Impact of nucleotide hydrophobicity on oligonucleotides separation in liquid chromatography

IF 3.8 2区化学 Q1 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography A Pub Date : 2025-04-14 DOI:10.1016/j.chroma.2025.465968

Martin Gilar , Nils Schomann , Saskia Schott , Michael Rühl

{"title":"Impact of nucleotide hydrophobicity on oligonucleotides separation in liquid chromatography","authors":"Martin Gilar , Nils Schomann , Saskia Schott , Michael Rühl","doi":"10.1016/j.chroma.2025.465968","DOIUrl":null,"url":null,"abstract":"<div><div>We investigated the contribution of nucleotide (nt) type and modifications on the retention and resolution of 22–24 nt long oligonucleotides in ion-pairing reversed-phase (IP RP) liquid chromatography (LC) and hydrophilic interaction chromatography (HILIC) methods. The nucleotides relative hydrophobicity affects the oligonucleotide LC retention and separation selectivity. The ion-pairing reagents of higher hydrophobicity (dipropylamine < dibutylamine < dipentylamine) require a greater organic solvents concentration to elute the oligonucleotides in IP RP LC. We observed that higher concentration of organic solvent reduced the contribution of (oligo)nucleotide hydrophobicity to the retention and led to more uniform separation of full-length oligonucleotide from its truncated products. We also investigated the diastereomeric resolution of 24 nt oligonucleotides with a single phosphorothioate modification at 5′-end. The diastereomeric separation is nucleotide-motif dependent; G (and modified G) nucleotides adjacent to the phosphorothioate linkage yield higher resolution of isomers than other types of nucleotides in IP RP LC and HILIC.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1753 ","pages":"Article 465968"},"PeriodicalIF":3.8000,"publicationDate":"2025-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Chromatography A","FirstCategoryId":"1","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0021967325003164","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}

引用次数: 0

Abstract

We investigated the contribution of nucleotide (nt) type and modifications on the retention and resolution of 22–24 nt long oligonucleotides in ion-pairing reversed-phase (IP RP) liquid chromatography (LC) and hydrophilic interaction chromatography (HILIC) methods. The nucleotides relative hydrophobicity affects the oligonucleotide LC retention and separation selectivity. The ion-pairing reagents of higher hydrophobicity (dipropylamine < dibutylamine < dipentylamine) require a greater organic solvents concentration to elute the oligonucleotides in IP RP LC. We observed that higher concentration of organic solvent reduced the contribution of (oligo)nucleotide hydrophobicity to the retention and led to more uniform separation of full-length oligonucleotide from its truncated products. We also investigated the diastereomeric resolution of 24 nt oligonucleotides with a single phosphorothioate modification at 5′-end. The diastereomeric separation is nucleotide-motif dependent; G (and modified G) nucleotides adjacent to the phosphorothioate linkage yield higher resolution of isomers than other types of nucleotides in IP RP LC and HILIC.

查看原文本刊更多论文

核苷酸疏水性对液相色谱中寡核苷酸分离的影响

研究了在离子配对反相液相色谱（IP RP）和亲水性相互作用色谱（HILIC）中，核苷酸（nt）类型和修饰对22-24 nt长寡核苷酸保留和分离的影响。核苷酸的相对疏水性影响寡核苷酸的LC保留和分离选择性。高疏水性离子配对试剂(二丙胺<；二丁胺& lt;二戊胺)需要较大的有机溶剂浓度来洗脱IP RP LC中的寡核苷酸。我们观察到，较高浓度的有机溶剂降低了（寡核苷酸）疏水性对保留的贡献，并导致全长寡核苷酸与其截短产物更均匀地分离。我们还研究了在5 '端进行单一硫代修饰的24nt寡核苷酸的非对映体分辨率。非对映体分离依赖于核苷酸基序；在IP RP LC和HILIC中，G（和修饰的G）核苷酸与其他类型的核苷酸相比，相邻的磷硫酸键产生更高的同分异构体分辨率。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Journal of Chromatography A 化学-分析化学

CiteScore

7.90

自引率

14.60%

发文量

742

审稿时长

45 days

期刊介绍： The Journal of Chromatography A provides a forum for the publication of original research and critical reviews on all aspects of fundamental and applied separation science. The scope of the journal includes chromatography and related techniques, electromigration techniques (e.g. electrophoresis, electrochromatography), hyphenated and other multi-dimensional techniques, sample preparation, and detection methods such as mass spectrometry. Contributions consist mainly of research papers dealing with the theory of separation methods, instrumental developments and analytical and preparative applications of general interest.