Confocal laser-induced fluorescence detector for narrow capillary system with yoctomole limit of detection.
Laser-induced fluorescence (LIF) detectors for low-micrometer and sub-micrometer capillary on-column detection are not commercially available. In this paper, we describe in details how to construct a confocal LIF detector to address this issue. We characterize the detector by determining its limit of detection (LOD), linear dynamic range (LDR) and background signal drift; a very low LOD (~70 fluorescein molecules or 12 yoctomole fluorescein), a wide LDR (greater than 3 orders of magnitude) and a small background signal drift (~1.2-fold of the root mean square noise) are obtained. For detecting analytes inside a low-micrometer and sub-micrometer capillary, proper alignment is essential. We present a simple protocol to align the capillary with the optical system and use the position-lock capability of a translation stage to fix the capillary in position during the experiment. To demonstrate the feasibility of using this detector for narrow capillary systems, we build a 2-μm-i.d. capillary flow injection analysis (FIA) system using the newly developed LIF prototype as a detector and obtain an FIA LOD of 14 zeptomole fluorescein. We also separate a DNA ladder sample by bare narrow capillary - hydrodynamic chromatography and use the LIF prototype to monitor the resolved DNA fragments. We obtain not only well-resolved peaks but also the quantitative information of all DNA fragments.
University of Oklahoma, Department of Chemistry and Biochemistry, Norman, OK 73019, USA.
Mitchell T
MT
Weaver
University of Oklahoma, Department of Chemistry and Biochemistry, Norman, OK 73019, USA.
Kyle B
KB
Lynch
University of Oklahoma, Department of Chemistry and Biochemistry, Norman, OK 73019, USA.
Zaifang
Z
Zhu
University of Oklahoma, Department of Chemistry and Biochemistry, Norman, OK 73019, USA.
Huang
H
Chen
University of Oklahoma, Department of Chemistry and Biochemistry, Norman, OK 73019, USA.
Joann J
JJ
Lu
Department of Chemistry, Lanzhou University, Lanzhou, Gansu 730000, China. Electronic address: puqs@lzu.edu.cn.
Qiaosheng
Q
Pu
University of Oklahoma, Department of Chemistry and Biochemistry, Norman, OK 73019, USA. Electronic address: Shaorong.liu@ou.edu.
Shaorong
S
Liu
D019075:Electrophoresis, Capillary / Q000379:methods*; D017022:Flow Injection Analysis / Q000379:methods*; D019793:Fluorescein / Q000032:analysis*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis*; D007834:Lasers; D057230:Limit of Detection; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D019793:Fluorescein
Confocal; DNA separation; Flow injection analysis; Hydrodynamic chromatography; Laser-induced fluorescence; Narrow capillary