The development of robust and reproducible synthetic strategies for the production of carbon dots (CDs) with improved fluorescence quantum yields and distinct emission profiles is of great relevance given the vast range of applications of CDs. The fundamental understanding at a molecular level of their formation mechanism, chemical structure and how these parameters are correlated to their photoluminescence (PL) properties is thus essential. In this study, we describe the synthesis and structural characterization of a range of CDs with distinct physico-chemical properties. The materials were prepared under three minutes of microwave irradiation using the same common starting materials (D-glucosamine hydrochloride 1 and ethylenediamine 2) but modifying the stoichiometry of the reagents. We show that small variation in reaction conditions leads to changes in the fluorescent behaviour of the CDs, especially in the selective enhancement of overlapped fluorescence bands. Structural analysis of the different CD samples suggested different reaction pathways during the CD formation and surface passivation, with the latter step being key to the observed differences. Moreover, we demonstrate that these materials have distinct reversible response to pH changes, which we can be attribute to different behaviour towards protonation/deprotonation events of distinct emission domains present within each nanomaterial. Our results highlight the importance of understanding the reaction pathways that lead to the formation of this carbon-based nanomaterials and how this can be exploited to develop tailored materials towards specific applications.