Determination of Pyrimidine and Purine Bases by Reversed-Phase Capillary Liquid Chromatography with At-Line Surface-Enhanced Raman Spectroscopic Detection Employing a Novel SERS Substrate Based on ZnS/CdSe Silver-Quantum Dots

We have developed a new SERS substrate based on the reduction of silver nitrate in the presence of ZnS-capped CdSe quantum dots. This substrate showed higher sensitivities as compared to a hydroxylamine-reduced silver sol. On the basis of this new substrate, at-line SERS detection was coupled with capillary liquid chromatography (cap-LC) for the separation and selective determination of pyrirnidine and purine bases. For this purpose, wells of a dedicated microtiter plate were loaded with 20 mu L of the SERS substrate and placed on an automated x,y translation stage. A flow-through microdispenser capable of ejecting 50 pL droplets, at a frequency 100 Hz, was used as the interface to connect the cap-LC system to the wells loaded with SERS substrate. A detailed study of the dependence of both the separation and the surface-enhanced Raman spectra of each base on the pH was performed to optimize the system for maximum sensitivity and selectivity. Highly satisfactory analytical figures of merit were obtained for the six investigated bases (cytosine, xanthine, hypoxanthine, guanine, thymine, and adenine) with detection limits ranging between 0.2 and 0.3 ng injected on the capillary LC column, and the precisions were in the range of 3.0-6.3%.