Two compounds have been isolated from the same fraction of a plant. On TLC they showed to have different Rf.
For the physical characteristics, one of them is close to being crystal-like, while the other is more amorphous.
On the HPLC both of them gave the same peak and same Rf.
Are they two different compounds or one compound?
Is there an explanation?
First, I would need to know what is the stationary phase you are using for each method? The most common stationary phase for TLC is silica, but the most common stationary phase for HPLC is silica substituted with octadecyl (C18) chains. Of course, in that case the resolution is likely to be different, even if you are using the same solvent system!
@ Sally Burr thank you for your answer and your suggestion
you are right the is difference between TLC stationary phase and the HPLC column and they are as you mentioned.
but the question still how to know if they are two different compounds or one compound ?
100% different, i am also agree with Sally burr's comment. but in choreography several factors are there for effect on separation, but if you got two differnt RF in TLC then you have to tuneup some parameters on HPLC if you required more help in this case please feel free to ask me
@vipul Davariya
Yes, So what can I do On HPLC to confirm that they are two different compounds ?
I am running the two fractions in the same HPLC run. which means the HPLC conditions are fixed. but the difference is the order of the fractions tubes in the HPLC tray ?
do you have any suggestions ?
It seems both the compounds are different. There wont be any doubt after you characterize those fractions.
Well you can analyze both the fractions by Spectroscopy using IR, NMR or mass spectrometry.
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