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-bonding. 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.
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ABSTRACT: The CE separation of twelve nucleotides (5′-mono-, di-, triphosphates of adenosine, guanosine, cytidine and uridine) was improved by adding cadmium ion to the ammonium citrate/citric acid buffer (pH 5, ionic strength 100 mM). Cadmium ion acts as a complexing agent for some nucleotides (ATP, CTP, GTP, UTP, GDP). In order to accelerate the separation, the electroosmotic flow was reversed by flushing the fused-silica capillary with 0.2 % aqueous solution of the polycationic surfactant hexadimethrine bromide. A good separation of the twelve nucleotides studied was then achieved on a dynamically coated capillary in less than 5 min by using an ammonium citrate/citric acid buffer (pH 5, ionic strength 100 mM) to which 2 mM cadmium ion has been added. High peak efficiencies were obtained (210 000 theoretical plates) and the resolution between two adjacent peaks was always greater than 1.5.Chromatographia 04/1999; 49(7):379-384. DOI:10.1007/BF02467610 · 1.37 Impact Factor
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ABSTRACT: An open-tubular wall-coated macrocyclic polyamine capillary column (70 cm x 75 microm ID) with 50 cm effective length for the separation of nucleoside monophosphates is described. Some parameters with respect to concentration, pH, composition of the buffer, and voltage in order to optimize the separation were studied. The coated capillary showed reversed electroosmotic flow (EOF), allowing anions to be separated in the co-EOF mode. Baseline separations were achieved for the eight nucleotides in less than 26 min using a background electrolyte consisting of H(3)PO(4)-NaH(2)PO(4) (30 mM, pH 3.10), an applied voltage of -15 kV, and detection at 254 nm. The macrocyclic polyamine on the capillary wall introduced anion coordination for the interaction with the analytes, the strength of which could be moderated by the type and concentration of the competing ion used in the background electrolyte (BGE). With a low concentration of the competing ion (phosphate ion), the migration behavior followed that obtained in the electrophoretic system. Increasing the concentration of the competing ion resulted in a faster migration and more complete elution of the analyte. The method established was also employed for the analysis of nucleotides in mushrooms. Aqueous extracts of mushrooms from different species and various extraction methods were injected directly for the analysis. Uridine 5'-monophosphate, guanosine 5'-monophosphate, adenosine 5'-monophosphate, and cytidine 5'-monophosphate, were found in the sample tested.Electrophoresis 05/2002; 23(9):1230-8. DOI:10.1002/1522-2683(200205)23:9<1230::AID-ELPS1230>3.0.CO;2-D · 3.16 Impact Factor