Ramsey R S, Chan V W, Dittmar B M, Row K H
Oak Ridge National Laboratory, Analytical Chemistry Division, TN 37831-6120.
J Chromatogr. 1989 May 12;468:167-79. doi: 10.1016/s0021-9673(00)96314-3.
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 five different metal ion additives in the mobile phase: K+, Mg2+, Mn2+, Ni2+ and Zn2+. Complexation of the 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 N7 of purine bases. All nucleotides were found to be eluted very close to the void volume of the high-performance 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 vs. linear), phosphate position (e.g., 2'- vs. 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.
反相液相色谱中的二级平衡已被研究，作为增强选择性和优化核酸成分分离的一种手段。研究了各种核苷酸、核苷和修饰化合物在流动相中作为五种不同金属离子添加剂（K⁺、Mg²⁺、Mn²⁺、Ni²⁺和Zn²⁺）的函数的保留行为。溶质分子与金属离子的络合改变了电子结构并改变了溶质 - 溶剂相互作用。碱金属和碱土金属主要与磷酸基团结合，而过渡金属也与嘌呤碱的N7相互作用。发现所有核苷酸在没有任何金属添加剂的情况下都在非常接近高效液相色谱柱的空体积处洗脱，但随着给定阳离子浓度的增加，保留增加。发现过渡金属的影响最大，对核苷酸单磷酸的亲和力比钾大100倍左右，是镁的10倍。还注意到基于磷酸结构（即环状与线性）、磷酸位置（例如；2'-与3'-单磷酸）和碱基修饰的亲和力差异。与带电化合物不同，大多数核苷的保留随着离子强度或阳离子类型的变化而保持相对恒定。此外，在钾缓冲流动相中添加二价离子后，一些寡核苷酸的分离度得到了改善。
