In this study, we have employed carbon dots (C-dots) derived from citric acid as a model system to demonstrate the use of high performance liquid chromatography (HPLC) in evaluating the dialysis time required for carbon dots. The time evolution of the HPLC chromatograms indicates that about ∼120 hours are required to completely remove the small molecular byproducts of C-dots from the solution. Our study clearly demonstrated that in many C-dot-related studies, the C-dots were dialyzed inadequately. Furthermore, HPLC analysis shows that at least three C-dots coexist in the dialyzed solution. These C-dots exhibit similar optical properties and size distributions, but they have different surface functional groups, Hg²⁺ ion sensitivities and photoreduction capabilities. These findings suggest that more attention should be paid to the purification of C-dots, and it is important to establish a standard protocol to purify and characterize C-dots.