Coil-globule transition of poly(N-tert-butylacrylamide-co-acrylamide) P(NTBAM-co-AM) copolymers is investigated in the aqueous solution and in the mixture of water-methanol by micro ATR-FTIR spectroscopy technique. In this study the microstructure and its changes in the hydration states of the distinct groups of these copolymers are investigated by micro ATR/FTIR technique. The results showed that by heating the solution above the LCST hydrogen bonding between C=O and water was decreased but the hydrogen bonding between polymeric chains increased, which prove the aggregation of polymer chain during phase separation. The chemical shifts of IR bands are also studied in the mixture of water-methanol. (C) 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48:356-363, 2010