Fu Xiaofang, Huang Ling, Gao Fei, Li Wei, Pang Nannan, Zhai Maolin, Liu Huwei, Wu Moutian
Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing, PR China.
Electrophoresis. 2007 Jun;28(12):1958-63. doi: 10.1002/elps.200600558.
A hydrophilic basic polysaccharide, carboxymethyl chitosan (CMC) as a capillary coating is presented with a simple preparation procedure. The CMC-coated capillary showed a long lifetime of more than 100 runs, and had good tolerance to some organic solvents, 0.1 M HCl, 0.1 M NaOH, and 5 M urea. The run-to-run, day-to-day, and capillary-to-capillary RSDs for the CMC-coated capillary were all below 2.0% for the determination of EOF. Moreover, the coatings with different concentrations and molecular weights of CMC were also investigated. The CMC-coated capillary was successfully applied to separate basic proteins and recombinant human erythropoietin (rhEPO). Furthermore, several experimental parameters, such as the concentration and pH of the running buffer, temperature, and applied voltage, were optimized for the separation of rhEPO glycoforms. Comparison of an uncoated capillary with chitosan- and CMC-coated capillaries for the separation of rhEPO glycoforms was also discussed. The results demonstrated that rhEPO glycoforms can be well separated by a CMC-coated capillary within 8 min with good reproducibility and resolution. Finally, the volatile BGE HAc-NH4Ac was utilized to separate rhEPO for its further application with CE-MS, achieving a satisfactory result.
本文介绍了一种制备过程简单的亲水性碱性多糖——羧甲基壳聚糖（CMC）作为毛细管涂层。涂覆有CMC的毛细管显示出超过100次运行的长使用寿命，并且对一些有机溶剂、0.1 M HCl、0.1 M NaOH和5 M尿素具有良好的耐受性。对于涂覆有CMC的毛细管，在测定电渗流时，运行间、日间和毛细管间的相对标准偏差均低于2.0%。此外，还研究了不同浓度和分子量的CMC涂层。涂覆有CMC的毛细管成功应用于分离碱性蛋白质和重组人促红细胞生成素（rhEPO）。此外，针对rhEPO糖型的分离，对几个实验参数进行了优化，如运行缓冲液的浓度和pH值、温度和施加电压。还讨论了未涂覆毛细管与壳聚糖和CMC涂覆毛细管在分离rhEPO糖型方面的比较。结果表明，涂覆有CMC的毛细管能够在8分钟内很好地分离rhEPO糖型，具有良好的重现性和分辨率。最后，利用挥发性背景电解质HAc-NH4Ac分离rhEPO，以便其进一步应用于毛细管电泳-质谱联用，获得了满意的结果。
