Abstract. Simultaneous separation of ionic and neutral compounds in micellar electrokinetic capillary chromatography (MECC) is a consequence of a combination of charge/mass ratios, hydrophobicity and charge interactions at the surface of the micelles. The type of resolution can be chosen mainly by modification of the buffer composition and pH. Reproducibility depends on the capillary tubing characteristics, operating conditions, and temperature effects. We report here on the different buffer compositions, pH, temperature, operating conditions, and capillary tubing treatments for the optimal separation and best reproducibility of deoxynucleosides and deoxynucleotide-3'-monophosphates. The effects of different buffer compositions were tested when operating in a constant current mode at 38 pAmps. Lithium phosphate buffer improved the separation speed with little loss in resolution. The best concentration was found to be 20 mM. A concentration of 5 % acetonitrile substantially improved the efficiency of the nucleoside peaks and therefore their resolution. The optimum SDS concentration was found to be at 100 mM. The pH has a significant influence on retention time and resolution, and was adjusted for maximum resolution at pH 9.6. Capillary surface treatments were also investigated. Effective heat dissipation occurred below an operating voltage of 15 kV, and a temperature of 35°C was found to give a good compromise between efficiency, resolution, and time of analysis. Time of analysis can be decreased by using higher operating voltages without compromising the resolution. Efficiencies were 350,000 and 235,000 plates m-l for the nucleosides and nucleotides, respectively. The reproducibility of retention times was good with coefficients of variation below 1 %, which allows the possibility for automatic fraction collection.

Summary 2 Materials and MethodsThe use of micellar electrokinetic capillary chromatography (MECC) for the analysis of the major nucleobases, nucleosides, and nucleotides, and their chemically modified derivatives, has been developed and refined.The dimensions of the separating capillaries, the compos~t~on of the buffering systems, and the conditions used for electrophoresis were investigated in order to obtain the best performance. ~~~~~~bphosphodlesterase, micrococcal nuclease, bases, nucleosides, and nuc!eotldes standards were Purchased from Sigma Sodium dodecyl sulfate (SDS) and other chemicals (of HPLC grade) were purchased from Aldrich thymus DNA, Particular emphasis was placed on the identification of the physiological constituents of nucleic acids and their chemically modified analogs: in vitm studies on calf thymus DNA exposed to genotoxic agents have demonstrated that adducted bases and nucleolides can be identified by MECC.