BioPHYSICAL chemistry

The relationship between emission and absorption spectra

The key features of absorbance spectra are
the wavelength of the peak of the band, the
width, and intensity (Figure 14.12). The
same features are used to describe fluores-
cence spectra. In comparing fluorescence
and absorbance, the peak of the fluores-
cence is always red-shifted, or equivalently
at a lower energy, with respect to the
absorbance spectrum. Second, the fluores-
cence spectrum looks rather like a mirror
image of the absorbance spectrum in terms
of width and intensity.
The reasons for this correlation can be
understood from a diagram of the elec-
tronic states (Figure 14.13). The majority
of the molecules absorb energy from the lowest vibrational level of the
ground state. However, the electrons can make transitions into any of a
number of vibrational levels of the excited state. Thus, several different
transitions are possible, and the absorption spectrum will have a width
that reflects these different possibilities but they are unresolved in the
spectrum. The transition to the lowest vibrational level of the excited state
is often referred to as the zero–zero transition energy. Fluorescence arises
from electrons in the excited state. Electrons in higher vibrational levels
of the excited state relax very rapidly, in picoseconds or less, to the lowest
vibrational level by losing heat to the surroundings. This means that
fluorescence occurs from the lowest vibrational level of the excited state
to any of a number of vibrational levels in the ground state. On average,
this transition energy is less than the zero–zero transition energy; in other
words, the emitted photons are at lower energy or to the red of the absorbed
photons used to generate the excited state. Assuming that the vibrational
bands in the ground and excited states have the same spacing, the two
spectra will be shifted but have comparable widths, and hence appear to
be mirror images.
The width evident in the spectra of biological molecules arises from
different contributions (Figure 14.14). A major contribution to the width
of the spectra is then from the presence of the vibrational states. It is
observed in some cases that changes in vibrational states accompany an
electronic transition, resulting in additional bands. In large organic mole-
cules, these vibrational bands often are unresolved and increase the width
of the band. A molecule may also have a distribution of structures at any
given time that broadens the spectrum. In other words, different mole-
cules in the sample have slightly different spectra because of the nuclear
conformation they happen to have at the time of light absorption. Such

CHAPTER 14 OPTICAL SPECTROSCOPY 303

Absorbance Fluorescence

1

0

500 550 600 650

Absorbance or fluorescence

intensity (a.u.)

Wavelength (nm)

Figure 14.12

Comparison of the

absorption and

fluorescence spectra

of a biological

molecule.