Fluorescence Spectrometry
451
excessive dark currents. In case of red/NIR fluorescence, the bialkali photocathode
is less helpful. Multi-alkali photocathode shows good sensitivity up 800 nm without the problem of dark current. PMT principle includes the counting of photons
and its function over a limited range of intensity above to linear count rate can be
the main disadvantages for steady-state measurements; therefore, the analog mode
is preferred in which individual pulses are averaged. At present, charge-coupled
detectors (CCDs) have obtained a growing interest in spectrofluorometers. CCDs
are imaging detectors with fantastic sensitivity and linear dynamic range having 10
6
or more pixels. Each pixel works as a detector.
2.2.4 Sample Stages
Most of the fluorescence measurements are performed in the solution state contained
in a cuvette. Cuvettes may be of different shapes like circular, square, or rectangular.
Square cuvettes are most precise for measurements since the parameters of pathlength
and parallel beam configurations are easier to maintain during the manufacturing.
In the recently developed Agilent Cary Eclipse fluorescence spectrophotometer, the
fiber optic-based remote measurement technology has been incorporated. With fiber
optics, one can measure a samples that would normally be too large to fit into the
sample compartment, providing the benefits of non-destructive analysis, and reduced
sample preparation. Using this arrangement, the fluorescence of solid samples can
also be measured.
2.3 Interfering Factors in Fluorescence Spectrometry
Several factors including the instrumentation influence the fluorescence emission.
The main interfering factors are discussed below.
2.3.1 Concentration of Sample
It is described in Sect. 1.1 (Eq. 1); that the fluorescence intensity varies linearly as
a function of path length and concentration of the solution. Particularly, the fluorescence intensity is directly proportional to the source power. Sometimes, we also
observed that on increasing the concentration of a solution, fluorescence intensity
decreases. This phenomenon is often termed as “concentration quenching” which is
attributed to the formation of non-fluorescent dimers due to an increase in the concentration of a solution. This effect is usually observed in the case of dyes wherein
there is sufficient overlap of their absorption and fluorescence spectra. The nonlinear
dependence of the fluorescence intensity on the concentration is caused by the inner
filter effect in which most of the excitation light is absorbed by the fluorophore that
reduces the fluorescence intensity. In an ideal case, the total fluorescence intensity
451
excessive dark currents. In case of red/NIR fluorescence, the bialkali photocathode
is less helpful. Multi-alkali photocathode shows good sensitivity up 800 nm without the problem of dark current. PMT principle includes the counting of photons
and its function over a limited range of intensity above to linear count rate can be
the main disadvantages for steady-state measurements; therefore, the analog mode
is preferred in which individual pulses are averaged. At present, charge-coupled
detectors (CCDs) have obtained a growing interest in spectrofluorometers. CCDs
are imaging detectors with fantastic sensitivity and linear dynamic range having 10
6
or more pixels. Each pixel works as a detector.
2.2.4 Sample Stages
Most of the fluorescence measurements are performed in the solution state contained
in a cuvette. Cuvettes may be of different shapes like circular, square, or rectangular.
Square cuvettes are most precise for measurements since the parameters of pathlength
and parallel beam configurations are easier to maintain during the manufacturing.
In the recently developed Agilent Cary Eclipse fluorescence spectrophotometer, the
fiber optic-based remote measurement technology has been incorporated. With fiber
optics, one can measure a samples that would normally be too large to fit into the
sample compartment, providing the benefits of non-destructive analysis, and reduced
sample preparation. Using this arrangement, the fluorescence of solid samples can
also be measured.
2.3 Interfering Factors in Fluorescence Spectrometry
Several factors including the instrumentation influence the fluorescence emission.
The main interfering factors are discussed below.
2.3.1 Concentration of Sample
It is described in Sect. 1.1 (Eq. 1); that the fluorescence intensity varies linearly as
a function of path length and concentration of the solution. Particularly, the fluorescence intensity is directly proportional to the source power. Sometimes, we also
observed that on increasing the concentration of a solution, fluorescence intensity
decreases. This phenomenon is often termed as “concentration quenching” which is
attributed to the formation of non-fluorescent dimers due to an increase in the concentration of a solution. This effect is usually observed in the case of dyes wherein
there is sufficient overlap of their absorption and fluorescence spectra. The nonlinear
dependence of the fluorescence intensity on the concentration is caused by the inner
filter effect in which most of the excitation light is absorbed by the fluorophore that
reduces the fluorescence intensity. In an ideal case, the total fluorescence intensity