12.14
Fluorescencia y fosforescencia: instrumentación
Los fluorómetros y espectrofluorómetros son dos tipos de instrumentos que se utilizan para medir la fluorescencia molecular. Estos instrumentos se dif…
Los fluorómetros y espectrofluorómetros son dos instrumentos utilizados para medir la fluorescencia molecular, cada uno con una fuente de luz, filtros o monocromadores, celda de muestra, transductor y procesador de señal.
Los fluorómetros utilizan lámparas de vapor de mercurio de baja presión como fuente de excitación, empleando filtros de absorción o interferencia para seleccionar las longitudes de onda de excitación y emisión.
Por el contrario, los espectrofluorómetros utilizan una lámpara de arco de xenón de alta presión y un monocromador para seleccionar las longitudes de onda de excitación y emisión.
Ambos dispositivos detectan la fluorescencia a través de un transductor, como un tubo fotomultiplicador o un dispositivo de carga acoplada, convirtiendo la señal en una medición eléctrica.
La fosforescencia molecular se mide utilizando un fosforímetro, que explica el retraso entre la excitación y la emisión de fosforescencia mediante el empleo de picadores giratorios fuera de fase.
Estos picadores bloquean alternativamente la emisión fluorescente durante la excitación y la fuente de excitación durante la medición de la emisión fosforescente.
En este caso, la muestra se disuelve en una mezcla de disolventes orgánicos y se congela a temperaturas de nitrógeno líquido o se inmoviliza sobre un sustrato sólido para evitar la desactivación del estado excitado por conversión externa.
View the full transcript and gain access to JoVE Core videos
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.