Phosphorothioate oligonucleotides separation in ion-pairing reversed-phase liquid chromatography: effect of temperature

Analysis of diastereomers of phosphorothioate oligonucleotides in ion-pairing reversed-phase liquid chro- matography is affected not only by the character and concentration of ion-pairing system, but also by the separation temperature. In this work, eight ion-pairing systems at two concentrations buffered with acetic acid were used with octadecyl column to investigate the effects of temperature (in the range from 20 degrees C to 90 degrees C) on retention, diastereomeric separation, resolution of mers of different length and reso- lution of oligonucleotides with different number of phosphorothioate linkages. It was observed that ele- vated temperature suppresses the diastereomeric separation and oligonucleotide peaks become narrower. This improves the resolution of n and n-1 mers at elevated temperature. Plots of ln k ( k=retention factor) versus reciprocal absolute temperature show that for 100 mM ion-pairing systems the increase in temperature does not lead to simple decrease in oligonucleotides retention as generally observed in reversed-phase liquid chromatography. The aim of this work is to improve chromatographic method for analysis of phosphorothioate oligonucleotides.(c) 2022 Elsevier B.V. All rights reserved.

Automated summary

The analysis of diastereomers of phosphorothioate oligonucleotides in ion-pairing reversed-phase liquid chromatography is influenced by the ion-pairing system, concentration, and temperature. This study investigated the effects of temperature on retention, diastereomeric separation, and resolution of mers of different length and phosphorothioate oligonucleotides with different linkages. The results showed that increased temperature suppresses diastereomeric separation but improves the resolution of n and n-1 mers.