Fluorescence and Phosphorescence: Instrumentation

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.

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.

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.

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.

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.

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.

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.