Fluorescence Spectrometry
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2.2.1 Excitation Light Source
A normal fluorescence spectrophotometer consists of a xenon lamp as a source of
excitation. Xenon lamps are effectively compatible with these spectrophotometers
due to their high and uniform intensity for a wide range of excitation wavelengths.
Currently, high-pressure xenon (Xe) arc lamps are commonly used as a light source in
fluorescence spectrophotometers. It emits a continuous light of wavelengths ranging
from 250 to 700 nm. In the Xe arc lamp, continuous light output is achieved due to the
recombination of electrons and ionized Xe atoms. The excited Xe atoms (not ionized)
provide a line spectrum rather than a broad emission band. The use of these lamps
doesn’t generate ozone in the surrounding environment. Some compact fluorometers
also use Xe flash lamp. The output of such lamps is higher in the UV region and
the peak intensity is higher than that of the continuous arcs. In addition to this,
high-pressure mercury lamps, Xe-Hg arc lamps, quartz-tungsten halogen lamps and
low-pressure Hg and Hg-Ar lamps can also be used according to necessity. In highpressure Hg lamps, the intensity is concentrated in lines and excitation wavelengths
are limited to Hg lines only. In the Xe-Hg arc lamps, the intensity is higher in the
UV region and output is dominated by the Hg lines. The operating temperature is
one of the drawbacks of such lamps. The quartz-tungsten lamps are getting attention
if fluorophores absorb red/NIR light. The recent development of LED light sources
has also been employed in the spectrofluorometer. Such light sources do not generate
infrared; therefore, additional heat filters are not needed and provide a wide range
of excitation wavelengths. Due to their long life and low power consumption, such
sources are first choices of instrumental companies nowadays. Contrary to LEDs,
laser diodes emit monochromatic light, which makes them a suitable light source for
the spectrofluorometer that has a wavelength range of 405–1500 nm.
2.2.2 Monochromaters and Filters
A monochromator is an optical device that separates polychromatic light, such as
sunlight or light coming from a lamp, into the constituent colors or wavelengths.
A light wave that contains a single wavelength is called a monochromatic light. A
monochromator allows a narrow band of these individual wavelengths to be chosen as excitation wavelength. A monochromator consists of a dispersive element,
an entrance slit and mirrors to create the parallel beam of light and an exit slit and
mirrors to extract the monochromatic light. Breaking down light into its constituent
wavelengths is known as “dispersion”, and an element that yields this property is
known as a “dispersive element”. The prism is a commonly used dispersive element.
The diffraction grating is another well-known dispersive element that is used in the
modern spectrophotometers. When white light is incident on a diffraction grating,
it splits into seven colors (violet to red) as shown in Fig. 8. The diffraction grating
can be rotated to change the color of the light extracted through a slit following a
similar mechanism like a prism. A monochromator should have low stray levels in
order to avoid the problems due to the scattering of light. Monochromators are able
449
2.2.1 Excitation Light Source
A normal fluorescence spectrophotometer consists of a xenon lamp as a source of
excitation. Xenon lamps are effectively compatible with these spectrophotometers
due to their high and uniform intensity for a wide range of excitation wavelengths.
Currently, high-pressure xenon (Xe) arc lamps are commonly used as a light source in
fluorescence spectrophotometers. It emits a continuous light of wavelengths ranging
from 250 to 700 nm. In the Xe arc lamp, continuous light output is achieved due to the
recombination of electrons and ionized Xe atoms. The excited Xe atoms (not ionized)
provide a line spectrum rather than a broad emission band. The use of these lamps
doesn’t generate ozone in the surrounding environment. Some compact fluorometers
also use Xe flash lamp. The output of such lamps is higher in the UV region and
the peak intensity is higher than that of the continuous arcs. In addition to this,
high-pressure mercury lamps, Xe-Hg arc lamps, quartz-tungsten halogen lamps and
low-pressure Hg and Hg-Ar lamps can also be used according to necessity. In highpressure Hg lamps, the intensity is concentrated in lines and excitation wavelengths
are limited to Hg lines only. In the Xe-Hg arc lamps, the intensity is higher in the
UV region and output is dominated by the Hg lines. The operating temperature is
one of the drawbacks of such lamps. The quartz-tungsten lamps are getting attention
if fluorophores absorb red/NIR light. The recent development of LED light sources
has also been employed in the spectrofluorometer. Such light sources do not generate
infrared; therefore, additional heat filters are not needed and provide a wide range
of excitation wavelengths. Due to their long life and low power consumption, such
sources are first choices of instrumental companies nowadays. Contrary to LEDs,
laser diodes emit monochromatic light, which makes them a suitable light source for
the spectrofluorometer that has a wavelength range of 405–1500 nm.
2.2.2 Monochromaters and Filters
A monochromator is an optical device that separates polychromatic light, such as
sunlight or light coming from a lamp, into the constituent colors or wavelengths.
A light wave that contains a single wavelength is called a monochromatic light. A
monochromator allows a narrow band of these individual wavelengths to be chosen as excitation wavelength. A monochromator consists of a dispersive element,
an entrance slit and mirrors to create the parallel beam of light and an exit slit and
mirrors to extract the monochromatic light. Breaking down light into its constituent
wavelengths is known as “dispersion”, and an element that yields this property is
known as a “dispersive element”. The prism is a commonly used dispersive element.
The diffraction grating is another well-known dispersive element that is used in the
modern spectrophotometers. When white light is incident on a diffraction grating,
it splits into seven colors (violet to red) as shown in Fig. 8. The diffraction grating
can be rotated to change the color of the light extracted through a slit following a
similar mechanism like a prism. A monochromator should have low stray levels in
order to avoid the problems due to the scattering of light. Monochromators are able