Fluorescence and Phosphorescence

Phosphorescence and fluorescence describe what happens to an electron after it absorbs radiation. In fluorescence, an electron emits a photon quickly after excitation and returns to the electronic ground state. This process occurs almost immediately within time frames of about 10-9 seconds. It is understood from Pauli’s exclusion principle that in an orbital with two electrons, the electrons will have opposite spins. In fluorescence, when an electron is excited into a higher energy orbital, the electron will maintain its spin—this is called the excited singlet state (Fig 1). The excited singlet state is unstable and short-lived because it is higher in energy than the ground state. Therefore fluorescence can be defined as the process that occurs when an electron returns from the excited singlet state to the ground state.

Figure 1: Different electronic states. In an excited singlet state, the electrons have opposite spins. In an excited triplet state, the electrons have the same spin. Both states return to the ground singlet state (fluorescence and phosphorescence, respectively).

Phosphorescence is a more complicated process, in which the electron changes configuration (i.e. changes spin) as it returns to the ground state. Although an excited electron goes into the singlet state after light absorption, there is also a possibility that the electron enters the excited state triplet, in which the electron spins are unpaired (Fig 1). A molecule can enter this state over time or after collisions. The excited triplet state has lower energy than the excited singlet by Hund’s rule, which states that electrons fill orbitals separately before pairing. In phosphorescence, the electron goes from the excited triplet state to the ground singlet state. This transition is very slow and can range up to as long as 10 seconds.

The Jablonski Diagram (pictured below) illustrates the different electronic states and transitions in a molecule. The various energy states are represented by short horizontal lines organized into different columns, from low energy at the bottom to high energy near the top, with different columns representing different spin states (i.e. singlet or triplet). There are four different types of electronic transitions caused by changes in energy when light is absorbed, which may be either radiative or non-radiative. Radiative transitions involve the absorption or emission of a proton. Non-radiative transitions do not involve protons; rather, they describe changes in electron spin or emissionless decrease in energy.

All transitions are represented by either straight (representing radiative) or squiggly (representing non-radiative) arrows.

To understand what sort of energy transitions are pictured on a Jablonski diagram, it is necessary to know what all the arrows mean.

·        Straight arrows pointing up toward higher energy states correspond to the absorption of a photon.
·        Straight arrows pointing down toward lower energy states correspond to the emission of a photon.
·        Squiggly arrows pointing vertically represent energy dissipated by internal conversion, that is, when energy is transferred from the higher electronic energy states into vibrational modes.
·        Horizontal (or diagonal) squiggly arrows correspond to intersystem conversion, which involves changing the spin-state of the electron. This is generally only applicable to phosphorescence.

The Jablonski Diagram is useful for understanding the differences between phosphorescence and fluorescence because it provides a visual explanation for the “time delay” (or lack thereof) due to the time spent by the electron changing spin or converting energy in luminescence.

There are several applications of both fluorescent and phosphorescent phenomenon. Direct applications include spectroscopic methods, types of microscopes, glow-in-the-dark paints and lamps, and the effect of bioluminescence. Similarities between fluorescence and phosphorescence concepts result in similar mechanistic functions within the applications listed above.

Fluorescent spectroscopy is a method that measures the fluorescent concentration within a sample using ultraviolet light. This measurement is achieved by exciting the electron in its singlet state at a particular wavelength then causing the electron to emitting a photon of light at a lower energy level. The lower energy level usually corresponds to the visible light range. Fluorescent spectroscopy can be used in conjunction with or in replacement of absorption methods. Phosphorescent spectroscopy has a very similar mode of operation except the excited electron is in its triplet state. UV radiation is also used as a measurement of phosphorescence in a sample.

A fluorescent microscope can be used to examine a component of a sample or an organism at a particular wavelength. The component of interest is highlighted through the injection of a fluorescent molecule, fluorophore. Once the fluorophore absorbs light shown by the microscope, light of a longer wavelength is emitted. This application is critical in the field of biology where specific proteins can be tagged and characterized. It is possible to observe phosphorescence using fluorescence microscopy considering how similar the two phenomena are. The adjustments include using two shutters that allow for short pulses of excitation compared to the fluorescent set up that uses one shutter to allow for long pulses of excitation.

Luminescent paints and lamps are a direct application of both fluorescence and phosphorescence concepts. They are commonly referred to as “glow-in-the-dark” paint. Both are able to absorb light and emit colors of light at a later time, however fluorescent paint has a greater ability to store light with longer periods of time of emission. Phosphorescent paint usually emits a pale green or blue color while fluorescent light can emit a variety of bright colors.

A biological implication of phosphorescent and fluorescent phenomena is the effect of bioluminescence. This is when a living organism produces and emits light and is commonly seen in fireflies or glowworms. It is a natural process by which a chemical reaction occurs between the enzyme luciferase and oxygen. Living organisms use their bioluminescent ability to attract or repulse other organism, as well as for camouflage.

Applications:
        Fluorescent spectroscopy measures the fluorescent concentration within a sample using ultraviolet light. This measurement is achieved by exciting the electron in its singlet state at a particular wavelength then causing the electron to emitting a photon of light at a lower energy level. The lower energy level usually corresponds to the visible light range. Phosphorescent spectroscopy has a very similar mode of operation except the excited electron is in its triplet state. Similar to fluorescent spectroscopy, phosphorescent spectroscopy also uses UV radiation to measure the concentration of the sample.
        A fluorescent microscope can be used to examine a component of a sample or an organism at a particular wavelength. This works by injecting a fluorescent molecule or fluorophore into the sample. Once the fluorophore absorbs the light shown by the microscope, light of a longer wavelength is emitted and the sample is highlighted. This application is critical in the field of biology where specific proteins can be tagged and characterized. It is possible to observe phosphorescence using fluorescence microscopy considering how similar the two phenomena are, but slight adjustments to the microscope need to be made. Adjustments to the experimental set up include using two shutters that allow for short pulses of excitation compared to the fluorescent set up that uses one shutter to allow for long pulses of excitation.
        Luminescent paints and lamps are another application of both fluorescence and phosphorescence concepts. They are commonly referred to as “glow-in-the-dark” paint because they contain phosphors. A phosphor is a transition metal compound that facilitates the effect of luminescence. The big difference between phosphorescent and fluorescent paint is that phosphorescent paint has a greater ability to store light with longer periods of emission times because of its triplet excitation. With the absence of a light source, fluorescent paint usually fades less than 1 millisecond. Phosphorescent paint usually emits a pale green or blue color while fluorescent light can emit a variety of bright colors.
        An application of phosphorescent and fluorescent phenomena in biology is the effect of bioluminescence. This is when a living organism produces and emits light and is commonly seen in fireflies or glowworms. The emission of light in living organisms is a natural process by which a chemical reaction occurs between the enzyme luciferase and oxygen. Living organisms use their bioluminescent ability to attract or repulse other organisms, as well as for camouflage. One example of this is the luminescent emission that squids use to ward off predators. Their glow is often viewed as a threat and deters the attack.
Basically, fluorescence and luminescence are different radiative ways that molecules deal with absorbed energy. Both allow the excited molecule to relax into a lower energy state and give off photons. While fluorescence quickly emits a photon after absorption, phosphorescence takes some time to allow the electron to change spin before emitting a photon. Both fluorescence and phosphorescence have many practical applications, from crafts to analysis to biological defenses.

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