Thermoresponsive hydrogels based on N-isopropylacrylamide, N-tertiary butylacrylamide (NTBA) and methacrylamide (MAAm) have been prepared by free radical copolymerization in water-dioxane mixture. Fourier transform infrared spectroscopy confirmed the presence of all monomers in the hydrogel system. Scanning electron microscopy revealed that the hydrogel having higher amount of NTBA had more smooth structure compared to others. Differential scanning calorimetry results showed that the free and interfacial water decreased but bound water increased with NTBA content in the hydrogel. Dynamic mechanical analysis results revealed increased storage modulus for the hydrogel having higher content of N-tertiarybutylacrylamide. Swelling studies of the hydrogels at different temperatures showed that all samples followed Fickian type of diffusion. The diffusion coefficient increases with increase in MAAm proportion in the hydrogel. The hydrogels were used in concentrating aqueous bovine serum albumin solution. The separation efficiency of hydrogels having higher content of MAAm were found up to 80 % at 5 degrees C but it decreased upon raising the temperature up to 30 degrees C.