dear Mohammad Bhuiyan, I have to make a thin layer chromatography can help. if the compound of interest, is in all the fractions, you get the same behavior on all plates, one (Rf) similar. The test is fast and need little sample.
And I agree with you, 20 compounds with the same activity ... highly unlikely.
I hope my comments can help.
Dear Mr. Arnau Serra-Cayuela, thank for your valuable suggestions. I think, my preparative column is ok because I always used guard column. I also run blank mobile phase, this is ok. Problem may be others. I concentrating my bioassay samples not HPLC injected samples. I used 10 fold higher conc. for bioassay rather than equivalent conc . I think, now you understand.
Dear Mr. Daniel, thank again. I am sure my compound is every faction. This is a great problem.
Same thing happened to me once, and surprise, the HPLC column was partially obstructed, I changed the column and everything returns to normal
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