色谱

色谱

• 研究论文 • 上一篇    下一篇

反相离子对色谱法分析3′, 5′反向寡核苷酸

骆雪芳1,陈蓉1,付静静1,胡育筑2

  

  1. 1. Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, China; 2.Key Laboratory of Drug Quality Control and Pharmacovigilance of Ministry of Education,China Pharmaceutical University, Nanjing 210009, China

  • 收稿日期:2007-06-14 修回日期:2007-08-19 出版日期:2007-11-30 发布日期:1984-12-25
  • 通讯作者: 骆雪芳

Analysis of 3′,5′ Reversed-Sequence Oligonucleotide Isomers by Reversed-Phase Ion-Pair Chromatography

LUO Xuefang1, CHEN Rong1, FU Jingjing1, HU Yuzhu2

  

  1. 1. Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, China; 2.Key Laboratory of Drug Quality Control and Pharmacovigilance of Ministry of Education,China Pharmaceutical University, Nanjing 210009, China

  • Received:2007-06-14 Revised:2007-08-19 Online:2007-11-30 Published:1984-12-25
  • Contact: [中]骆雪芳 [英]LUO Xue-fang

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摘要：

3′,5′反向寡核苷酸是碱基组成和长度完全相同、碱基顺序相反的两个寡核苷酸序列。以三乙胺为离子对试剂,研究了缓冲液浓度(0.025～0.15 mol/L)、pH (5.0～6.8)、柱温(25～45 ℃)、流速(0.3～0.7 mL/min)以及不同初始洗脱强度和洗脱梯度条件下,6个3′,5′反向寡核苷酸模拟样品保留和分离的变化特点。三组反向序列在缓冲液浓度为0.05 mol/L,pH 6.8和流速0.4 mL/min条件下获得最大分离,温度对分离的影响不大,而初始洗脱强度对反向序列的影响远大于洗脱梯度。实验结果表明3′,5′反向寡核苷酸的分离和保留趋势不完全一致,色谱条件的优化应有利于实现样品在柱上的弱保留。研究结果还显示寡核苷酸序列中5′末端的保留强于3′末端。

关键词: 3′,5′反向寡核苷酸, 保留行为 , 反相离子对色谱法

Abstract:

3′, 5′ Reversed-sequence oligonucleotides are isomers with the same length and base composition, except with reversed base sequence. In the current work, 6 oligonucleotide model samples were designed to study chromatographic behaviour of 3′,5′ reversed-sequence isomers by optimizing effects on retention and separation. The retention factor and resolution of isomers were investigated under triethylamine-acetic acid (TEAA) concentrations of 0.025-0.15 mol/L, pH values of 5.0-6.8, temperatures of 25-45 ℃ and flow rates of 0.3-0.7 mL/min by reversed-phase ion-pair chromatography (RP-IPC). The best resolution was observed under TEAA concentration of 0.05 mol/L, pH 6.8 and flow rate of 0.4 mL/min. While the effect of temperature on the separation was not apparent, effect of initial organic strength was stronger than that of the elution gradient. The retention and separation trends of the model samples were different, and weak retention of the samples on the solid phase contributes to good separation. It is concluded that 5′end of oligonucleotide sequence showed stronger interactions with the stationary phase than the 3′end did. This research might help to understand the retention mechanism of oligonucleotides by RP-IPC.

Key words: 3′,5′ reversed-sequence oligonucleotides, retention behavior , reversed-phase ion-pair chromatography (RP-IPC)