The Effects of Stationary Phases on Retention and Selectivity of Oligonucleotides in IP-RP-HPLC

There is a growing demand for the separation and identification of short nucleic acid fragments, such as oligonucleotides. There were two main goals of the present investigation, namely, evaluation of the impact of stationary phase type and the influence of various ion-pair reagents on the retention behavior of oligonucleotides in ion-pair liquid chromatography. Three types of ion-pair reagents were studied: triethylammonium acetate, dimethylbuthylammonium acetate and mixtures of 1,1,1,3,3,3-hexafluoro-2-propanol and triethylamine. Two novel types of packing materials, namely, cholesterol and alkylamide were used for this purpose for the first time. The results indicate that the mechanism of oligonucleotides retention is determined by the hydrophobicity of ion-pair reagents and polar ligands localized on the surface of stationary phases. Oligonucleotides were most effectively separated with the use of alkylamide and cholesterol packings. These two stationary phases reduce the time of analysis in comparison with the octadecyl packing material. Moreover, separation was achieved under non-denaturating conditions.