I have mixture of two organic compounds one with molecular weight 226.41g/mol and other is 166.22 g/mol. Can I use size exclusion chromatography for the difference of 60 g/mol? Kindly do let me know how to separate.
That's rather small for size exclusion to have much of an effect. Silica gel or reverse phase chromatography would purify them, but there isn't enough information in the question to suggest a more detailed purification method.
If your compounds are volatile and thermally stable, try GC, if your compound has chromophore in structure then try HPLC UV, TLC for separation. Size exclusion chromatography technique is well suitable for higher molecular wt compounds such as a polymer, protein peptides etc.