Analysis of 3′, 5′ Reversed-Sequence Oligonucleotide Isomers by Reversed-Phase Ion-Pair Chromatography

3′, 5′ Reversed-sequence oligonucleotides are isomers with the same length and base composition, except with reversed base sequence. In the current work, 6 oligonucleotide model samples were designed to study chromatographic behaviour of 3′, 5′ reversed-sequence isomers by optimizing effects on retention and separation. The retention factor and resolution of isomers were investigated under triethylamine-acetic acid (TEAA) concentrations of 0.025–0.15 mol/L, pH values of 5.0–6.8, temperatures of 25–45°C and flow rates of 0.3–0.7 mL/min by reversed-phase ion-pair chromatography (RP-IPC). The best resolution was observed under TEAA concentration of 0.05 mol/L, pH 6.8 and flow rate of 0.4 mL/min. While the effect of temperature on the separation was not apparent, effect of initial organic strength was stronger than that of the elution gradient. The retention and separation trends of the model samples were different, and weak retention of the samples on the solid phase contributed to good separation. It is concluded that 5′ end of oligonucleotide sequence showed stronger interactions with the stationary phase than the 3′ end did. This research might help to understand the retention mechanism of oligonucleotides by RP-IPC.