Chromatographic separation was conducted on a Waters instrument comprising a 2695 separations
module capable of gradient elution and a 2475 multi-wavelength
fluorescence detector (FD). Fluorescence chromatogram was
obtained at excitation/emission wavelengths (λex/λem) of 300/450
nm. An Agilent TC-C8 chromatographic column (4.6 mm i.d. × 150 mm) packed with 5 µm octyl-bonded silica (C8) particles (130 Å pore size) was used for
separation of C-dots. 10 mg C-dots were dissolved in 10 mL methanol and the C-dots solution was pre-filtered through an Alltech 0.2 µm cellulose acetate membrane syringe filter before injections. The mobile phase containing MeOH and Milli-Q water was filtered through 0.45 µm cellulose acetate membrane filters (Alltech) prior to use. The injection volume was 10 µL and the
column temperature was maintained at 25 oC. A gradient elution programme was applied at a flow rate of 0.80 mL/min as follows: 45% v/v MeOH from 0.0 to 10 min, linearly increased to 50% v/v
MeOH from 10 to 20 min, then linearly increased to 80% v/v
MeOH from 20 to 50 min and finally linearly increased to 100%
v/v MeOH from 50 to 60 min. The selected HPLC fractions were collected manually based on the appropriate fluorescence signal threshold.