Hernandez L, Joshi N, Murzi E, Verdeguer P, Mifsud J C, Guzman N
Los Andes University, Merida, Venezuela.
J Chromatogr A. 1993 Oct 22;652(2):399-405. doi: 10.1016/0021-9673(93)83259-u.
Experiments with capillary electrophoresis using a laser-induced fluorescence detector with a colinear optical arrangement demonstrated several important points. First, increasing the numerical aperture of the microscope objective that is used simultaneously for focusing the excitation laser light as well as collection of emitted fluorescence enhances the signal used for the measurement of the emitted fluorescence and at the same time decreases the noise of interfering light. Second, detection of fluorescein-labelled amphetamine was performed at high-picomolar (10(-10) M) levels. Third, the signal-to-noise ratio of 280 found at the above-mentioned picomolar concentrations indicates that the measurement of low-picomolar concentrations (10(-12) M) of this compound in biological samples should be possible. Fourth, narrow-bore capillaries (5-10 microm internal diameter) were used to detect the neurotransmitters glutamic acid and aspartic acid as their naphthalene-2,3-dicarboxaldehyde derivatives in brain dialysates obtained from a freely moving rat. A mathematical model was developed to explain the relationship between numerical aperture, working distance, magnification of the lens, noise due to laser scattering and signal due to fluorescence. The model correctly predicted the observed values of photomultiplier tube current due to both laser scattering and fluorescence. The potential of the application of capillary electrophoresis with laser-induced fluorescence detection in the neurosciences is discussed.
使用具有共线光学装置的激光诱导荧光检测器进行的毛细管电泳实验证明了几个要点。首先，增加同时用于聚焦激发激光以及收集发射荧光的显微镜物镜的数值孔径，可增强用于测量发射荧光的信号，同时降低干扰光的噪声。其次，在高皮摩尔（10⁻¹⁰ M）水平下对荧光素标记的苯丙胺进行了检测。第三，在上述皮摩尔浓度下测得的280的信噪比表明，在生物样品中测量该化合物的低皮摩尔浓度（10⁻¹² M）应该是可行的。第四，使用内径为5 - 10微米的窄孔毛细管，在从自由活动大鼠获得的脑透析液中检测神经递质谷氨酸和天冬氨酸，它们以萘-2,3-二甲醛衍生物的形式存在。建立了一个数学模型来解释数值孔径、工作距离、透镜放大倍数、激光散射引起的噪声和荧光引起的信号之间的关系。该模型正确地预测了由于激光散射和荧光引起的光电倍增管电流的观测值。讨论了毛细管电泳与激光诱导荧光检测在神经科学中的应用潜力。
