12.14
Fluorescência e Fosforescência: Instrumentação
Fluorômetros e espectrofluorômetros são dois tipos de instrumentos usados para medir a fluorescência molecular. Esses instrumentos diferem na forma…
Fluorômetros e espectrofluorômetros são dois instrumentos usados para medir a fluorescência molecular, cada um apresentando uma fonte de luz, filtros ou monocromadores, célula de amostra, transdutor e processador de sinal.
Os fluorômetros usam lâmpadas de vapor de mercúrio de baixa pressão como fonte de excitação, empregando filtros de absorção ou interferência para selecionar comprimentos de onda de excitação e emissão.
Por outro lado, os espectrofluorômetros usam uma lâmpada de arco de xenônio de alta pressão e um monocromador para selecionar os comprimentos de onda de excitação e emissão.
Ambos os dispositivos detectam fluorescência através de um transdutor, como um tubo fotomultiplicador ou um dispositivo de carga acoplada, convertendo o sinal em uma medição elétrica.
A fosforescência molecular é medida usando um fosforímetro, que leva em conta o atraso entre a excitação e a emissão de fosforescência empregando helicópteros rotativos fora de fase.
Esses helicópteros bloqueiam alternadamente a emissão fluorescente durante a excitação e a fonte de excitação durante a medição da emissão fosforescente.
Aqui, a amostra é dissolvida em uma mistura de solvente orgânico e congelada a temperaturas de nitrogênio líquido ou imobilizada em um substrato sólido para evitar a desativação do estado excitado por conversão externa.
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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.