Fluorescent detection, instrument photodiode

After optimization of the correct capillary parameters (ID, OD, Lj), detection at the microscale level became the next major challenge for the survival of CE. Despite the challenges, many of the common HPLC detectors have a CE complement, e.g., absorbance, fluorescence, conductivity, photodiode array, and mass spectroscopy. Small dimensions mean universal detectors such as refractive index cannot be used. A sample of detectors will be discussed. The technical aspects of each detector will not be covered except in relation to the CE instrument. Readers are advised to consult an instrumentation textbook for more details on theory of operation. 

Samples are introduced into the capillary by either electrokinetic or hydrodynamic or hydrostatic means. Electrokinetic injection is preferentially employed with packed or monolithic capillaries whereas hydrostatic injection systems are limited to open capillary columns and are primarily used in homemade instruments. Optical detection directly through the capillary at the opposite end of sample injection is the most employed detection mode, using either a photodiode array or fluorescence or a laser-induced fluorescence (LIF) detector. Less common detection modes include conductivity [1], amperometric [2], chemiluminescence [3], and mass spectrometric [4] detection. 

A laboratory-built scanning densitometer using a fiber optic bundle for illumination of the layer and collection of reflected light (or fluorescence) in conjunction with a photodiode array detector affords some attractive features that conventional scanning instruments lack [186,206,207]. These include the simultaneous recording of spectra from 198-612 nm with a spectral resolution of 0.8 nm simultaneous detection at multiple wavelengths a spatial resolution <0.16 mm and implementation of a wide... 

Boyer and coworkers were the first to develop instrumentation for near-infrared fluorescence immunoassays [117]. WiUiams and coworkers also developed instrumentation for detection of near-infrared fluorescence in sohd-phase immunoassays [118]. The instrument consists of a semiconductor laser coupled with a fiber-optic cable, a silicon photodiode for detection, a sample stage coupled to a motor drive, and a data acquisition device. The instrument could detect 500 pM concentrations of human immunoglobulin G (IgG) on a nitrocellulose matrix. The assay was performed in roughly two hours. The detection limits obtained on this instrument were comparable to that obtainable with ELISA. The assay developed by WiUiams suffers from excessive scatter generated from the membrane, nonspecific binding, and incompatibility with conventional microtiter plate immunoassay formats [140]. Patonay and coworkers developed a NIR fluorescence immunoassay apparatus that overcame many of these limitations. Baars and Patonay have evaluated a novel NIR dye NN382 (Fig. 14.25) for the ultrasensitive detection of peptides with capilary electrophoresis [141]. A solid-phase, NIR fluorescence immunoassay system was... 

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