19 Characterization of Fluorescence Emission

Dr. Ajit K. Mahapatro

Learning Objectives

From this module students may get to know about the following

i. The fundamental physics of emission of light.

ii. Various processes for emission of light from materials exposed to different external energy sources.

iii. Rules to understand the emission of light.

1. Origin of Energy States in Materials

All materials consist of atoms of various elements that contain movable electrons and localized nucleus. The dynamic properties of the materials depend mostly on the activities of the electron and their responses towards the externally applied energies at different environments. The available energy states permissible for electrons are the molecular states in the energy spectrum. The negatively charged particle, electron, has a specific orbital with definite energy defined in the atom, where they can stay. This could be understood by considering electron as a particle experiencing the confinement potential in the dimension of the atom, and the resulting quantum mechanical system yields discrete energy levels due to the confinement effect and referred as electronic energy states. The energy spectrum of the quantum mechanical (QM) systems with such discrete energy levels is said to be quantized. In equilibrium, the atom, ion, or molecule as QM system, the electrons like to be in a low energy state referred as ground state (solid blue line in Figure 1), and if the electron stays at a higher energy level than the ground state energy level, then the energy level is referred as excited energy state.

The energy levels of electrons in atoms, ions, or molecules, which are bound by the electric fie ld of the nucleus, can also refer to energy levels of nuclei, or vibrational or rotational energy levels in molecules. The vibrational ground states of each electronic state are indicated with thick and dark lines, and the higher vibrationa l states with light colored lines (as in Figure 1).

Figure 1. Energy levels for a quantum mechanical system (an electron bounded in an atom) with lo west energy ground state (solid blue line), higher possible excited states (solid line),and vibrational or energy levels (light color lines).

2. Emission of Light

The transition of electrons within the allocated energy states give rise to two different kinds of processes during artificial production of light.

2.1 Incandescence: This is the light emitted from the heat energy produced by the excessive temperature within a material. When a material is heated to a high temperature, it begins to glow and the process is termed as incandescence. For example, electric heater or metal in flame begin to glow red hot, and the glow of sun and stars.

2.2 Luminescence: Luminescence is the light emission due to an excited energy. If some external energy is supplied to any luminescent material, the supplied external energy moves the electron from a certain baseline energy known as the ground state (E₀) to a higher energy state called excited state (E₁), then the electron falls back to its ground state by emitting its energy in the form of light. Generally, the excitation energy is greater than the energy emitted by electron. Luminescence is described by light absorption and emission, and could be defined as below:

(a) Absorption: The process in which e lectron absorbs the incident photon and moves to an excited state is called absorption. The energy of incident photon must be equal to the energy difference between ground state and excited state. The absorption is shown in figure 2(a). Absorption spectrum is also referred as excitation spectrum.

(b) Emission: The process in which the excited electron loses its energy by emission of photon and comes back to its ground state is called e mission. It is shown in figure 2 (b).

3. Types of Luminescence

Luminescence process could be defined in various types depending on the source of the excitation energy, as discussed below:

Luminescence process	Activation Process: Luminescence is produced during	Emission as an effect from
(i) Chemiluminescence	Chemical reaction
(a) Bioluminescence		biochemical reaction by a living organism
(b) Electrochemiluminescence		Electrochemical reaction
ii) Crystalloluminescence	Crystallization
iii) Electroluminescence	Electric current passed through a material.
Cathodoluminescence		Luminescent material being struck by the electrons.
iv) Mechanoluminescence	Mechanical action on a solid.
(a) Triboluminescence		Produced by breaking of bonds in a material when it is scratched, crushed, or rubbed.
(b) Fractoluminescence		Due to bonds in certain crystals are broken by fractures.
(c) Piezoluminescence		By the action of pressure on certain solids.
(d) Sonoluminescence		By imploding bubbles in a liquid when excited by sound.
v) Radioluminescence		Bombardment by ionizing radiation.
vi) The rmoluminescence	Due to heating of a substance.
(a) Cryoluminescence		When an object is cooled.
vii) Photoluminescence	Laser light exposure	When the material is excited with monochromatic light.

3.1 Photoluminescence

Photoluminescence is the process in which light is emitted by absorption of photons. After exciting the electron from ground to the excited state, the relaxation occurs and the electron falls back to its ground state by emitting light. During this process, if there is no “allowed transition” possible for an electron to transit from an excited state to any lower energy state, it remains in the higher energy state for a relatively longer period. Such states are termed as “metastable states.” Depending on the nature of the emitted electron, the photoluminescence process can be defined in two ways:

Figure 3. Different energy states in photoluminescence

a) Fluorescence: Luminescence produced as a result of singlet–singlet electronic relaxation

b) Phosphorescence : Luminescence produced as a result of triplet–singlet electronic relaxation

4. Fluorescence

In this process, the materials absorb light at a particular wavelength and emit light at longer wavelengths than the incident light. This process can also be explained as absorbance of light of electromagnetic radiation at a frequency ν and emits light at lesser frequency ν’ (ν > ν’). Fluorescence refers to immediate release of light, where light is emitted within a fraction of second after excitation. The fluorescence process is sketched in Figure 4.

Figure 4. Fluorescence process with excitation energy of hν and emitted energy of hν’

The materials showing fluorescence behaviour are called fluorophores. Depending on the difference in energies between the ground and excited states, the fluorophores absorb lights of different wavelengths. Hence it has specific absorption spectra and similarly they have specific emission spectra.

4.1 Possible Spin States

Absorption of ultraviolet or visible photon promotes a valence electron from its ground state to an excited state and the phenomenon is called “excitation”. The excited state where an electron retains its spin is called singlet excite d state . When the spin of the excited electron changes with respect to ground spin state then it is called triple t excite d state. The schematic view of these states is shown in Figure 5.

But the excited electron is not stable and can’t stay in the excited state indefinitely and returns to the ground state with the emission of a photon. This process is called fluorescence. The average lifetime of the electron in the excited state is only 10^-5 -10^-8 sec. Here, the wavelength of emitted photon is longer than the wavelength of absorbed photon (λ’ > λ). Fluorescence decays rapidly after the excitation source is removed.

4.2 Basic Principles for Fluorescence

The materials showing fluorescence follows certain rules

• Franck Condon Princ iple

• Stokes shift

• Mirror image rule

4.2.1 Franck Condon Principle

i) Nucleus has much greater mass than the electrons so that during the electronic transition the nucleus remains stationary but electronic configuration changes.

ii) Therefore, the inter-nuclear separation remains same after absorption.

iii) The most intense electronic transition occurs between the vibrations states that arrange vertically in the different electronic state.

iv) Classically the Frank-Condon principle says that the transition of electrons from the ground to the excited state occurs without any change in the position of the nucleus. This promotes the ground state electrons to the excited state through vertical transitions and plotted as vertical lines in Figure 6.

Figure 6. Transition between different electronic states

4.2.2 Stokes shift

In the process of luminescence, after absorption of incident light the electron goes to an excited state and falls back to its ground state with the emission of light having lower energy than the absorbed light. Therefore, the emission spectra move towards the longer wavelength and a shift is observed in absorption and emission spectra. This shift in wavelength (Δλ) is called Stokes shift or governed by stokes law.

Figure 7. Shift in absorption and emission spectra in fluorescence spectroscopy

The fluorophore emission intensity peak is usually less in magnitude than that exhibited by the excitation peak. As Stoke shift value increases, it becomes easier to separate excitation from emission light using fluorescence filters. The Stokes shift occurs due to the loss of energy to bring the excited electrons to the lowest vibrational energy level of the excited state from the highest vibrational energy levels. In addition, fluorescence emission is usually accompanied by transitions to higher vibrational energy levels of the ground state, resulting in further loss of excitation energy to regain the lowest vibrational energy level of the ground state. The total loss of energy observed during the emission process is a combined loss of energy of electron in the excited state (S₁) before emission and in the ground state (S₀) after emission. Other events, such as solvent orientation effects, excited-state reactions, complex formation, and resonance energy transfer can also contribute to longer emission wavelengths.

In practice, the Stokes shift is measured as the difference between the wavelengths at the maximum peak intensities in the excitation and emission spectra of a particular fluorophore. The amount of the shift varies with molecular structure and can range from few nanometers to over several hundred nanometers. For example, the Stokes shift for fluorescein is approximately 20 nm, the shift for quinine is 110 nm, and for the porphyrins is over 200 nm.

4.2.3 Mirror Image Rule

Generally, the emission spectra are the mirror image of absorption spectra during the transition from the ground state (S₀) to the first excited state (S₁). Same transition occurs between the ground state and first excited state shows the similarities of vibrational levels of ground and first excited states.

Figure 8. Mirror image of absorption and emission spectra

Exceptions of the Mirror Image Rule

Deviation from the mirror image is observed for transitions to excited states higher than the next excited state, viz., S₀ to S₂ or S₃. Excitation by the high energy photons leads to transition of electrons from the ground state S₀ to the higher electronic and vibrational levels S₂ and S₃, etc. The resulting population of electrons in the higher energy states rapidly looses the excess energy and relaxes to the lowest vibrational level of that specific excited state followed by transition to the first excited state S₁ and finally comes back to the ground state S₀. So that the emission spectra are not similar to the excitation spectra and the higher excitation states do not follow the mirror image rule.

Figure 9. Difference in absorption and emission spectra

4.3 Uses of Fluorescence:

• The marks made by fluorescent highlighters quickly draw our attention to important points in a document

• Fluorescence is also useful in a number of safety applications, as a scientific research tool, and in investigative medicine.

• Binding studies in bio-sensing: The use of fluorescence spectroscopy can provide important information about the process of ligand binding and ligand dependent conformational changes in receptors.

• Fluorescence based techniques are widely used to address the fundamental and applied questions in the biological and biomedical sciences.

• Current use of fluorescence-based technology include assays for biomolecules, metabolic enzymes, DNA sequencing, research into biomolecule dynamics, cell signa ling, and adaptation, and observing the in-situfluorescence of hybridisations to identify specific DNA and/or RNA sequences in tissues.

• In safety equipments including sign-boards in marine buoys and roads, which also using of fluorescent paints.

• Fluorescence is used to enhance the color image of the gene and cell structure s using fluorescence microscopy, to locate useful mineral resources.

• Medical research makes widespread use of fluorescence to understand biological process and in diagnosis.

5. Phosphorescence

Phosphorescence is a type of luminescence where the emission process continues for noticeable time after taking off the incident excited radiations. When a substance exposed to radiation, it emits light and persists to glow for sometime after the exciting radiation is removed. In fluorescence, the absorbed light is spontaneously emitted within 10-8 sec. after excitation as compared to the phosphorescence, which requires about 10-3 sec., or days or years, depending on the circumstances.

Figure 10. Transitions observed in the phosphorescence process

5.1 Working of Phosphorescence

In some materials, there exist an intermediate energy state between the ground and excited states, referred as metastable state or electron trap. Once an electron falls from the excited level to the metastable level (by radiation or by energy transfer to the system), it remains there until it makes a forbidden transition or until it comes back to ground level by losing energy through thermal agitation among the neighboring atoms or molecules (called thermoluminescence) or through optical emission. This makes slow transition of the electrons in the metastable state to come to the original ground state by spending more time in the metastable state (or electron trap) that determines the length of time for phosphorescence.

The total spin of the electrons in the excited state is zero while the total spin of the electrons in the metastable state is one (as in Figure 10). The multiplicity can be calculated by the formula M=2S+1, which for the excited state is one (termed as singlet state) and the multiplicity of the metastable state is three (termed as triplet state). The transition of the electrons from ground state to a metastable state is forbidden because it violates the selection rule for conservation of spin, i.e., ΔS=0, and can transit from the ground state to an excited state. Most of the electrons immediately hop back to the ground state by radiating /emitting energy in the form of photons but non-radiative processes takes the electrons to a less energetic triplet state. Once these molecules get to the lowest triplet state, they are stuck there, at least for a while. The electron first flips its spin and then come back to its ground state.

5.2 Uses of phosphorescence

• Phosphorescent materials are used in a wide variety of applications and products. For example , phosphorescent powder and gel can be found in many different toys, such as glow sticks and other glow – in-the-dark products.

• The majority of phosphorescence is often used in drugs in pharmaceutical field. Some common drugs that have phosphorescence property includeaspirin, benzoic acid, morphine, and dopamine. Phosphorescence is also used to analyze water, air and chemical pollutions.

• Television tubes also use phosphorescence. The tube itself is coated with phosphor, and a narrow beam of electrons causes excitation in a small portion of the phosphor. The phosphor then emits red, green, or blue light, the primary colors of light and continues to do so even after the electron beam has moved on to another region of phosphor on the tube. As it scans across the tube, the electron beam is turned rapidly on and off, creating an image made up of thousands of glowing, colored dots.

• Phosphorescent materials store light for several minutes or even for several hours and re-emit a significant amount of light for over a long period of time.

6. Difference between fluorescence and phosphorescence

Generally, the fluorescence and phosphorescence were distinguished only by the criterion of an observable after glow: if the luminescence did not last longer than the irradiation, it was called fluorescence; if it was visible for an appreciable length of time after the end of the excitation, it was called phosphorescence. Modern experimental technique however, permits the measurement of the finite duration of any emission process, even if it is as short as 10-9 sec, and, on the other hand, spontaneous transition probabilities, even in atomic process, correspond to lifetimes which vary continuously from 10-8 sec to several seconds. Therefore, it is no longer possible to define some arbitrary duration of emission process as the boundary between fluorescence and phosphorescence. Fluorescence and phosphorescence are the first order processes and follow exponential laws of decay.

Summary

The various transitions after absorption of light can be shown by a common diagram.

In the diagram below several energy levels of an atom or a more complicated system are represented by horizontal lines. The vertical distance between two of these lines is proportional to the corresponding difference in the energy. The level N represents the ground state. By the absorption of light, the atom is raised to the level F and if no other energy level exists between N and F, the atom can return to N only by re-emission of light of the same frequency as incident light. It is called “resonance radiation.” Several levels C, D… are located between N and F. Under these conditions other transitions from F to C, D…, can occur, resulting in the emission of spectral lines of frequency smaller than incident frequency.

The Figure 11 shows the following processes:

(1) Resonance radiation,

(2) Phosphorescence,

(3) Fluorescence, and

(4) and (5) Anti- stokes fluorescence

Figure 11. Energy states for representing different phenomenon.

References

• B.I. Stepanov and V.P. Gribkovski, The theory of luminescence, ILIFFE Publishers, 1968.
• Cornellis R. Ronda, Luminescence: from theory to applications, Willey-VC H, 2008.
• Adrian K itai, Luminescent materials and applications, John Wiley & Sons, 2008.