Fluorescence and Phosphorescence Instrumentation

The basic instrumentation for monitoring fluorescence and phosphorescence—a source of radiation, a means of selecting a narrow band of radiation, and a detector—are the same as those for absorption spectroscopy. The unique demands of both fluorescence and phosphorescence, however, require some modifications to the instrument designs for a filter photometer, single-beam spectrophotometer, double-beam spectrophotometer, or diode array spectrometer. The most important difference is that the detector cannot be placed directly across from the source. As shown here, if we place the detector along the source’s axis it will receive both the transmitted source radiation, PT, and the fluorescent, If, or phosphorescent, Ip, radiation. Instead, we rotate the director and place it at 90o to the source.


The illustration below shows two basic instruments for measuring fluorescence, each of which includes two wavelength selectors: one for selecting an excitation wavelength from the source and one for selecting the emission wavelength from the sample. A fluorimeter uses absorption or interference filters to select the excitation and emission wavelengths. The excitation source for a fluorimeter is usually a low-pressure Hg vapor lamp, which provides intense emission lines distributed throughout the ultraviolet and visible region (254, 312, 365, 405, 436, 546, 577, 691, and 773 nm). When a monochromator is used to select the excitation and emission wavelengths, the instrument is called a spectrofluorimeter. With a monochromator the excitation source is usually high-pressure Xe arc lamp, which has a continuous emission spectrum.


Instrumentation for molecular phosphorescence must discriminate between phosphorescence and fluorescence. Because the lifetime for fluorescence is shorter than that for phosphorescence, discrimination is achieved by incorporating a delay between exciting the sample and measuring phosphorescent emission. As illustrated here, we can use two out-of-phase choppers to block emission from reaching the detector when the sample is being excited, and to prevent source radiation from reaching the sample while we are measuring the phosphorescent emission.


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