My sample dissolves only on chloroform and methanol but neither on pure chloroform nor pure methanol.... so, would like to know if i can subject it to sephadex Lh-20 using chloroform and methanol as solvent system,,, if so, how to do it?
Yes definitely. You can go for purification of your compounds using MeOH:Chloroform in Sephadex LH-20.
Start from chloroform to methanol by different polarity. Chcl3:MeOH (95:5), 80:20, 70:30, 50:50, 40:60, 20:80, 100%MeOH
gudluck
thank you @Robin.... so, packing has to be done with chloroform?? n while changing the polarity of the solvent how long do i have to wait to equilibrate the sephadex??
Pack and start your column with chloroform and increase polarity slowely. For equilibration it depends upon your compound of intrest. Dont forget to check TLC of each fraction from column. When you will run the column you will know automatically.
You can use methanol Chloroform(50:50) and pack with the same and run. For more
details see the instructions attached.
chloroform/methanol 2:1 has worked in many cases, you may need to try that.
Dear Lalrinzuali Khawlhring, It is not bad to have a glance to this point which using methanol and chloroform for sephadex column causes column to be packed not very well and this reduces column efficacy so you maybe need to repeat sephadex column running several times. But considering your solubility limitation there is no escaping way.
thank you all for ur answers, the thing is, TLC of my sample showed only two spots and i have tried many solvent systems to get good separation between these spots but to no avail, so i want try sephadex, but am doubting if i ill be able to separate them using chloroform and methanol since i dont have much experience with sephadex.
sephadex usually used for separation of glycosides, as sephadex separates molecule based on molecular weight and intial starting material should be two band mixture.
The sephadex should be loaded with methanol and mobile phase should be also methanol only.
no need to use chloroform.
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