450
D. P. Singh et al.
to detect low light levels resulting in high efficiency; however, the transmission efficiency depends upon the polarization of the light. It is observed that the transmission
efficiency for vertically polarized light is higher at shorter wavelengths. The selection of resolution in monochromator is a secondary parameter because the emission
spectrum rarely exhibits peaks having linewidth less than 5 nm. The intensity of light
that passes via a monochromator is proportional to the square of the slit width. Larger
slit width provides an increased signal level and thus, a high signal-to-noise ratio;
whereas, the narrow slit width yields higher resolution. Monochromators include
planar or concave gratings. Planar gratings are produced mechanically, whereas, concave gratings can be obtained by holographic or photoresist methods. If the employed
grating is imperfect in monochromator; it induces stray light transmission and ghost
images. In fluorescence spectrophotometer, two monochromators i.e., excitation and
emission, are used. Different types of monochromators are listed below:
• Prism monochromators
• Grating monochromators
• Single, double and quadruple monochromators.
In spectrofluorometer, the use of optical filters is preferred to compensate for the
less-than-ideal behavior of monochromator. Optical filters provide maximum sensitivity. The nomenclature of filters is assigned according to their colors, like blue
glass, green glass, etc. During the last decade, immense interest has been generated
in designing the thin-film optical filters. Long-pass filters have a sharp cut on the
transmission above 325 or 488 nm. The maximum sensitivity can be achieved above
the cut-on wavelength as the transmission is close to 100%. One should remember
that the polarization or anisotropy measurements are preferred using filters in comparison to the monochromator. As we know that the scattered light is 100% polarized;
therefore, a small scattering leads to serious errors.
2.2.3 Detectors
Usually, photomultiplier tubes (PMTs) are used as detectors in almost all spectrofluorometers. A PMT is composed of a photocathode and a series of dynodes that act as
an amplification stage. PMTs are considered as current sources in which current is
directly proportional to the intensity of light. The photocathode is a metallic thin film
which ejects electrons when photons are incident on it. The emission of electrons
and their efficiency depend upon the incident wavelength of light. The photocathode
is held at a very high negative voltage (i.e., −1000–−2000 V). The dynodes are also
held at negative voltage and the potential difference between the photocathode and
the dynode and between successive dynodes is constant (≈ −50–200 V), and fixed by
using a Zener diode. These emitted electrons reach the anode in the form of a current
pulse. PMTs are very useful for low-light intensity detection. The anode current follows direct proportionality with the intensity of light; however, at high-intensity, the
potential of the photocathode decreases due to its limited current-carrying capacity.
The excess photocurrent can destroy the photocathode resulting in loss of gain and
D. P. Singh et al.
to detect low light levels resulting in high efficiency; however, the transmission efficiency depends upon the polarization of the light. It is observed that the transmission
efficiency for vertically polarized light is higher at shorter wavelengths. The selection of resolution in monochromator is a secondary parameter because the emission
spectrum rarely exhibits peaks having linewidth less than 5 nm. The intensity of light
that passes via a monochromator is proportional to the square of the slit width. Larger
slit width provides an increased signal level and thus, a high signal-to-noise ratio;
whereas, the narrow slit width yields higher resolution. Monochromators include
planar or concave gratings. Planar gratings are produced mechanically, whereas, concave gratings can be obtained by holographic or photoresist methods. If the employed
grating is imperfect in monochromator; it induces stray light transmission and ghost
images. In fluorescence spectrophotometer, two monochromators i.e., excitation and
emission, are used. Different types of monochromators are listed below:
• Prism monochromators
• Grating monochromators
• Single, double and quadruple monochromators.
In spectrofluorometer, the use of optical filters is preferred to compensate for the
less-than-ideal behavior of monochromator. Optical filters provide maximum sensitivity. The nomenclature of filters is assigned according to their colors, like blue
glass, green glass, etc. During the last decade, immense interest has been generated
in designing the thin-film optical filters. Long-pass filters have a sharp cut on the
transmission above 325 or 488 nm. The maximum sensitivity can be achieved above
the cut-on wavelength as the transmission is close to 100%. One should remember
that the polarization or anisotropy measurements are preferred using filters in comparison to the monochromator. As we know that the scattered light is 100% polarized;
therefore, a small scattering leads to serious errors.
2.2.3 Detectors
Usually, photomultiplier tubes (PMTs) are used as detectors in almost all spectrofluorometers. A PMT is composed of a photocathode and a series of dynodes that act as
an amplification stage. PMTs are considered as current sources in which current is
directly proportional to the intensity of light. The photocathode is a metallic thin film
which ejects electrons when photons are incident on it. The emission of electrons
and their efficiency depend upon the incident wavelength of light. The photocathode
is held at a very high negative voltage (i.e., −1000–−2000 V). The dynodes are also
held at negative voltage and the potential difference between the photocathode and
the dynode and between successive dynodes is constant (≈ −50–200 V), and fixed by
using a Zener diode. These emitted electrons reach the anode in the form of a current
pulse. PMTs are very useful for low-light intensity detection. The anode current follows direct proportionality with the intensity of light; however, at high-intensity, the
potential of the photocathode decreases due to its limited current-carrying capacity.
The excess photocurrent can destroy the photocathode resulting in loss of gain and