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/PAAA)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c_p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(ESIMS)的兼容性,有利于寡核苷酸的定性分析.
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 c p.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.
作者
乔俊琴
梁超
曹兆明
练鸿振
QIAO Junqin;LIANG Chao;CAO Zhaoming;LIAN Hongzhen(State Key Laboratory of Analytical Chemistry for Life Science,School of Chemistry&Chemical Engineering and Center of Materials Analysis,Nanjing University,Nanjing 210023,China)
出处
《高等学校化学学报》
SCIE
EI
CAS
CSCD
北大核心
2018年第9期1893-1903,共11页
Chemical Journal of Chinese Universities
基金
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.
作者简介
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
