The rapid synthesis techniques and interesting multidisciplinary applications make carbon nanodots (CNDs) stand out from semiconductor quantum dots. Moreover, CNDs derived from green precursors have gained more importance beyond chemically derived CNDs due to sustainable synthesis opportunities. However, the presence of molecular impurities or intermediates or fluorophores was neglected during the entire process. Herein, we illustrate the sustainable synthesis of CNDs from Hemigraphis alternata plant leaves with extended carbonization procedure (3 and 9 min) along with simultaneous ethylene glycol and diethyl ether solvent treatment method for the successful removal of interfering fluorophores. To unravel the distinction between purified CNDs (P-CNDs) and organic fluorescent carbon nanostructures (org-FCNs), we carried out photophysical, structural, and morphological studies. A quantum yield (QY) of 69 and 42% was observed for crude org-FCNs, and crude P-CNDs; however after purification, QY of 1% and absence of one component from the fluorescent decays curve suggest the removal of fluorophores. Further, HR-TEM and DLS studies showed the quasi-spherical amorphous particles having < 10 nm particle size for P-CNDs. Besides, in vitro biocompatibility investigation and cellular uptake assay (1–100 μg/mL) against the MDA-MB 468 cell lines proves the ≥ 95% cell viability and good internalization for both org-FCNs and P-CNDs. Hence, our study shows the presence of fluorophore impurities in plant-derived CNDs, the removal and resemblance in biocompatibility properties. Hence, this information can be considered during the synthesis and isolation of CNDs. Simple and effective removal of impurities to harvest pure carbon nanodots (CNDs) through solvent-based selective separation method, and revelation of the cocktail flourphores similar to biocompatible blue fluorescent CNDs were studied.

Revelation of fluorophore impurities among biocompatible blue fluorescent carbon nanodots derived from Hemigraphis alternata plant and bioimaging
    Abstract
        Graphical abstract
    1 Introduction
    2 Experimental section
        2.1 Materials
        2.2 Instrumentation
        2.3 Synthesis of org-FCNs and P-CNDs, and purification
        2.4 In vitro biocompatibility (MTT) assay
        2.5 In vitro bioimaging studies
        2.6 Photoluminescence quantum yield calculation
        2.7 Bandgap calculation
    3 Result and discussion
        3.1 Synthesis and purification of carbon nanodots
            3.1.1 Photophysical and morphological properties of CNDs in solution
            3.1.2 X-ray photoelectron spectroscopy (XPS)
            3.1.3 HR-TEM and particle size analysis
        3.2 In vitro bioimaging and biocompatibility studies
    4 Conclusion
    Anchor 20
    Acknowledgements 
    References

• H. Manisha(Nanomaterial Research Laboratory (NMRL), Nano Division, Yenepoya Research Centre, Yenepoya (Deemed to Be University), Deralakatte, Mangalore 575 018, India)
• M. Velayudham(Department of Chemistry, Thiagarajar College of Engineering, Madurai 625 015, India)
• B. N. Kumara(Nanomaterial Research Laboratory (NMRL), Nano Division, Yenepoya Research Centre, Yenepoya (Deemed to Be University), Deralakatte, Mangalore 575 018, India)
• M. H. Naveen(Department of Chemistry, Pusan National University, Busan, South Korea)
• Yoon‑Bo Shim(Department of Chemistry, Pusan National University, Busan, South Korea)
• K. Sudhakara Prasad(Nanomaterial Research Laboratory (NMRL), Nano Division, Yenepoya Research Centre, Yenepoya (Deemed to Be University), Deralakatte, Mangalore 575 018, India, Centre for Nutrition Studies, Yenepoya (Deemed to Be University), Deralakatte, Mangalore 575 018, India)