Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 31
1. The light intensity from the excitation source
is a function of wavelength. Even if the intensity
of the exciting light is monitored via the
beam splitter shown in Figure 2.1, and corrected
by division, the response of the reference
solution or detector may be dependent
upon wavelength.
2. The transmission efficiency of the excitation
monochromators is a function of wavelength.
3. The optical density of the sample may exceed
the linear range, which is about 0.1 absorbance
units, depending upon sample geometry.
Emission spectra are recorded by choosing an appropriate
excitation wavelength and scanning wavelength with
the emission monochromator. In addition to the factors discussed
above, the emission spectrum is further distorted by
the wavelength-dependent efficiency of the emission monochromator
and photomultiplier. The emission spectrum can
also be distorted by absorption of the sample.
2.2. LIGHT SOURCES
We now describe the individual components of a spectrofluorometer.
The general characteristics of these components
are considered along with the reason for choosing specific
components. Understanding the characteristics of these
components allows one to understand the capabilities and
limitations of spectrofluorometers. We will first consider
light sources.
2.2.1. Arc and Incandescent Xenon Lamps
At present the most versatile light source for a steady-state
spectrofluorometer is a high-pressure xenon (Xe) arc lamp.
These lamps provide a relatively continuous light output
from 250 to 700 nm (Figure 2.5), with a number of sharp
lines occurring near 450 nm and above 800 nm. Xenon arc
lamps emit a continuum of light as a result of recombination
of electrons with ionized Xe atoms. These ions are generated
by collisions of Xe atoms with the electrons that flow
across the arc. Complete separation of the electrons from
the atoms yields the continuous emission. Xe atoms that are
in excited states but not ionized yield lines rather than broad
emission bands. The peaks near 450 nm are due to these
excited states. The output intensity drops rapidly below 280
nm. Furthermore, many Xe lamps are classified as being
ozone-free, meaning that their operation does not generate
Figure 2.5. Spectral output of a continuous xenon arc lamp and a
xenon flash lamp. Revised from [3]. Courtesy of Newport Corp.
ozone in the surrounding air. The quartz envelope used in
such ozone-free lamps does not transmit light with wavelengths
shorter than 250 nm, and the output of such lamps
decreases rapidly with decreasing wavelength.
The wavelength-dependent output of Xe lamps is a
major reason for distortion of the excitation spectra of compounds
that absorb in the visible and ultraviolet. To illustrate
this effect Figure 2.6 shows corrected and uncorrected
excitation fluorescein spectra. The uncorrected spectra are
recorded emission intensities with no correction for wavelength-dependent
excitation intensity. The uncorrected
excitation spectrum displays a number of peaks near 450
nm. These peaks are due to the output of the Xe lamp,
which also displays peaks near 450 nm (Figure 2.5). Also
shown in Figure 2.6 is the excitation spectrum, corrected for
the wavelength-dependent output of the Xe arc lamp. A
Figure 2.6. Corrected and uncorrected excitation spectra of fluorescein.
From [4].