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The role of hydrogen bonds to the micro-phase separation process of poly(polyamide 12-block-polytetrahydrofuran) alternating block copolymer upon cooling (170?15 °C) was investigated by in situ FTIR spectra combined with scaling moving-window two-dimensional correlation spectroscopy (scaling-MW2D) and the generalized 2D correlation analysis. The micro-phase separation process of polyamide elastomer was found to be composed of two processes via the combination of scaling-MW2D and differential scanning calorimetry (DSC). The first process (process A, 138?80 °C) is the initial process of the micro-phase separation of the polyamide elastomer, and the second process (process B, 80?35 °C) is the completion process of the micro-phase separation. The method of calculating the generation enthalpy of hydrogen bonds from in situ FTIR was also successfully established via Van''t Hoff analysis, and the above two processes were further confirmed through the enthalpy of hydrogen bonding. The generalized 2D correlation analysis was performed for these two processes. The results of 2D correlation analysis revealed that the impetus for the process A was the generation of the disordered hydrogen bonds between the C=O and N?H groups in the PA12 blocks, the PA12 crystallization induced by the formation of the ordered hydrogen bonds is the key step of the process B.