Synthetic oligonucleotide separations by mixed-mode reversed-phase/weak anion-exchange liquid chromatography

Synthetic oligonucleotides gain increasing importance in new therapeutic concepts and as probes in biological sciences. If pharmaceutical-grade purities are required, chromatographic purification using ion-pair reversed-phase chromatography is commonly carried out. However, separation selectivity for structurally closely related impurities is often insufficient, especially at high sample loads. In this study, a “mixed-mode” reversed-phase/weak anion exchanger stationary phase has been investi- gated as an alternative tool for chromatographic separation of synthetic oligonucleotides with minor sequence variations. The employed mixed-mode phase shows great flexibility in method develop- ment. It has been run in various gradient elution modes, viz. one, two or three parameter (mixed) gradients (altering buffer pH, buffer concentration, and organic modifier) to find optimal elution con- ditions and gain further insight into retention mechanisms. Compared to ion-pair reversed-phase and mere anion-exchange separation, enhanced selectivities were observed with the mixed-mode phase for 20–23 nucleotide (nt) long oligonucleotides with similar sequences. Oligonucleotides differing by 1, 2 or 3 nucleotides in length could be readily resolved and separation factors for single nucleotide replacements declined in the order Cytosine (C)/Guanine (G) > Adenine (A)/Guanine ∼ Guanine/Thymine (T) > Adenine/Cytosine ∼ Cytosine/Thymine > Adenine/Thymine. Selectivities were larger when the mod- ification was at the 3 terminal-end, declined when it was in the middle of the sequence and was smallest ̊ when it was located at the 5 terminus. Due to the lower surface area of the 200 A pore size mixed- ̊ mode stationary phase compared to the corresponding 100 A material, lower retention times with equal selectivities under milder elution conditions were achievable. Considering high sample loading capac- ities of the mixed-mode anion-exchanger phase, it should have great potential for chromatographic oligonucleotide separation and purificatio