Application of phenyl-based stationary phases for the study of retention and separation of oligonucleotides

- Different phenyl-bonded stationary phases were applied for oligonucleotide analysis.
- Propyl-phenyl, hexyl-phenyl and polar embedded phenyl stationary phases were used.
- The retention and separation of homo- and heterooligonucleotides was studied.
- QSRR allowed for development of models for prediction of oligonucleotide retention.

The main goal of our work was to apply three different phenyl-bonded stationary phases in ion pair chromatography for the analysis of synthetic oligonucleotides of various sequences. The influence of the stationary phase structure and the impact of ion-pairing reagent concentration on the retention of oligonucleotides were tested. Moreover the influence of oligonucleotide sequence on their interactions with phenyl-based stationary phases was also investigated. Such complex studies for analysis of oligonucleotides with these adsorbents were done for the first time. Investigations were implemented in the Quantitative Structure Retention Relationships analysis in order to improve the discussion on the retention mechanism of analyzed compounds. The retention of oligonucleotides was the lowest for polar embedded phenyl stationary phase, however its selectivity was high and allowed for complete separation of studied compounds in the shortest time. It was shown that the low retention factor value was observed for oligonucleotides forming secondary structures, such as hairpin loops. Moreover obtained data showed that except for electrostatic and hydrophobic interactions, π-π also influences on the retention mechanism. These interactions cause higher retention factor values for phenyl-based stationary phases compared to octadecyl ones.