Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
J Chromatogr A. 2012 Apr 6;1232:52-8. doi: 10.1016/j.chroma.2011.09.032. Epub 2011 Sep 17.
To obtain high sensitivity in capillary electrophoresis of oligosaccharide without reducing the high resolution with an easy experimental procedure, large-volume sample stacking with an electroosmotic flow pump (LVSEP) was investigated. As a fundamental study, effect of the conductivity of a sample solution in LVSEP was examined. It was revealed that LVSEP was successfully carried out even in using a sample solution with the ionic strength of 150 μM and the conductivity ratio of 20, indicating a good applicability of LVSEP to the analysis of real samples containing salts. When glucose oligomer was analyzed as a model sample in LVSEP-capillary zone electrophoresis (CZE), all peaks were well resolved with decreasing only 5% of the peak-to-peak distance, which suggested 95% of the whole capillary could be used for the effective separation. In the analysis of maltoheptaose, a good calibration line with correlation coefficient of 0.9995 was obtained. The limit of detection was estimated as 2 pM, which was 500-fold lower than that in the conventional CZE. N-linked glycans released from three glycoproteins, bovine ribonuclease B, bovine fetuin, and human α(1)-acid glycoprotein were also analyzed by LVSEP-CZE. By the sample purification with a gel filtration column, further sample dilution to reduce the sample conductivity for LVSEP was not needed. All glycan samples were well concentrated and separated with up to a 770-fold sensitivity increase. The run-to-run repeatabilities of the migration time, peak height, and peak area were good with relative standard deviations of 0.1-1.3%, 1.2-1.7%, and 2.8-4.9%, respectively.
为了在不降低分辨率的情况下提高寡糖毛细管电泳的灵敏度，同时采用一种简单的实验程序，研究了利用电渗流泵进行大体积样品堆积（LVSEP）的方法。作为基础研究，考察了 LVSEP 中样品溶液电导率的影响。结果表明，即使使用离子强度为 150μM、电导率比为 20 的样品溶液，LVSEP 也能成功进行，表明 LVSEP 可很好地应用于含有盐的实际样品分析。当用 LVSEP-毛细管区带电泳（CZE）分析葡萄糖低聚物作为模型样品时，所有峰均得到很好的分离，峰峰间距仅降低了 5%，这表明整个毛细管的 95%可用于有效分离。在分析麦芽七糖时，得到了相关系数为 0.9995 的良好校准曲线。检测限估计为 2pM，比常规 CZE 低 500 倍。还通过 LVSEP-CZE 分析了从三种糖蛋白（牛核糖核酸酶 B、牛胎球蛋白和人α(1)-酸性糖蛋白）释放的 N-连接糖链。通过凝胶过滤柱进行样品纯化，无需进一步稀释样品以降低样品电导率用于 LVSEP。所有糖链样品均得到很好的浓缩和分离，灵敏度提高了 770 倍。迁移时间、峰高和峰面积的运行间重复性良好，相对标准偏差分别为 0.1-1.3%、1.2-1.7%和 2.8-4.9%。
