What are the key differences between Absorbance and Luminiscence spectroscopic measurements?
The basic spectroscopic measurement system components are same for both the techniques. However, due to inherent differences between the two techniques some orientation and other changes become necessary in the system configuration.
The present article highlights the need for such differences.
Beam Direction
It Is essential to realize that in absorbance measurements both incident and transmitted beam after absorption by sample move in the same direction. In case of luminiscence the sample molecules emanate light universally in all directions. Absorbance measurements require the detector to be aligned in the same direction as the incident beam. However, in case of luminiscence the detector is aligned at right angles to the direction of the incident beam. This alignment ensures freedom of interference of luminescence light from transmitted light component.
Light Source
The fluorescence emission of a molecule increases with the intensity of the light source. A powerful light source contributes to enhancement of sensitivity. Tungsten filament and deuterium lamps are not suitable for fluorescence applications and the required source power is achieved through use of mercury vapour or xenon arc lamps. Luminiscence spectrometers also use laser excitation sources which produce a coherent monochromatic light beam which does not require the excitation monochromator. Certain light-sensitive samples require pulsed xenon sources to prevent sample degradation or photo bleaching
Excitation and Emissions Monochromators
Absorbance spectrophotometers require only a single monochromator in the beam path to isolate the required wavelength. The detector sees the same wavelength after absorption by the sample. However, in case of fluorescence measurements sample gives rise to a range of emitted wavelengths and with the help of the emission monochromator it is possible to isolate the wavelength for optimum sensitivity.
Sample Cells
Both absorbance and luminiscence measurements can be carried out using cuvettes or flow through cells. Glass cells can be used in absorbance and luminiscence measurements in the visible region but for both UV and visible regions quartz cells should be used. Absorbance measurements require precisely machined square cells. However, circular cells which are less expensive can be used for fluorescence measurements without sacrificing sensitivity.
Phosphorescence Measurements
Phosphorescence emissions take place after a significant time delay after the sample is irradiated. Higher sensitivity is achieved at sub ambient temperatures of liquid nitrogen at 77° K. At such low temperature of the solvent should exist as a clear rigid glass.
The sample is taken in a narrow quartz tube and in a liquid nitrogen dewar. However, room temperature studies can be carried out on some inorganic materials which show significant phosphorescence without any pre-treatment
It is possible to record simultaneously fluorescence and phosphorescence as such emissions occur at different wavelengths. Common interference between such measurements can be eliminated by using excitation and emission shutter mechanisms. When the incident light is cut off fluorescence ceases but phosphorescence continues. The mechanical movement of shutter can help differentiate between the two emissions.
Subsequent articles will cover key areas where fluorescence measurements have found major applications.
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