Separation of 5′-ribonucleoside monophosphates by ion-pair reverse-phase high-performance liquid chromatography

The isocratic separation of six naturally occurring 5′-ribonucleoside monophosphates by reverse-phase high-performance liquid chromatography has been achieved using solvents containing tetramethylammonium, tetraethylammonium, or tetrabutylammonium ions. Their effectiveness in resolving the nucleoside monophosphates increases with increasing akyl chain length (butyl > ethyl > methyl). In an acidic aqueous solvent containing methanol, these ion-pair reagents effect discrete separations of all the nucleoside monophosphates. The systems described also provide for virtually quantitative recovery of the separated nucleotides and utilize phosphate-free solvents comprised of volatile components easily removed by evaporation.