Impact of nucleotide hydrophobicity on oligonucleotides separation in liquid chromatography

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.