Oligonucleotide separation in ion-pairing reversed-phase (IR-RP) liquid chromatography is typically performed with shallow gradients. Recently, a novel ion-pairing buffer compatible both with UV and electrospray MS detection has been described.2,3 The buffer is comprised of triethylamine (TEA, an ion-pairing agent) and aqueous hexafluoroisopropanol (HFIP, a volatile weak acid used as buffering component to bring the pH to ~ 8).
While triethylammonium acetate (TEAA) is useful for oligonucleotide analysis, it is not compatible with MS detection. The LC retention behavior of oligonucleotides strongly depends on their nucleotide composition, requiring careful optimization of gradient elution conditions for each specific oligonucleotide.4 TEA-HFIP ion-pairing buffer yields more consistent and predictable oligonucleotide retention behavior over a broad range of compositional differences as compared to TEAA.
Oligonucleotide retention also varies depending on its length (charge). Differences are also observed for different classes of oligonucleotides and chimeric oligonucleotides (DNA, RNA, LNA, phosphorothioates, morpholino backbone, 2’O-methylated species, and combination of all above).
Waters UPLC method development guidelines for oligonucleotide analysis can be summarized in three steps:
- Identify a suitable initial gradient strength. If elution behavior of the oligonucleotide is unknown, start with a scouting gradient. Recommended flow rate for a 2.1 x 50 mm OST Column is 0.2 mL/min and separation temperature is 60 °C.
- Adjust the gradient slope to achieve a desirable separation. In general, shallower gradients provide increased resolution.
- Faster analyses can be achieved by increasing the flow rate while maintaining gradient column volumes. If the gradient range and the gradient slope in column volumes remain constant, the separation selectivity is not negatively affected.
Figure 1 illustrates method development for a 30 to 60 nt oligonucleotide using TEA-HFIP. The high resolution separation in chromatogram Figure 1A has an initial mobile strength of 22.5% MeOH and slope 0.25% MeOH/min. The resolution can be further improved by using a shallower gradient (0.15% MeOH/min, Figure 1B), but at the expense of analysis time – which will negatively impact throughput.
The analysis time can be shortened by adjusting the initial gradient conditions (Figure 1C). Since the gradient slope was unchanged, the high resolution separation was preserved, with the possible exception of the early eluting peaks.
