In traditional capillary electrophoresis (CE) with fluorescence detection, the difference of the migration time of analytes leads to the inequivalent photobleaching, therefore the quantitative determination of the analytes is usually difficult. Especially, when an internal standard (IS) is used, the fluorescent intensities of analytes are hardly comparable to that of the IS if the inequivalent photobleaching is not restricted. We propose a practical approach to reduce the effect of inequivalent photobleaching via an online fluorescence imaging system coupled with the IS method. The detection was operated by the acquisitions of the fluorescent intensities from the divided small section array in the detection window and the electropherograms were obtained from the integrated each acquisition. Compared with the traditional CE detection method, the proposed method demonstrates a real-time detection to collect the fluorescence signals of different analytes under the definite exposure time, rather than the same traversing length. Experimental demonstration for the proposed method was carried out by using DNA-dye conjugations. As a result, our method provided a lower relative error of the peak area ratio of 0.3–3.2 % and a more practical operation because the efforts to restrict the photobleaching are exempted. Revealed in this paper also includes the fact that the photobleaching process manipulated the determination of the peak width and separation resolution.
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