Optimization for the separation of ribonucleotides by capillary electrophoresis at high pH

A versatile method for the rapid capillary electrophoresis (CE) separation of ribonucleotides using a carbonate buffer was optimized. The separation of twelve 5\u27-ribonucleotides was obtained in 25 min using a voltage of 18 kV and a 30 mM sodium carbonate/bicarbonate buffer at a pH of 10. For the separation of the monophosphates or the diphosphates, a lower buffer concentration and pH could be used. However, for the triphosphates a 50 mM buffer at pH 11 gave the best peak sharpness and resolution. Therefore, optimal conditions for the separation of the twelve ribonucleotides were a compromise between those best for the mono- and diphosphates and those needed for the triphosphate nucleotides. The sodium carbonate/bicarbonate buffer was inexpensive and easy to prepare. In addition, the high alkalinity of the buffer resulted in a shorter analysis time and the capillary equilibration time with NaOH was minimal compared to an acidic buffer. Since bare capillaries were used, the time and expense of preparing and maintaining coated capillaries was eliminated