Study on Retention Behaviour of Homo-Oligonucleotides in IP-RPLC Using Dual-Point Retention Time Correction on “Standard Column” with Internal Standards

The retention behavior of homo-oligonucleotides was investigated in ion-pair reverse-phase high-performance liquid chromatography (IP-RPLC). Rectification of retention time was performed by introducing the concept of “standard column” with double-point retention time correction (DP-RTC) using two anchor oligonucleotides as internal standards to ensure the accuracy of the retention measurement. Through polynomial fitting, a quadric relationship was adopted between normalized retention time and chain length of (dA)10-21 and (dT)10-21. The retention times of homo-oligonucleotides depended on the number of bases and increased with increasing hydrophobicity, namely, A<T. Moreover, the impact of column temperature was also investigated on retention of homo-oligonucleotides. It was found that retention time of (dA)n first increased and then decreased with increase of the temperature due to the destruction of the coil structure in the mobile phase. This chromatographic behaviour study has the potential to reveal the existing state of nucleic acids in various solutions, and to elucidate their conformational transition and biological activity.