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Issue 16, 2013
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Improving the sensitivity of confocal laser induced fluorescence detection to the sub-picomolar scale for round capillaries by laterally shifting the laser focus point

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Abstract

This paper describes a simple and efficient approach to reduce the background level of confocal laser induced fluorescence (LIF) detection for round capillaries by laterally shifting the laser focus point. A phenomenon of spontaneous separation of the fluorescence and reflected laser beams at the pinhole of a confocal LIF system when the laser focus point deviates from the center of a capillary channel to the sides was observed for the first time. On the basis of this phenomenon, the reflected laser light from the capillary–air interfaces could be mostly eliminated with a spatial filtering pinhole. A comprehensive study on the phenomenon and optimization of the shift distance was carried out using both experimental and simulation methods. A best shift distance of ±20 μm was obtained, with which background intensity could be significantly reduced by 98.9%, while fluorescence intensity was only reduced by 25.7%, resulting in an improvement of signal-to-noise ratio of 8.3 times, compared with that at a shift distance of 0 μm usually used in most of the confocal LIF systems for round capillaries. A limit of detection of 66 fM was obtained for sodium fluorescein. To demonstrate its potential as an on-column sensitive detector for microscale separation systems, the present system was coupled with a capillary electrophoresis system for separation of four fluorescein isothiocyanate labeled amino acids with concentrations of 100 pM.

Graphical abstract: Improving the sensitivity of confocal laser induced fluorescence detection to the sub-picomolar scale for round capillaries by laterally shifting the laser focus point

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Publication details

The article was received on 19 Feb 2013, accepted on 17 May 2013 and first published on 17 May 2013


Article type: Paper
DOI: 10.1039/C3AN00345K
Analyst, 2013,138, 4642-4648

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    Improving the sensitivity of confocal laser induced fluorescence detection to the sub-picomolar scale for round capillaries by laterally shifting the laser focus point

    Y. Zhu, N. Chen, Q. Li and Q. Fang, Analyst, 2013, 138, 4642
    DOI: 10.1039/C3AN00345K

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