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In order to understand the structure of asphaltene after its disaggregating by chemical processing, a comprehensive study for exploring the structural changes of asphaltene after acylation as a case of chemical processing was conducted. Functional groups, crystal parameters, hydrogen types, and micromorphology of acylated asphaltenes were analyzed by FT-IR, XPS, XRD, 1H NMR, SEM and IFM (inverted fluorescence microscope) methods. Additionally, fluorescence spectroscopy methods were performed to analyze the effect of acylation on the aggregation ability of asphaltene. Experimental results indicated that the C=O double bond was enhanced and the content of O–C=O was increased on the surface of acylated asphaltene. The number of stacking layers and the aggregate size of asphaltene decreased obviously after acylation. Fluorescence spectral analysis showed that the critical aggregation concentration of acylated asphaltenes increased compared to the raw asphaltenes. These results indicate that acylation reaction can disaggregate asphaltenes and hinder their re-aggregation in a solution. The disaggregation effect of asphaltene acylation can be attributed to the weakening of a hydrogen bonding and enhancement of a steric hindrance in the asphaltene molecule. This study provided the further understanding of the structural changes of asphaltene after the chemical treatment. [ABSTRACT FROM AUTHOR]