Monochromator, fluorescence

Luminescence. Both excitation and emission spectra were recorded using a Spex Fluorolog 202 B double monochromator fluorescence spectrometer. The spectrometer was operated in the front face mode with bandpass slits of 1.0 nm. 

Figure 9. Micrographs of 1S7 in polystyrene (50 wt%). Monochrome fluorescence image of the pattern (left) and close up ofa single dome (right), in transmission andfluorescence mode. The diameter of the domes are...

Fluorescence spectrometers for in vivo diagnostics are commonly based on fibre optic systems [30-33], The excitation light of a lamp or a laser is guided to the tissue (e.g. some specific organ) via glass fibre using appropriate optical filters (instead of an excitation monochromator). Fluorescence spectra are usually measured either via the same fibre or via a second fibre or fibre bundle in close proximity to the excitation fibre. Scanning monochromators or OMA systems as reported above are used for emission spectroscopy. 

An instrument for measuring fluorescence that uses a monochromator to select the excitation and emission wavelengths. 

Figure 8.28 shows how the X-rays fall on the solid or liquid sample which then emits X-ray fluorescence in the region 0.2-20 A. The fluorescence is dispersed by a flat crystal, often of lithium fluoride, which acts as a diffraction grating (rather like the quartz crystal in the X-ray monochromator in Figure 8.3). The fluorescence may be detected by a scintillation counter, a semiconductor detector or a gas flow proportional detector in which the X-rays ionize a gas such as argon and the resulting ions are counted.

Fluorometry and Phosphorimetry. Modem spectrofluorometers can record both fluorescence and excitation spectra. Excitation is furnished by a broad-band xenon arc lamp foUowed by a grating monochromator. The selected excitation frequency, is focused on the sample the emission is coUected at usuaUy 90° from the probe beam and passed through a second monochromator to a photomultiplier detector. Scan control of both monochromators yields either the fluorescence spectmm, ie, emission intensity as a function of wavelength X for a fixed X, or the excitation spectmm, ie, emission intensity at a fixed X as a function of X. Fluorescence and phosphorescence can be distinguished from the temporal decay of the emission. 

Fluorescence spectra were measured at wavelength scanning of tunable dye-laser. In spite of the monochromic excitation the fluorescence spectmm has quite complex composition. Such variety of wavelengths allows to optimize fluorescence excitation and registration for any technological conditions. 

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. 

When recording excitation and fluorescence spectra it must be ensured that monochromatic light falls on the detector This can best be verified in instruments built up on the kit principle or in those equipped with two monochromators (spectrofluonmeters) The majority of scanners commercially available at the moment do not allow of such an optical train, which was realized in the KM3 chromatogram spectrometer (Zeiss) So such units are not able to generate direct absorption or fluorescence spectra for the charactenzation of fluorescent components... 

Some commercial spectrophotometers have fluorescence attachments which allow the sample to be irradiated from an ancillary source and the resulting fluoresence to pass through the monochromator for spectral analysis. 

The ultimate in fluorescence detection is a detector that uses a monochromator to select the excitation wavelength and a second monochromator to select the wavelength of the fluorescent light. This instrument is ideal, giving the maximum versatility and allowing the... 

Figure 2. Schematic of the SLM 8000 fluorometer. Excitation occurs through the excitation monochromator, and light emitted from the sample is observed in as many as four different positions. Photomultiplier tubes (PMTs) A, B, and C can be used to monitor fluorescence or right-angle light scatter through the monochromator (PMT A) or through filters (PUT B and C), and position D measures transmittance. Three channels can be monitored simultaneously with measurements being acquired at intervals of 1 s or less. The data are stored by the computer for subsequent manipulation.

Procedures for calibrating both monochromators in a fluorescence spectrometer using narrow line sources have been discussed (IS,18) care must be taken with placement of the calibration source. 

In the equation, the subscripts 1 and 2 refer to the reference compound and the compound of interest, respectively, is the intensity of the fluorescent signal of each compound measured as peak height in centimeters, 8 is the molar absorptivity, c is the concentration in moles per liter, and is the fluorescence quantum yield. In this application, i is set at 1.00. The concentrations of the solutions that were tested ranged from 10 to 10 M. The solutions run at the higher concentrations were all checked for self-quenching, but none was found. All measurements, except the fluorescence-versus-solvent study, were made in 0.1-N phosphate buffer, pH 7.4. Slit settings on the Perkin-Elmer MPF-2A were 10 mp (nm) for both emission and excitation monochromators. 

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