高等学校化学学报 ›› 2018, Vol. 39 ›› Issue (9): 1893-1903.doi: 10.7503/cjcu20180262

• 分析化学 • 上一篇    下一篇

混合离子对试剂体系下寡核苷酸的IP-RPLC保留行为

乔俊琴, 梁超, 曹兆明, 练鸿振()   

  1. 生命分析化学国家重点实验室, 南京大学化学化工学院和现代分析中心, 南京 210023
  • 收稿日期:2018-04-04 出版日期:2018-09-07 发布日期:2018-06-26
  • 作者简介:

    联系人简介: 练鸿振, 男, 博士, 教授, 博士生导师, 主要从事色谱分离和分析方面的研究. E-mail: hzlian@nju.edu.cn

  • 基金资助:
    国家自然科学基金(批准号: 21275069, 21577057, 91643105)、 江苏省自然科学基金(批准号: BK20171335)和南京大学分析测试基金资助.

Retention Behavior of Oligonucleotides under System Containing Mixed Ion-pair Reagents by IP-RPLC

QIAO Junqin, LIANG Chao, CAO Zhaoming, LIAN Hongzhen*()   

  1. State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry & Chemical Engineering and Center of Materials Analysis, Nanjing University, Nanjing 210023, China
  • Received:2018-04-04 Online:2018-09-07 Published:2018-06-26
  • Contact: LIAN Hongzhen E-mail:hzlian@nju.edu.cn
  • Supported by:
    † Supported by the National Natural Science Foundation of China(Nos.21275069, 21577057, 91643105), the Natural Science Foundation of Jiangsu Province, China(No.BK20171335) and the Analysis & Test Fund of Nanjing University, China.

摘要:

利用离子对反相液相色谱(IP-RPLC)对寡核苷酸在混合离子对试剂三乙胺/丙胺-乙酸盐(TEA/PA-AA)体系下的保留行为进行了研究, 并与经典离子对试剂三乙胺乙酸盐(TEAA)体系下的保留行为进行了对比. 实验结果表明, 相同离子对试剂浓度下, 寡核苷酸在TEA/PA-AA体系下的保留均弱于TEAA体系下的保留, 且寡核苷酸的保留均随着离子对试剂浓度(20~120 mmol/L)的增加而增强. 同型寡核苷酸(dC)n作为特例, 当n>10时, 保留基本趋于稳定, 这是由于(dC)n随着离子对试剂浓度的增加保留增长较快, 在较低的离子对试剂浓度下即可达到最大保留. 同时发现, 短链同型寡核苷酸(dT)n和异型寡核苷酸在TEA/PA-AA体系下的分离均优于TEAA体系, 而同型寡核苷酸(dA)n和(dC)n的分离则在TEAA体系下更优. 通过研究流动相中离子对试剂总浓度cp与寡核苷酸保留因子k之间的关系, 推断出2种体系下寡核苷酸的保留机理均以离子对模型占主导地位. 总体而言, 分离相同长度的异型寡核苷酸时, TEA/PA-AA混合离子对体系尤其在中等离子对浓度下比TEAA体系具有明显优势, 低的离子对试剂浓度可增加与后续电喷雾质谱(ESI-MS)的兼容性, 有利于寡核苷酸的定性分析.

关键词: 离子对反相液相色谱(IP-RPLC), 寡核苷酸, 混合离子对试剂, 保留行为, 保留机理

Abstract:

The retention behaviors of oligonucleotides were investigated under mobile phase containing mixed ion-pair reagents of triethylamine/propylamine-acetate(TEA/PA-AA) and compared with that under classical mobile phase containing triethylamine acetate(TEAA) as ion-pair reagent by ion pair reversed-phase liquid chromatography(IP-RPLC). It is found that the retention of oligonucleotides under TEA/PA-AA system was always weaker than that under TEAA system when the concentration of ion-pair reagents was kept the same. In addition, retention time for most oligonucleotides became longer and longer as the increasing concentration of ion-pair reagents(20—120 mmol/L). As the special case, the retention time of (dC)n(n>10) was relative stable. The reason should be that the retention of (dC)n increases fast with the enhanced concentration of ion-pair reagents, and the maximum retention can be achieved under relative low concentration of ion-pair reagents. Additionally, short chain (dT)n and hetero-oligonucleotides both had better resolution under TEA/PA-AA system than TEAA system. Nevertheless, (dA)n and (dC)n both had better resolution under TEAA system. What’s more, the dominated separation mechanism of oligonucleotides under the two systems both were ion-pair model, deduced from the relationship between retention factor k and ion-pair reagents concentration cp. In general, TEA/PA-AA especially under moderate concentration is superior to TEAA in the separation of hetero-oligonucleotides having same chain length. Ion-pair reagent with low concentration improves the compa-tibility with electrospray ionization-mass spectrometry(ESI-MS), which benefits the qualitative analysis of oligonucleotides.

Key words: Ion pair reversed-phase liquid chromatography(IP-RPLC), Oligonucleotide, Mixed ion-pair reagent, Retention behavior, Retention mechanism

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