Lowering the UV absorbance detection limit and increasing the sensitivity of capillary electrophoresis using a dual linear photodiode array detector and signal averaging

Abstract

We have recently described a new spatial signal-averaging approach to improve the UV absorbance detection limits for capillary electrophoresis (CE) using a single photodiode array (PDA) detector. (Culbertson, C. T.; Jorgenson, J. W. Anal Chem 1998 70, 2629: Culbertson, C. T. PhD Dissertation, University of North Carolina, Chapel Hill, 1996). At low analyte concentrations, however, source flicker noise interfered with the signal-averaging process. This noise was due to inadequate compensation of the spatial and temporal lamp fluctuations that occurred over the course of a run in this single-beam instrument. To better compensate for the source lamp fluctuations and drift, a double-beam detector has been constructed using a dual PDA to continuously monitor the output of the source. The addition of the reference channel significantly reduces the source flicker noise. To demonstrate the capabilities of this new detector, separations of four nucleic acids at six different concentrations were performed. The signal-averaged electropherograms generated using the double-beam instrument are compared with electropherograms obtained from individual diodes in the array, a single-beam detector, and a commercial single-point detector. The detection limits for the double-beam instrument were 35(±3) times better than that obtained using an individual diode in the array, ca. 4 times better than that obtained using a single-beam detector, and 6.3(±0.4) times better than that obtained using a commercial single-point detector. The linearity of the new detector is found to be equal to or better than that of the commercial single-point detector, and a separation of analytes at mixed concentrations corresponding to a two order of magnitude dynamic range is shown. ©1999 John Wiley & Sons, Inc. J Micro Sep 11: 652–662, 1999