Hydrophobic labeling of amino acids: Transient trapping–capillary/microchip electrophoresis
Hydrophobic labeling of amino acids: Transient trapping–capillary/microchip electrophoresis
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DOI：
10.1002/elps.201000567
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发表时间：
2011-05
期刊：
影响因子：
2.9
通讯作者：
K. Sueyoshi;K. Hashiba;T. Kawai;F. Kitagawa;K. Otsuka
中科院分区：
文献类型：
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作者：
K. Sueyoshi;K. Hashiba;T. Kawai;F. Kitagawa;K. Otsuka
瞬时捕获（tr-trapping）是一种在线样品预富集技术，用于改善微芯片电泳（MCE）中低浓度灵敏度的问题，基于捕获-释放机制提供高效的预富集和分离。然而，对亲水性分析物的不良性能限制了TR捕获的适用性。为了克服这一缺点，在CE中使用疏水试剂将tr-捕获与样品标记相结合。测试了三种市售荧光染料，异硫氰酸荧光素、Alexa Fluor 488的琥珀酰亚胺酯和BODIPY FL-X，作为衍生化试剂，以增加由于无荧光/UV吸光度而无法检测到的氨基酸（AA）的疏水性。结果，证实了BODIPY标记允许通过疏水性的增加在反捕集-胶束电动色谱（tr-trapping-MEKC）中分析各种AA。在tr-trapping-MEKC中，相对于传统的毛细管区带电泳，实现了分辨率的提高和标记AA的检测能力的106-125倍增强。通过应用tr-trapping-MEKC，标记苯丙氨酸的检测限从800 pM提高到5 pM。此外，在tr-trapping-微芯片MEKC中，峰强度和基线分离也在30秒内实现了80-160倍的增强。这些结果清楚地表明，疏水标记的tr-trapping技术将使CE/MCE对各种分析物更敏感。
Transient trapping (tr‐trapping) was developed as one of the on‐line sample preconcentration techniques to improve a low concentration‐sensitivity in microchip electrophoresis (MCE), providing highly effective preconcentration and separation based on the trap‐and‐release mechanism. However, a poor performance to hydrophilic analytes limited the applicability of tr‐trapping. To overcome this drawback, tr‐trapping was combined with a sample labeling using a hydrophobic reagent in CE. Three commercially available fluorescent dyes, fluorescein isothiocyanate, succinimidyl esters of Alexa Fluor 488 and BODIPY FL‐X, were tested as derivatization reagents to increase the hydrophobicity of amino acids (AAs) that were undetectable due to no fluorescence/UV‐absorbance. As a result, it was confirmed that BODIPY labeling allowed various AAs to be analyzed in tr‐trapping–micellar electrokinetic chromatography (tr‐trapping–MEKC) by the increase in the hydrophobicity. In tr‐trapping–MEKC, both the improvement of the resolution and 106–125‐fold enhancements of the detectability of labeled AAs were achieved relative to the conventional capillary zone electrophoresis. The limit of detection of labeled phenylalanine was improved from 800 to 5 pM by applying tr‐trapping–MEKC. In tr‐trapping–microchip MEKC, furthermore, an 80–160‐fold enhancement of the peak intensity and a baseline separation was also achieved within 30 s. These results clearly demonstrate that the tr‐trapping technique with hydrophobic labeling will make CE/MCE more sensitive for various analytes.