Fluorescence detector laser-based

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. 

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. 

DNA samples are introduced into the 96-capillary array. When the samples are separated through the capillaries, the fragments are irradiated with laser hght. A charge coupled device measures the fluorescence and acts as a multichannel detector. The bases are identifled in order in accordance to the time required for them to reach the laser-detector region. 

The quantity and volume of samples required for impurity determination by CZE are very small probably less than 5 uL of volume is required for a well-designed injector, and only a few nanoliters (i.e., a few nanograms) are actually injected. However, it is experimentally simpler if that sample is present in a relatively concentrated solution, 0.05-2 mg/mL, when UV detection is being used. Our focus was not to achieve ultra-low detection limits such as might be required for trace level contaminants or for quantitation of trace levels of natural products. For those applications, the most common approach has been the use of a laser-based detector, preferably combined with a fluorescent label on the analyte. With this combination, extremely low limits of detection can be achieved (9, 22-25). 

During the last two decades, there has been an enormous increase in the use of photophysical methods in supra-molecular chemistry. Until recently, photophysical methods, such as transient spectrometry and time-resolved fluorescence spectrometry, were primarily research tools in the arenas of photokinetics of small molecules, materials physics, and biophysics. This situation changed dramatically with the introduction of commercial, user-friendly electro-optical components such as charge-coupled detector (ED)-based spectrometers, solid-state pulsed lasers, and other instrumentation necessary for time-resolved measurements. As a result, time-resolved spectrometry became more available to the community of supramolecular chemists, who now reached the level of sophistication that can benefit from the new horizons offered. 

Fluorescence detectors for HPLC are similar in design to the fluorometers and spectrofluorometers described in Section 15B-2. In most, fluorescence is observed by a photoelectric transducer located at 90° to the excitation beam. The simplest detectors use a mercury excitation source and one or more filters to isolate a band of emitted radiation. More sophisticated instruments are based on a xenon source and use a grating monochromator to isolate the fluorescence radiation. Laser-induced fluorescence is also used because of its sensitivity and selectivity. 

Since TIRF produces an evanescent wave of typically 80 nm depth and several tens of microns width, detection of TIRF-induced fluorescence requires a camera-based (imaging) detector. Hence, implementing TIRF on scanning FLIM systems or multiphoton FLIM systems is generally not possible. To combine it with FLIM, a nanosecond-gated or high-frequency-modulated imaging detector is required in addition to a pulsed or modulated laser source. In this chapter, the implementation with of TIRF into a frequency-domain wide-field FLIM system is described. 

In conventional chip experiments, fluorescence scanners are used for chip read-out. In the case of laser scanners, HeNe lasers are used as excitation sources and photomultiplier tubes as detectors, whereas CCD-based scanners use white light sources. The optical system can be confocal or non-confocal. Standard biochip experiments are performed using two fluorescent labels as... 

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