The separation by column chromatography methods depend on the nature of your crude extract. You should first tested with TLC to know the no. of ingredients from the no of spots. then after that you can select the best solvent of the mobile phase for separation by column.therefore the ratio between your solvents determined by Rf of TLC. Try to start from non polar to polar gradually .
THanks alaa but in Knew that this extract contain five active substances so is important to do TLC
Dear Fatima :as you know the good separation between components depends on different things one of them is the mobile phase ratio. if you have a good spaces between your spots then you can separate your crude by CC .please see this link;
http://www.chemistryviews.org/details/education/2345141/Tips_and_Tricks_for_the_Lab_Column_Troubleshooting_and_Alternatives.html
I agree with Alaa, I think the best way to do CC, is by do separation using TLC (with same stationary phase); by TLC you can optimize the separation of your compounds by modification the polarity of your mobile phase. After you get best separation by TLC than you can apply to your CC.
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