This application note describes the use of temperature to enhance oligonucleotide mass transfer and improve resolution in ion-pair reversed phase HPLC. Temperature enhancement of mass transfer can improve oligonucleotide separation resolution, enabling the use of smaller particle columns and reduced column lengths. Temperature enhancement of resolution is particularly useful for oligonucleotide analysis, as oligonucleotides have relatively low retention in reversed phase chromatography. Temperature also accelerates oligonucleotide separations, increasing laboratory throughput.
This document explores the use of elevated temperatures to enhance oligonucleotide mass transfer and improve resolution in ion-pair reversed phase HPLC. It addresses the limitations of traditional silica-based columns at high temperatures and compares their performance against hybrid and polymeric columns.
| Column A Type | PLRP-S 100A, 3 ym, PS/DVB HPLC column |
|---|---|
| Column B Type | 2.5 um, 125A Hybrid silica-methylsiloxane C18 column |
| Column C Type | 1000A, 3 ym, Thermally stable bonded silica C18 column |
| Column D Type | 100A, 5 um, Traditional silica C18 column |
| Eluent A Composition | 100 mM Triethylammonium acetate (TEAA) in HPLC-grade water |
| Eluent B Composition | 100 mM TEAA in 25:75 Acetonitrile:water |
| Flow Rate | 1.0 mL/min or 0.45 mL/min |
| Detection Wavelength | UV, 254 nm |
The PLRP-S column provides superior performance at 80 °C compared to other columns.
Elevated temperatures improve the mass transfer of denatured oligonucleotides between the stationary and mobile phases, enhancing resolution.