The analysis of nucleotides by capillary electrophoresis with sodium carbonate buffers

The analyses of nucleotides are important for a variety of biological samples, including blood, plasma, tissue and DNA and RNA hydrosylates. High performance liquid chromatography (HPLC) has been used in the past for nucleotide separations but is limited by the microliter volume sample requirement, resolution and the speed and high cost of the analyses. Capillary electrophoresis (CE) is a useful alternative to HPLC for the analysis of nucleotides. CE separations of nucleotides have been reported for both the CZE and MECC modes. Several different types of buffers, such as phosphate, borate and organic buffers, including CAPS have successfully been used to separate nucleotides. MECC separations have used SDS to provide a partitioning phase within the buffer for the simultaneous separation of nucleosides and nucleotides. Researchers have used both methods to separate nucleotides quickly and with plate counts above 100,000 plates/m. However, while CE separations are faster, more selective and less expensive than comparable HPLC methods, the mechanism of separation in CE analyses is not fully understood, thus method optimization is performed on a trial and error basis. In addition, the nanoliter size sample used in many CE analyses limits the sensitivity of most detection methods, including UV, fluorescence, and MS. In the present work, CZE methods for nucleotides, nucleosides and bases were optimized using bare capillaries and sodium bicarbonate/carbonate buffer systems. The parameters of concentration, voltage and pH were investigated to determine their effects on the separations. The data collected from these optimizations was used to correlate the experimental mobilities of the analytes to their ionic charge to mass ratios. A linear relationship would permit the prediction of the migration order of the analytes before analysis, thus minimizing the time and expense of method optimization. In addition to method optimization, the sample preconcentration method of whole capillary stacking was applied to nucleotides to lower the detection limits of the CE-UV technique from the micromolar range to nanomolar quantities. After optimization of both the stacking and separation methods with ribonucleotides, the technique was applied to a separation of carbovir triphosphate, a nucleoside analog with anti-HIV properties, from dATP and dGTP