Retention modification of nucleic acid constituents in reversed-phase high-performance liquid chromatography

✍ Scribed by R.S. Ramsey; V.W. Chan; B.M. Dittmar; K.H. Row

• Publisher: Elsevier Science
• Year: 1989
• Tongue: English
• Weight: 815 KB
• Volume: 468
• Category: Article
• ISSN: 1873-3778
• DOI: 10.1016/s0021-9673(00)96314-3

✦ Synopsis

Secondary equilibria in reversed-phase liquid chromatography have been investigated as a means of enhancing selectivity and optimizing separations of nucleic acid constituents. The retention behavior of various nucleotides, nucleosides and modified compounds has been examined as a function of live different metal ion additives in the mobile phase: K+, Mg2+ Mn2+, Ni2+ and Zn'+. Complexation ofthe solute molecules with the metal ions 'changes the electronic structure and alters solute-solvent interactions. Alkali and alkaline earth metals bind primarily to phosphate groups while transition metals also interact with the N' of purine bases. All nucleotides were found to be eluted very close to the void volume of the highperformance liquid chromatographic column without any metal additive, but retention increased as the concentration of a given cation increased. The transition metals were found to have the greatest effect, with affinities for nucleotide monophosphates on the order of 100 times greater than potassium, and 10 times that of magnesium. Differences in affinity based upon phosphate structure (i.e., cyclic W. linear), phosphate position (e.g., 2'-W. 3'-monophosphates), and base modification were also noted. The retention of most nucleosides, unlike the charged compounds, remained relatively constant as the ionic strength or type of cation was varied. Also, improvements were obtained in the resolution of some oligonucleotides with the addition of divalent ions to a potassium buffer mobile phase.