Rapid Separation of the Components of Nucleic Acids and Urine by High-Resolution Liquid Chromatography

Abstract

Abstract The improved separations of nucleic acid components obtained with two recently developed liquid chromatography systems are presented. An ion-exchange system operating at 3000 lb/in.2, developed for use with pellicular ion-exchange resins, separates the 2',3'-ribonucleotides of the four common bases in 55 min, the 5'-deoxynucleotides in 10 min, a mixture of four dinucleotides in 30 min, and a mixture of AMP, ADP, and ATP in 3.5 min. The difficult separation of the mono-, di-, and triphosphates of the four nucleosides requires 2.5 h with the pellicular anion-exchange resin. The four bases, or their nucleosides, are separated in less than 15 min with pellicular cation-exchange resin. The system has been modified to allow separation of more than 90 uv-absorbing constituents in human urine. A versatile, nonpulsating system, operated at 5000 lb/in.2, separates the ribonucleosides in less than 5 min on small-particle, conventional cation-exchange resin. Resins from three separate sources performed comparably, but parameters such as pH, temperature, and linear velocity must be optimized for each. Both systems are designed with a minimum of dead volume and use a sensitive uv photometer. The 0.02 absorbance unit full-scale sensitivity and 1-cm pathlength of the uv photometer allow analysis of picomole quantities of nucleic acid components.