Spectrofluorometer uncorrected

Fluorescence Instrumentation and Measurements. Fluorescence spectra of the FS samples were obtained on a steady state spectrofluorometer of modular construction with a 1000 W xenon arc lamp and tandem quarter meter excitation monochromator and quarter meter analysis monochromator. The diffraction gratings In the excitation monochromators have blaze angles that allow maximum light transmission at a wavelength of 240 nm. Uncorrected spectra were taken under front-face Illumination with exciting light at 260 nm. Monomer fluorescence was measured at 280 nm and exclmer fluorescence was measured at 330 nm, where there Is no overlap of exclmer and monomer bands. 

Optical rotations, accurate within 0.003°, were measured on Perkin-Elmer 241 and 141 polarimeters using a 1 dm cell. All reported rotations (from which residual rotations from solvent impurities have been subtracted) are the difference between solution and pure solvent measurements. Uncorrected steady-state emission spectra were obtained from room temperature samples on a Perkin-Elmer MPF-2A or Spex Fluorolog spectrofluorometer. 

Medium-priced uncorrected spectrofluorometers, inexpensive enough to be used for routine analytical work. (2) Uncorrected research spectrofluorometers, more expensive and adaptable for many different types of investigation one such instrument, for example, can be fitted with a phosphorescence attachment (including a rotating can) that converts it into a spectrophosphorimeter. (3) Corrected, or absolute, spectrofluorometers that directly record fluorescence excitation and emission... 

Components of an Uncorrected Spectrofluorometer. The design of filter fluorometers and spectrofluorometers has already been discussed here, we shall describe in more detail the components of an uncorrected spectrofluorometer (see Fig. 9.7). 

Figure 9.7. Schematic diagram of an uncorrected spectrofluorometer. A high-pressure xenon arc is the usual source. The gratings can be adjusted manually, or driven by a motor for recording. The usual detector is the 1P21 photomultiplier tube. The response of the tube is displayed on a meter and is sometimes recorded. From G. H. Schenk, Absorption of Light and Ultraviolet Radiation, Boston Allyn and Bacon, 1973, p 278, by permission of the publisher.

A xenon-arc lamp is used as the source in most spectrofluorometers, since it emits continuously over the range 200-700 nm (see Fig. 9.4C) and hence can be used to obtain fluorescence excitation spectra as well as emission spectra. (Emission spectra for many substances could be obtained with a mercury-arc source, but excitation spectra could not, because the emission is discontinuous and the frequency range is so limited.) However, in uncorrected instruments using a xenon-arc lamp, no correction is made for the variation in intensity of the source with changing wavelength. 

All spectrofluorometers are equipped with high-gain photomultipliers as detectors this partly compensates for the smaller amount of energy that gratings transmit compared to filters. However, uncorrected spectrofluorometers do not compensate for the variable response of the photomultiplier at varying wavelengths, so the measured relative intensities of two fluorescence-emission bands of a given species are not a correct indication of the true intensities. 

Medium-Priced Uncorrected Spectrofluorometers. There are now available a number of so-called medium-priced spectrofluorometers. Some are equipped with a mercury source for optimum trace analysis, but a xenon source is also available. The important advantage of these instruments is that the variable-slit-width emission grating allows one to measure fluorescence at the wavelength of maximum emission using as narrow or wide a bandwidth as allowed by the instrumental design. 

Uncorrected Spectrofluorometers for Research. This category of instrument is adaptable to all kinds of research, and is generally much more expensive than the medium-priced spectrofluorometers. The best known example is the Aminco-Bowman SPF instrument. The latter can be used as a spectrofluorometer, but with the attachment of an Aminco-Keirs phosphoroscope, it can also be used as a spectrophosphorimeter. When used as a spectrofluorometer, it consists of essentially the same components as those shown in Figure 9.7,... 

Corrected Spectrofluorometers. To obtain absolute spectra, the spectra obtained on uncorrected spectrofluorometers must be corrected point by point for instrumental parameters that vary with wavelength. Such corrections are tedious, and it is desirable to obtain spectra on a corrected spectrofluorometer if possible. These instruments correct for variations in the intensity of the xenon source so that the sample is excited at constant energy at all wavelengths, and for variations with wavelength in the response of the photomultiplier emission spectra are presented directly in quanta per unit bandwidth. 

As an example of the first technique, consider the determination of the aromatic hydrocarbon, anthracene, in the presence of its isomer, phenanthrene. Phenan-threne does not absorb in the ultraviolet at wavelengths longer than 360 nm. Since anthracene has an excitation band above 360 nm, it is possible to excite only anthracene. In an experimental study [24] of this type of mixture, the actual wavelength used was 365 nm. As can be seen from Figure 9.8, the best wavelength at which to measure the fluorescence of anthracene on an uncorrected spectrofluorometer would be about 400 nm. It is also possible to determine phenanthrene by use of the second technique. Phenanthrene and anthracene are excited intensely at 265 nm, but phenanthrene fluoresces at 350 nm where anthracene does not (Fig. 9.8). 

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