Key result
Higher concentrations of organic solvent reduced the contribution of nucleotide hydrophobicity to retention, and diastereomeric separation was found to be nucleotide-motif dependent.
The relative hydrophobicity of nucleotides and the choice of ion-pairing reagents significantly affect the retention and separation selectivity of oligonucleotides in liquid chromatography.
Refines LC method development for oligonucleotides; leaves open generalizability across motifs and reagents.
We investigated the contribution of nucleotide (nt) type and modifications on the retention and resolution of 22-24 nt long oligonucleotides in ion-pairing reversed-phase (IP RP) liquid chromatography (LC) and hydrophilic interaction chromatography (HILIC) methods. The nucleotides relative hydrophobicity affects the oligonucleotide LC retention and separation selectivity. The ion-pairing reagents of higher hydrophobicity (dipropylamine < dibutylamine < dipentylamine) require a greater organic solvents concentration to elute the oligonucleotides in IP RP LC. We observed that higher concentration of organic solvent reduced the contribution of (oligo)nucleotide hydrophobicity to the retention and led to more uniform separation of full-length oligonucleotide from its truncated products. We also investigated the diastereomeric resolution of 24 nt oligonucleotides with a single phosphorothioate modification at 5'-end. The diastereomeric separation is nucleotide-motif dependent; G (and modified G) nucleotides adjacent to the phosphorothioate linkage yield higher resolution of isomers than other types of nucleotides in IP RP LC and HILIC.
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Gilár et al. (2025) studied this question. Ion-pairing reversed-phase liquid chromatography and hydrophilic interaction chromatography was evaluated on Retention and resolution of 22-24 nt long oligonucleotides. Higher concentrations of organic solvent reduced the contribution of nucleotide hydrophobicity to retention, and diastereomeric separation was found to be nucleotide-motif dependent.
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