Sr. No. Parameter Initial condition Changed condition Effect 1. Mobile phase Tolune: ethyl acetate: formic acid (5:4:0.2) Tolune: ethyl acetate: formic acid (6:2:0.8) Rf increased slightly but no effect on resolution 2. Development Distance 8 cm 4 cm No effect on resolution 3. Temperature 25°C 40°C Rf of components increased 4. Tank Saturation Time 25 min 30 min Plate develops slowly 5. Extracting solvent Ethanol Petroleum ether Bad resolution

Table 4: Ruggedness and robustness studies.