You will have to choose the correct conditions to separate diastereomers on a TLC plate. It takes lots of trial and error to get the separation conditions right. On some of the larger macromolecules like peptides you will not be able to separate some of the diatereomers; keep in mind the smaller the molecule the larger the differences in the diastereomers and the easier their separation. What is the nature of the molecules you are working with? With this information I could suggest a solvent system to try based on my experiences. A literature search may help you find the right conditions for the resolution of your compounds.

Hi, I agree with the comments of Bruce above. I found some publications on TLC of diastereomers in TLC Camag Data base, maybe you can take a look, Best regards

http://www.camag.com/en/tlc_hptlc/customer_magazine_cbs/ccbs_database.cfm?method=query

If TLC is not working, you might want to consider other tools like HPLC.

Thank you Bruce Levison sir,

                I am working with a small oxindole molecules, having molecular weight below 500 da. Please suggest me a solvent system.  

I used methanol plus 1% formic acid to separate various oxidized tryptophan's (Oia, DiOia, NFK)  on silica gel TLC plates.

the smaller the molecule the larger the differences in the diastereomers and the easier their separation. What is the nature of the molecules you are working with

Thank you Bruce Levison sir,

                 Ok. I will use methanol plus 1% formic acid as a mobile phase for TLC. Please tell me a mobile phase for such separation through simple column chromatography. In simple column chromatography we can not use a methanol plus 1% formic acid as a mobile phase. Please suggest. Thank you.  

Thank you Teklit Gebregiorgis Ambaye sir,

                I am working with a small oxindole molecules, having molecular weight below 500 da. Please suggest me a solvent system. Molecule is of polar nature which shows a Rf value of 0.56 on 40 : 60 :: EtOAc : n-Hexane as a mobile phase on TLC. Please suggest me a method to for such separation through simple column chromatography. Thank you.

What do you mean by "simple column chromatography"? Please let me know the details stationary phase, column dimensions, detector, flow rate. I consider Flash chromatography to be simple column chromatography visit: http://pubs.acs.org/doi/abs/10.1021/jo00408a041, is that what you are referring to?  

Did the 1% formic acid in methanol work?  You should to try 15:85 :: EtOAc : n-Hexane as a mobile phase based on your response above.

Whether you get a single TLC spot for a mixture of two diastereomers or not depends on how differently the diastereomers interact with the stationary phase and the mobile phase. If you get two spots, you have two diastereomers. If you get one, you have to keep trying. One spot does not mean you have only one diastereomer. Predictions are difficult.