Laser fluorescence detector

A schematic diagram of the liquid chromatography system and laser fluorescence detector Is shown in Figure 1. The key components of this analytical system are described sequentially In what follows ... 

A variable-wavelength UV-absorbance detector (Model Ovldec 100-V, Jasco Inc., Tokyo, Japan) was modified to permit "on-column" detection with packed and open tubular fused-slllca microcolumns, as described previously (3,45,47). The UV—absorbance detector was placed In series before the laser fluorescence detector (see Figure 1), and was used for comparisons of sensitivity, selectivity, and dead volume (44). 

Laser Fluorescence Detector. A helium-cadmium laser (Model 4240B, Llconlx, Sunnyvale, CA) was (diosen as the excitation source because of Its stability and convenient wavelengths (325 and 442 nm). The UV laser radiation (325 nm, 5-10 mH cw) was Isolated with a dielectric mirror and was focused on the miniaturized flowcell with a quartz lens. Sample fluorescence, collected perpendicular to and coplanar with the excitation beam, was spectrally Isolated by appropriate Interference filters and then focused on a photomultiplier tube (Centronlc Model Q 4249 B, Bailey Instruments Co., Inc., Saddle Brook, NJ). The resulting photocurrent was amplified with a plcoammeter (Model 480, Kelthley Instruments,... 

Sugarman J, Pmd homme R (1987) Effect of photobleaching on the output of an on-column laser fluorescence detector. Ind Eng Chem Res 26 1449-1454... 

A new cyanide dye for derivatizing thiols has been reported (65). This thiol label can be used with a visible diode laser and provide a detection limit of 8 X 10 M of the tested thiol. A highly sensitive laser-induced fluorescence detector for analysis of biogenic amines has been developed that employs a He—Cd laser (66). The amines are derivatized by naphthalenedicarboxaldehyde in the presence of cyanide ion to produce a cyanobenz[ isoindole which absorbs radiation at the output of He—Cd laser (441.6 nm). Optimization of the detection system yielded a detection limit of 2 x 10 M. 

The synthesis of a new near-infrared cyanine dye was monitored by CE and fluorescence detection. The chemicals structure of the dye and its synthetic precursor are depicted in Fig. 3.165. The analysis of the dye was realized in fused-silica capillaries of 75 and 100 /an i.d. The total and effective lengths of capillaries were 75 and 60 cm, respectively. The separation voltage was 30 kV and separations were carried out at ambient temperature. The running buffer was 2.5 mM Na2B407 (pH = 9.2). A near-infrared laser-induced fluorescence detector was applied. Electropherograms illustrating the separation of the dye are shown in Fig. 3.166. 

Fluorescence spectroscopy and its applications to the physical and life sciences have evolved rapidly during the past decade. The increased interest in fluorescence appears to be due to advances in time resolution, methods of data analysis and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of macromolecules, to probe the structures of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry. 

When compared to fluorescence detectors for HPLC, the design of a fluorescence detector for CE presents some technical problems. In order to obtain acceptable sensitivity, it is necessary to focus sufficient excitation light on the capillary lumen. This is difficult to achieve with a conventional light source but is easily accomplished using a laser. The most popular source for laser-induced fluorescence (LIF) detection is the argon ion laser, which is stable and relatively inexpensive. The 488-nm argon ion laser line is close to the desired excitation wavelength for several common fluorophores. The CLOD for a laser-based fluorescence detector can be as low as 10 12 M. 

The number of cellular parameters that can be evaluated simultaneously depends on the optical and electronic configuration of a given instrument. A flow cytometer may be a single-laser analyzer that can measure three fluorescence colors and two hght scatter parameters (five parameters), or a complex instrument with multiple lasers and detectors capable of recording signals for as many as 13 fluorescence colors (15 parameters) in individual cells. For example, two widely used single-laser instruments, the BD... 

FIGURE 7.6 Schematic of a laser-induced fluorescence detector. A lamp with focusing optics and an appropriate band-pass filter could be used in place of the laser excitation when tightly collimated light is not required. The emitted fluorescence is detected by a PMT that can be operated in current mode or photon counting mode. Inset shows the mutually perpendicular arrangement of excitation, capillary, and detection optics. 

Limits of detection become a problem in capillary electrophoresis because the amounts of analyte that can be loaded into a capillary are extremely small. In a 20 Jim capillary, for example, there is 0.03 LL/cm capillary length. This is 1 /100 to 1 /1000 of the volume typically loaded onto polyacrylamide or agarose gels. For trace analysis, a very small number of molecules may actually exist in the capillary after loading. To detect these small amounts of components, some on-line detectors have been developed which use conductivity, laser Doppler effects, or narrowly focused lasers (qv) to detect either absorbance or fluorescence (47,48). The conductivity detector claims detection limits down to 1 O molecules. The laser absorbance detector has been used to measure some of the components in a single human cell (see Trace and residue analysis). 

Musenga et al. [55] described a capillary electrophoresis method for determination of vigabatrin in human plasma after precolumn derivatization with 6-carbox yfluorescein-N-s ucc i n i m i d i d yl ester. Optimal separation and detection were obtained with 50 mM borate buffer (pH 9.0) containing 100 mM N-methylglucamine with laser-induced fluorescence detector (Aexc = 488 nm). The assay was rectilinear over the concentration... 

Abbreviations AOD, Acousto-optical deflection BCB, bisbenzyocyclobutadiene CCD, indirect contact conductivity detection CL, chemiluminescence ECD, electron capture detector FCS, fluorescence correlation spectroscopy FRET, fluorescence resonance energy transfer ICCD, integrated contact conductivity detection GMR, giant magnetoresistive LED-CFD, light emitting diode confocal fluorescence detector LIF, laser-induced fluorescence LOD, limit of detection MALDI, matrix-assisted laser desorption ionization PDMS, poly(dimethylsiloxane) PMMA, poly(methylmetha-crylate) SPR, surface plasmon resonance SVD, sinusoidal voltammetric detection TLS, thermal lens spectroscopy. 

Fig. 3 GPC setup for functional group profiling. RI refractive index detector Fluorescence fluorescence detector MALLS multi-angle laser light scattering detector...

The innovative thermostated separation system published by de Bokx et al. [17] represents an interesting example and comprises a capillary cross intersection for sample injection and a 100 pi fluorescence detector cell based on fiber optics. This apparatus shows basically all features that are required to perform automated fast and efficient electrophoretic separations and has been used to separate a mixture of laser dyes in 35 seconds with moderate efficiency. However, in order to keep all dead volumes at the junctions sufficiently small, the connections had to be done by tedious laser-based drilling of holes through the capillary walls. A similar approach to interconnect capillaries was described for a postcolumn derivatization reactor for CE [18], and many more inventive capillary coupling devices have been designed. 

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