Real-time fluorescence imaging in analytical chemistry

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Abstract
A detection system for capillary electroseparation methods based on fluorescence imaging has been developed. In capillary electrophoresis (CE) the detection unit is normally placed near the outlet part of the fused silica column where a window is opened in the coating and the fluorescence is recorded over a short distance to maintain a high resolution. Our method employs fluorescence imaging of the whole column during separation of various samples. The column is positioned in a straight holder and the outer protective coating of the column is removed to get optical access to the sample. An excimer/dye laser is used for excitation of the sample and the fluorescence is recorded with an image-intensified CCD detector and displayed in real-time. The CCD detector is read out with a rate of about 5 frames per second and the corresponding full fluorescence line profiles along the column are displayed. Thus, full electropherogram are displayed showing the propagation and gradual separation of the sample fractions. The main advantage of this method is that parameters such as sample concentrations, diffusion, wall interaction and sample-to-sample interaction can be studied in real-time over the full length of the column, which is crucial for efficient system optimization. Among several applications, isoelectric focusing, isotachophoresis and enzyme-substrate interactions can be mentioned. Methods for increasing the collection efficiency, such as fiber optic arrays, have been investigated as well as different methods for computer-assisted signal integration and filtering. A fiber array consisting of 500 optical quartz fibers has been constructed that gives a substantial improvement of the optical collection efficiency.

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