Carbon dots (CDs) are an astonishing class of fluorescent materials with many applications in bioimaging, drug delivery, photovoltaics and photocatalysis due to their outstanding luminescence properties and low toxicity. However, the internal CD structure of bottom-up synthesized CDs is still the subject of considerable debate. Unambiguous analysis of the internal CD composition is hampered by the fact that reaction products usually contain mixtures of several CD fractions as well as molecular intermediate and side products. Therefore, purification and careful separation of the various CD fractions is vital for structural analysis and isolation of pure CDs possessing optimized optical properties. In this study, CD solutions were synthesized from citric acid and cysteine via a one-pot hydrothermal synthesis. A simple column chromatography unit was used to systematically study the influence of the molar precursor ratios and synthesis conditions (temperature, reaction time) on the CD solution composition. By investigating the structural and optical properties of the chromatographically separated fractions, three different fluorescent species could be identified. Freely floating molecular fluorophores left the column first, followed by highly fluorescent CDs with fluorophores bound to the carbon core, finally followed by low-fluorescent carbon particles without fluorophores. We demonstrate that the CD solution composition and the internal structure of the individual fluorescent components can be clarified via chromatographic separation. This information can be further applied to isolate pure CDs with optimized optical properties.