Department of Decommissioning and Waste Management, Japan Atomic Energy Agency, 2-4 Shirakata, Tokai-mura, Naka-gun, Ibaraki, 319-1195, Japan.
Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama, 338-8570, Japan.
Electrophoresis. 2020 Jul;41(13-14):1152-1159. doi: 10.1002/elps.201900399. Epub 2020 Apr 28.
A novel combination of CE-based separation techniques was used for the precise fractionation of ionic compounds from impurities. The combination of on-capillary concentration and separation using transient isotachophoresis, with multiple injections and a two-point detection system provided higher efficiency, and accuracy at a microliter-scale injection volume, than when CE was individually used for purification. In this paper, we present successful applications of the CE fractionation techniques for the purification of fluorescein, fluorescein-4-isothiocyanate, two fluorescent metal ion probes, and a fluorescein-modified DNA aptamer. The purity of the isolated fluorescent probes ranged from 95 to 99%. Such high purity could not be achieved using chromatographic purification techniques. With relatively low dilution factors of 6-9, the purified probe solutions were practical for use as purified stock solutions. In addition, the fluorescein-modified DNA aptamer purified by our method was successfully used in a thrombin binding assay. The method developed was useful for the purification of anionic fluorescent reagents to be of ultratrace analytical grade for use with CE-LIF.
一种新型的基于 CE 的分离技术组合被用于从杂质中精确分离离子化合物。在毛细管上浓缩和使用瞬态等速电泳进行分离的组合，通过多次进样和两点检测系统，在微升进样体积下提供了更高的效率和准确性，比单独使用 CE 进行纯化时更为有效。在本文中，我们成功地应用 CE 分离技术对荧光素、荧光素-4-异硫氰酸酯、两种荧光金属离子探针以及荧光素修饰的 DNA 适体进行了纯化。分离出的荧光探针的纯度范围为 95%至 99%。这种高纯度是无法通过色谱纯化技术实现的。通过相对较低的 6-9 倍稀释因子，得到的纯化探针溶液可实际用作纯化储备溶液。此外，我们方法纯化的荧光素修饰的 DNA 适体可成功用于凝血酶结合测定。所开发的方法可用于阴离子荧光试剂的纯化，以达到用于 CE-LIF 的痕量分析级别的纯度。
