Understanding the mechanistic pathways underlying carbon nanodot (CND) formation is essential for the rational design of synthesis strategies and the development of well-defined nanomaterials. In this work, we combine chromatographic isolation, spectroscopic characterization, and synthetic validation to identify key molecular intermediates in the early stages of CND formation from arginine (Arg) and ethylenediamine (EDA) under hydrothermal conditions. Using reverse-phase high-performance liquid chromatography, 1H/13C nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry, we isolate and structurally confirm multiple reaction intermediates, including species arising from Arg cyclization and EDA-derived condensation products. Each proposed intermediate was validated via direct synthesis and comparative analysis. Fully formed CNDs were then isolated and extensively characterized through several techniques. Multi-detection gel permeation chromatography enabled the determination of absolute number average molecular weight (Mn = 4,400 ± 458 g mol−1), dispersity (Đ = 1.34), and hydrodynamic diameter (2.8 ± 0.2 nm). Quantification of amine functionalities using Kaiser test and 19F NMR, upon condensation with a fluorinated aldehyde probe, established the presence of 6–7 amines per particle. Together, this systematic approach not only elucidates the mechanistic origin of CND formation but also establishes a robust framework for molecular-level CND design and functionalization.
