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Fluorescence Spectroscopy
FAQs

An Introduction to the Fundamentals of Fluorescence Spectroscopy—

Including Fluorescence, Phosphorescence, Bioluminescence &
Chemiluminescence
Learn about the fundamentals of fluorescence spectroscopy, with frequently asked questions on the principle
of fluorescence, how fluorescence spectrophotometers work, fluorescence applications, accessories, and
measurement techniques. Plus, find out about other emission spectroscopy techniques—phosphorescence,
bioluminescence, and chemiluminescence.

The Fundamentals of Fluorescence Spectroscopy - FAQs

What is fluorescence?
Luminescence

Understanding the phenomena of luminescence helps explain fluorescence and phosphorescence.

Luminescence is the emission of light while an emitting system goes from a state of higher energy into a
state of lower energy. An object can be excited—creating a state of higher energy—by applying an electric
current (electroluminescence), because of radioactive bombardment (radioluminescence), and by
illuminating the object with a light source (photoluminescence), which is the most common case in a
spectroscopy lab. Sometimes the emission of light is the result of a chemical reaction (chemiluminescence),
or a biochemical reaction (bioluminescence).

Some phenomena do not fall under the term luminescence. The emission of light due to heating
(incandescence) is not a luminescence process and needs to be well separated from the process of
thermoluminescence, the re-emission of light upon heating.

Photoluminescence, emission, and absorption

When a chemical compound is illuminated with electromagnetic radiation, some of this radiation will be
absorbed. Depending on the energies involved, this absorption can trigger several different processes:

The lowest energy radiation—microwave radiation—induces the compound to rotate along its axes.
The absorption of infrared (IR) and near-infrared (NIR) radiation induces vibrations in the compound.
Absorption of visible (Vis) and ultraviolet (UV) radiation results in electrons reaching higher states
states.

For all these processes, it is essential that the absorbed light has exactly the energy the system needs to
reach the higher energy state. Different compounds have very specific “gaps” between their energy states,
generating unique emission and absorption spectra (Figure 1). Absorption spectroscopy measures these
energy state profiles, giving us information about the electronically excited state.

Figure 1. Processes that take place in a molecule during photoluminescence.

An “excited” compound can release the energy to the molecule’s environment by, for example, collisions with
solvent molecules. Or it can return to its ground state by sending out the energy in the form of light. This is
the process of luminescence.

The emission of light occurs without any external trigger (spontaneous emission), which is very different to
the stimulated emission that takes place (e.g., in lasers). The energy of the emitted light (fluorescence) is
generally lower than the energy of the absorbed light. As a result, the absorption and emission bands often
appear as image and mirror image (Figure 1). The energy difference between the maximum of lowest energy
absorption band and the maximum of the highest energy emission band is called “Stokes shift” (Figure 2).

Figure 2 . The term Stokes shift describes the energy difference between absorption and emission.

Fluorescence or phosphorescence?

In most cases, photoluminescence is a fluorescence process. Luminescence is called fluorescence if the

“transition” (the jump from the higher energy state into the lower energy state) is an “allowed process”.
Luminescence is called phosphorescence if the transition is a “forbidden process”. Forbidden does not mean
it cannot happen, but it is not as likely as the allowed process. And the forbidden process takes more time,
which is why the lifetime of a phosphorescent compound is much longer than of a fluorescent compound.
The next FAQ has more information on fluorescence lifetimes.

Note: Chemiluminescence, bioluminescence, and electroluminescence can be fluorescence or

phosphorescence, but this differentiation is rarely important.

For more detailed explanations of the energy state transitions mentioned here, including “allowed” and
Mechanics, Peter. W. Atkins, Ronald S. Friedman, Oxford
“forbidden” transitions, see Molecular Quantum Mechanics
University Press.

What is fluorescence spectroscopy?

What is fluorescence spectroscopy used for?

What instruments are used to detect fluorescence?

How does a fluorescence spectrophotometer work?

What does a fluorescence spectrophotometer measure?

What are the advantages of fluorescence spectroscopy?

What is phosphorescence?

What is chemiluminescence?

What is bioluminescence?

Additional Resources
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