Fluorescence measurements instrumentation

Photomultipliers are appreciably more sensitive sensors than the eye in their response to line or continuum sources. Monochromators are fitted to the light beam in order to be able to operate as substance-speciflcally as possible [5]. Additional filter combinations (monochromatic and cut-off filters) are needed for the measurement of fluorescence. Appropriate instruments are not only suitable for the qualitative detection of separated substances (scanning absorption or fluorescence along the chromatogram) but also for characterization of the substance (recording of spectra in addition to hR and for quantitative determinations. 

Note. It is good practice to make the fluorescence measurements for samples and standards as close together as possible to minimise any drift in instrument response. 

Within the confines of the present volume it is not possible to provide a detailed discussion of instrumentation for atomic fluorescence spectroscopy. An instrument for simultaneous multi-element determination described by Mitchell and Johansson53 has been developed commercially. Many atomic absorption spectrophotometers can be adapted for fluorescence measurements and details are available from the manufacturers. Detailed descriptions of atomic fluorescence spectroscopy are to be found in many of the volumes listed in the Bibliography (Section 21.27). 

The instrumentation required for atomic fluorescence measurements is simpler than that used for absorption. As the detector is placed so as to avoid receiving radiation directly from the lamp, it is not strictly necessary to use a sharp-line source or a monochromator. Furthermore, fluorescence intensities are directly related to the intensity of the primary radiation so that detection limits can be improved by employing a high-intensity discharge lamp. 

Instruments. A schematic diagram of the aicrocoaputer-controlled system for the fluorescence measurement just upon ablation is shown in Figure 1. The excitation light source was a 351 nm laser (Lumonics He-400, 15 ns) and fresh surface of PVCz film was examined in air. 

A wide variety of X-ray fluorescence spectrometers may be used, depending on the nature and complexity of the sample, and on the number of samples to be analysed. To prove this and to indicate the substantial influence which the sample has on the choice of measuring instrument, let us consider some of the main characteristics of some X-ray fluorescence instruments used today [38]. These are shown in Table 14.11. 

Obtain fluorescence measurements (F) on all of your standards, the unknown, and control. Use 5% acetic acid for a blank. Your instructor will demonstrate the use of the instrument, and explain how it was set up to measure riboflavin. 

Commercially available instrumentation for absorbance and fluorescence measurements in the NIR region is scarce. Conventional absorbance spectrophotometers... 

In this section we will review the application of near-IR system instrumentation to the most commonly encountered fluorescence measurements such as steady-state spectra, excited state lifetimes, anisotropy, microscopy, multiplexing, high-performance liquid chromatography (HPLC), and sensors. 

Bradshaw, J. D., M. O. Rodgers, S. T. Sandholm, S. KeSheng, and D. D. Davis, A Two-Photon Laser-Induced Fluorescence Field Instrument for Ground-Based and Airborne Measurements of Atmospheric NO, J. Geophys. Res., 90, 12861-12873 (1985). 

This paper reports on research involved the design, construction, and evaluation of a portable instrument, a "luminoscope", for detecting skin contamination by coal tars via induced fluorescence. The instrument has been used in the laboratory to measure the fluorescence of various coal tars and recycle solvents from liquefaction processes spotted on filter paper on rat and on hamster skin. The practical use of the devices in field test measurements to monitor skin contamination of workers at coal gasifier is discussed. The paper also discusses the practicality and usefulness of the luminescence method for detecting skin contamination. 

For sensors that are truly mass sensitive and for which the mass flow of sample through the sensing element is held constant as a function of pressure (for example, by use of electronic mass-flow controllers), instrument response is proportional to the mixing ratio independent of the pressure. For concentration-sensitive detectors, such as simple spectrophotometric instruments measuring absorbance or fluorescence, instrument response is a function of the absolute concentration, and the response will decrease for a constant mixing ratio as the pressure decreases. For example, the response of a pulsed fluorescence SO instrument sampling air containing a fixed... 

Figure 6. Instrumental schematic for vacuum UV photofragmentation-laser induced fluorescence measurement of ammonia SHGC, second harmonic generation crystal SFMC, sum frequency mixing crystal BS, beam splitter BD, beam dump TP, turning prism CL, cylindrical lens R, reflector TD, trigger diode OSC, oscillator cell AMP, amplifier cell BE, beam expander G, grating OC, output coupler M, mirror BC, beam combiner L, lens A, aperture PD, photodiode SC, sample cell RC, reference cell FP, filter pack SAM.PMT, sample cell photomultiplier REF.PMT, reference cell photomultiplier PP, additional photomultiplier port EX, exhaust and CGI, calibration gas inlet to flow line. (Reproduced with permission from reference 15. Copyright 1990 Optical Society of America.)...

In the direct competitive ELISA, the analyte specific antibodies are first coated on a solid phase. The sample or standard solution of analyte is generally incubated simultaneously with the analyte enzyme conjugate or incubated separately in two steps. The amount of enzyme bound to the plate is then determined by incubation with a chromogenic substrate solution. The resulting color/fluorescence, which is inversely proportional to the analyte concentration present in the sample, is then measured instrumentally or by visual comparison with the standards. 

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