Separation of isomeric and nonisomeric monoribonucleotides by isocratic ion pair high performance liquid chromatography

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Abstract
A simple, isocratic, high performance liquid chromatographic procedure is described for the first time for the separation of nine monoribonucleotides using the ion‐pairing technique. An aqueous mobile phase containing 100 mM KH2PO4 and 12.5 mM tetramethylammonium hydroxide as the solvophobic ion, pH 3.9, was used with a reverse phase RP‐18 column. The nine monoribonucleotides studied were separated and eluted in the following order: cytidine‐5′ ‐phosphate, uridine‐5′ ‐phosphate, cytidine‐3′ ‐phosphate, guanosine‐5′ ‐phosphate, uridine‐3′ ‐phosphate, uridine‐2′ ‐phosphate, adenosine‐5′ ‐phosphate, guanosine‐3′ ‐phosphate, and adenosine‐3′ ‐phosphate. Generally the 5′ nucleotides eluted faster than the 3′ and the order of elution within each series was: cytidine, uridine, guanosine, and adenosine. The only nucleotide where three isomers were studied was uridine, and the 2′ eluted later than the 3′. Baseline separation was attained for a mixture containing four 3′ nucleotides and uridine‐2′ ‐phosphate. When the four 5′ nucleotides were chromatographed, baseline separation was also obtained except between cytidine‐5′ ‐phosphate and uridine‐5′ ‐phosphate. The coefficient of variation of the retention characteristics, which reflected day‐to‐day variation, averaged 6.4%.