High-performance size exclusion chromatography with online fluorescence and multi-wavelength absorbance detection for isolation of high-purity carbon dots fractions, free of non-fluorescent material

•Synthesis of CDs mixture by oxidation of natural nanographite.•Obtaining of CDs fraction by ultrafiltration/dialysis procedures.•Absorbance and EEM fluorescence analyses of CDs mixture and its fraction.•HPSEC with online fluorescence and absorbance detections of CDs mixtures.•HPSEC revealed some fluorescent and non-fluorescent CDs fractions. The carbon dots (CDs) from natural nanographite oxide mixture (NGO-MIX) and from its fraction NGO (3.5-10K) recovered after ultrafiltration and dialysis were analyzed by 3D-excitation/emission matrix and high-performance size exclusion chromatography (HPSEC) combined with online fluorescence and absorbance detections. HPSEC chromatograms obtained simultaneously with absorption within the wavelength range 200-500 nm and fluorescence detection at λexc/λem = 270/450 nm/nm showed that NGO-MIX sample is not homogeneous and consist of well resolved CDs fractions with different sizes, absorption spectra and distinct fluorescence and non-fluorescence properties. Despite the twice higher fluorescence intensity of fraction NGO (3.5-10K) compared to the NGO-MIX, some impurity of non-fluorescent components was detected by HPSEC. The absorbance spectra of chromatographic peaks, extracted from the data of multi-wavelength absorbance detector, demonstrated different combinations of absorbance maxima. It means that different chromatographic peaks correspond to sized and chemically different CDs fractions. This study demonstrated for the first time the possibility of separating oxidized nanographite into homogeneous free from non-fluorescent material CDs fractions with their simultaneous spectroscopic characterization.