12.14
Fluorescence and Phosphorescence: Instrumentation
Fluorometers and spectrofluorometers are two types of instruments used for measuring molecular fluorescence. These instruments differ in how they sele…
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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Q1: What is the main difference between a fluorometer and a spectrofluorometer?
Fluorometers use low-pressure mercury vapor lamps and absorption or interference filters to select excitation and emission wavelengths, making them suitable for quantitative measurements but unable to record spectra. Spectrofluorometers employ high-pressure xenon arc lamps and monochromators to select wavelengths, allowing them to record excitation or emission spectra for more detailed fluorescence analysis.
Q2: How do transducers in fluorometers and spectrofluorometers convert light signals?
Both instruments use transducers such as photomultiplier tubes or charge-coupled devices to detect fluorescence emission and convert the light signal into an electrical measurement. The signal processor then analyzes this electrical output, enabling quantitative determination of fluorescence intensity from the sample.
Q3: Why is a phosphorimeter different from a fluorometer?
A phosphorimeter measures phosphorescence, which has a longer lifetime than fluorescence. It uses out-of-phase rotating choppers to discriminate between the two: one blocks fluorescent emission during excitation, while the other blocks the excitation source during phosphorescent emission measurement, allowing accurate phosphorescence detection.
Q4: What sample preparation methods are used for phosphorescence measurements?
Samples for phosphorescence are typically dissolved in an organic solvent mixture and frozen at liquid nitrogen temperatures to form an optically clear solid matrix that minimizes external conversion, a radiationless relaxation process. Alternatively, samples can be immobilized on a solid substrate, enabling phosphorescence measurement at room temperature.
Q5: What type of light source does a spectrofluorometer use and why?
Spectrofluorometers use high-pressure xenon arc lamps that produce a continuum emission spectrum across a wide range of wavelengths. This continuous spectrum allows the monochromator to select any desired excitation or emission wavelength, enabling detailed spectral analysis and flexibility in fluorescence measurements.
Q6: How do choppers in a phosphorimeter prevent fluorescence interference?
Phosphorimeters employ two out-of-phase rotating choppers that work sequentially: one blocks fluorescent emission when the excitation source illuminates the sample, and the other blocks the excitation source when measuring phosphorescent emission. This temporal separation allows accurate phosphorescence detection without fluorescence interference.
Q7: What is external conversion and how does it affect phosphorescence measurements?
External conversion is a radiationless relaxation process where energy transfers to the solvent or sample matrix instead of being emitted as light, causing loss of the excited state. To prevent this deactivation, phosphorescence samples are frozen at liquid nitrogen temperatures or immobilized on solid substrates, preserving the excited state long enough for phosphorescence emission.