Fluorometers and spectrofluorometers are two types of instruments used for measuring molecular fluorescence. These instruments differ in how they select excitation and emission wavelengths and the type of light sources they utilize. Fluorometers use absorption interference filters to choose excitation and emission wavelengths. The excitation source in a fluorometer is typically a low-pressure mercury vapor lamp that emits intense lines distributed throughout the ultraviolet and visible regions. While fluorometers are suitable for quantitative work and can measure fluorescence intensity, they cannot record excitation or emission spectra.
In contrast, spectrofluorometers use a monochromator to select excitation and emission wavelengths. These instruments employ a high-pressure xenon arc lamp as the excitation source, which produces a continuum emission spectrum. Spectrofluorometers can record excitation or emission spectra, allowing for a more detailed fluorescence analysis. They are also suitable for quantitative measurements.
In the case of molecular phosphorescence, specialized instrumentation, known as phosphorimetery, is required to discriminate between phosphorescence and fluorescence. Phosphorescence has a longer lifetime than fluorescence, so a delay is incorporated between excitation and measuring phosphorescent emission. This can be achieved using two choppers that rotate out of phase. The choppers block the fluorescent emission when the excitation source is focused on the sample and block the excitation source when measuring phosphorescent emission. To prevent deactivation of the excited state by external conversion, a form of radiationless relaxation in which energy is transferred to the solvent or sample matrix instead of being emitted as light. Samples for phosphorescence measurements are typically dissolved in a suitable organic solvent and frozen at liquid nitrogen temperatures. This forms an optically clear solid matrix that minimizes external conversion. Alternatively, the sample can be immobilized on a solid substrate, enabling phosphorescence measurement at room temperature.
Fluorometers and spectrofluorometers are two instruments used to measure molecular fluorescence, each featuring a light source, filters or monochromators, sample cell, transducer, and signal processor.
Fluorometers use low-pressure mercury vapor lamps as the excitation source, employing absorption or interference filters to select excitation and emission wavelengths.
Conversely, spectrofluorometers use a high-pressure Xenon arc lamp and a monochromator to select excitation and emission wavelengths.
Both devices detect fluorescence through a transducer, such as a photomultiplier tube or a charge-coupled device, converting the signal into an electrical measurement.
Molecular phosphorescence is measured using a phosphorimeter, which accounts for the delay between excitation and phosphorescence emission by employing out-of-phase rotating choppers.
These choppers alternately block fluorescent emission during excitation and the excitation source during phosphorescent emission measurement.
Here, the sample is either dissolved in an organic solvent mixture and frozen at liquid nitrogen temperatures or immobilized on a solid substrate to prevent deactivation of the excited state by external conversion.
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