Consider C18 HPLC on a 10 mm ID column using the same solvent system as your TLC plate, or use a focused gradient from 40 to 60% methanol in water to allow for differences between your TLC plate and your column.

thank you so much .. can i run another column instead .. not HPLC ?

What do you have available to you? I don't know how you got to this stage, since I read "another column".

You can try packing a C18 column, if you have loose material. Alternatively, try silica gel. you'll need to run silica TLC to develop a method. The compound should elute with hexane/ethyl acetate or dichloromethane/methanol, given it runs on C18 TLC with 1:1 methanol:water.

isn't c18 TLC is the same as reversed phase TLC ?

@Sherif, C18 TLC is a reversed phase TLC

Hi!

We can apply the small fraction of sample to the TLC and by using the same solvent the components can be separated and collected by scraping on minor scale.

This is not very authentic method,but in case of scarcity of sample amount one can apply it.

Good Luck.

@Sherif, C18 TLC is a subset of reverse phase TLC since diol, cyano, and amine TLC plates can exhibit reverse phase character as well depending on the polarity of the analyte and the concentration of water.

for example, here is a study I did for diol (in a column, but the principles hold for TLC too):  http://www.teledyneisco.com/liquidChromatography/Chromatography%20Documents/Application%20Notes/Chimeric%20Diol%20Column%20Behavior%20using%20Aqueous%20Solvents%20App%20Note.pdf#search=chimeric

thank you so much ..one of my colleagues asked me to use sephadex to purify this fraction