title: Improving the sensitivity of confocal laser induced fluorescence detection to the sub-picomolar scale for round capillaries by laterally shifting the laser focus point.
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.
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou, 310058, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhu
authors.forename: Niannian
authors.initials: N
authors.lastname: Chen
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
authors.forename: Qun
authors.initials: Q
authors.lastname: Fang
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods / Q000592:standards; D005456:Fluorescent Dyes / Q000737:chemistry / Q000592:standards*; D007834:Lasers / Q000592:standards*; D018613:Microscopy, Confocal / Q000379:methods / Q000592:standards; D008856:Microscopy, Fluorescence / Q000379:methods / Q000592:standards; D036103:Nanotechnology / Q000379:methods* / Q000592:standards*
chemical_list: D005456:Fluorescent Dyes