Charge-Coupled Device Fluorescence Detection for Capillary-Zone Electrophoresis (CCD-CZE)

Abstract

This paper describes a new instrument system combining capillary-zone electrophoresis (CZE) separation and charge-coupled device (CCD) fluorescence detection. The performance of this CCD-CZE system is evaluated and compared with the performance of a photomultiplier tube (PMT) detector, with the use of signal-to-noise (S/N) ratio and acquisition time as measures of performance. The feasibility of on-column fluorescence spectrometric detection is demonstrated, and a three-dimensional electrophorogram is presented to show the spectral and time resolution obtained by injection of 2 femtomoles (5 nanoliters of 4.0 × 10⁻⁷ M) of fluorescein into the capillary column. The software uses a “peak sum” method to achieve optimum S/N ration. The limit of optical detection (LOD) for this system is 4 attomoles (4 × 10⁻¹⁸ mole) of fluorescein, with a CCD integration time of only 0.2 s. If the fluorescence signal were integrated for 4 s on the CCD, an LOD of less than 1 attomole could be achieved for fluorescein. A 4-s integration time would still permit three complete spectra during the 30-s elution time of the sample.