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

被引:22
作者
Kadlecova, Zuzana [1 ]
Kalikova, Kveta [1 ]
Tesarova, Eva [1 ]
Gilar, Martin [2 ]
机构
[1] Charles Univ Prague, Fac Sci, Dept Phys & Macromol Chem, Hlavova 8, Prague 12800, Czech Republic
[2] Waters Corp, 34 Maple St, Milford, MA 01757 USA
关键词
Phosphorothioate oligonucleotides; Ion-pairing reversed-phase chromatography; Diastereomeric separation; Temperature; Resolution; SENSITIVITY; REAGENTS; COLUMNS;
D O I
10.1016/j.chroma.2022.463473
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
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页数:7
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