Construction And Evaluation Of Several Novel And Sensitive Fluorescence Detection System

High separation efficiency and high detection sensitivity are the eternal goals of liquid chromatography. Benefit by the development of various kinds of advanced separation and detection methods, most samples can be well analyzed. But the analysis of ultra-low concentration materials needs further improvement of detection methods sensitivity. Fluorescence detection is sensitive and selective. Novel analysis systems with high separation efficiency and sensitivity can be built by coupling fluorescence detection with high performance liquid chromatography (HPLC) or capillary electrophoresis (CE), and the systems shall be effective tools for complex samples analysis.Multiple fluorescence detectors were built using473nm laser,365nm and335nm light emitting diode (LED) as light source in this paper. Then a novel HPLC-LIFD system was constructed by coupling laser induced fluorescence detector (LIFD) with HPLC. Since a three-dimensional adjustable supportor of reflector and optimized structure were used in the LIFD, a10"12mol/L LOD of FITC was achieved when using the novel LIFD as detector. The trace bioamines in water were analyzed by the HPLC-LIFD system, and LODs of spermidine, putrescine and histamine were0.13、0.22and0.23nmol/L respectively, the recoveries were in the range of95%-105%. The HPLC-LIFD system was also used to analy fluorescent materials and amines in tea, about15～35fluorescent materials and more than48amines were detected in7teas, the type and content differences of the fluorescent materials and amines among the7teas were discovered.Base on the advantage of shape emission profile and large stokes shift of Eu fluorescence probes, a HPLC-LED-IFD system special for Eu fluorescence probes was built, using a universal and modularized confocal optical system. The BSA, which was derivatized by BHHCT-Eu, was analyzed by the HPLC-LED-IFD system, and a10-8mol/L LOD was achieved. Then several materials derivatized by BHHCT-Eu were analyzed by the HPLC-LED-IFD system with different HPLC modes. It was confirmed that the BHHCT-Eu derivatized proteins might be analyzed by the reversed phase HPLC-LED-IFD system. Furthermore, base on the Eu fluorescence probes specialized LED-IFD, a365nm LED-IFD was built by changing the LED and optical filters, and the365nm LED-IFD might be used to detect benzopyrene.Base on the macromolecules holding and small molecules exchange characteristic of the hollow fiber dialysis membrane, a novel post-column derivatization membrane reactor for protein analysis in CE has been designed. And a protein post-column derivatization membrane reactor based CE-LIFD system was constructed by combining the reactor, CE and LIFD. The membrane length, capillary inner diameter, separation buffer and derivatization solution were optimized. Under the optimized conditions, BSA was detected and a3.3nmol/L LOD was achieved. The system was furtherly successfully applied in the real protein samples analysis.The LIFD built in this paper was sample and sensitive. The structure of the LED-IFD was universal, and other LED-IFD can be built for other samples analysis by changing the LED and filters. The foundation of the large scale production and broaden application range of LIFD and LED-IFD was strengthened by this paper. The chemical conditions in the post-column derivatization membrane reactor could be adjusted by the derivatization solution without sample loss, and other membrane reactors can be built using different membranes to satisfy the requirements of different samples analysis. The membrane reactor can be used in the HPLC, μHPLC or CE system for analysis of macromolecules, such as proteins and DNA.