Separation of ribonucleotides by capillary electrophoresis with multifunctional electrophoretic media of phosphate ammonium salts

Abstract

Baseline separation of all common ribonucleotides by capillary electrophoresis (CE) was accomplished with bare fused-silica capillaries of 30 cm effective length and multifunctional electrophoretic media of simple composition, i.e., only phosphate ammonium salts, of moderate pH values in less than 10 min. A separation efficiency of 8·105/m in terms of theoretical plate number and a resolution between all adjacent peaks greater than 2 were obtained in capillaries of 75.0 cm effective length. Buffers containing phosphate anions resulted in good peak shape in the CE separation of the ribonucleotides. Among the phosphate buffers with different cations, potassium phosphate buffer showed better resolution than sodium phosphate buffer, but ammonium phosphate buffer gave the best resolution for the ribonucleotides. Inorganic ammonium cations affected the mobilities of ribonucleotides and enhanced their resolution during separation by ionic interaction and hydrogen-bending. The migration behavior of the ribonucleotides was examined over the pH range from 5.5 to 9.0. In addition, the effects of organic solvents, such as acetonitrile, on the migration order of the ribonucleotides were studied. The CE method developed has proven to be a fast and simple means for the separation of the ribonucleotides with high efficiency and resolution. The results obtained demonstrated that buffers of phosphate ammonium salts have unique characteristics and may be excellent electrophoretic media for the separation of the ribonucleotides and other biochemical compounds by CE.