Owing to its highly branched structure, bPEI is comprised of a mix of primary, secondary, and tertiary amines. Previous studies suggest an equal distribution of these amines in commercial bPEI products. Although it is often assumed that PEI amines primarily undergo conjugation reactions at primary amines due to less steric hindrance, secondary amines are more nucleophilic than primary amines. NMR is a powerful tool for characterization of the structure and properties of complex macromolecules. To determine where PEI modification occurred under our reaction conditions, we performed 2D-NMR experiments. The modified PEI polymers, acPEI and zPEI, were analyzed using 1 H- C heteronuclear single-quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) experiments. Representative HMBC spectra for zPEI20 and acPEI40 are shown in Figure , and additional HMBC and HSQC spectra are provided in the supporting information (Figure ). Figure shows two distinct peaks in the HMBC spectra corresponding to the 2°-amide and 3°-amide carbonyl carbon in both modified PEIs. For zPEI20, the HMBC spectra demonstrated three-bond 1 H- Taken together, the HMBC spectra indicate a distinct chemical environment attributable to succinyl and acetyl functionalities. HSQC spectra further validated the differences in chemical environments, evidenced by the attachment of three types of methylene groups (Figure ). The 2D-NMR experiments demonstrate a relative preference for secondary amine reactions of bPEI for both zPEI and acPEI. Especially at low percent modification, we observed a predilection for conjugation on secondary amines, forming tertiary amides. A significant reaction of anhydrides with primary amines to form secondary amides under our reaction conditions was noticeable only at higher degrees of modification.