**1. Introduction**

208 Macro to Nano Spectroscopy

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Spectrofluorometric methods of analysis are the most commonly analytical techniques and continue to enjoy wide popularity. The wide availability of the instrumentation, the simplicity of procedure, sensitivity, selectivity, precision, accuracy, and speed of the technique still make the spectrofluorometric methods attractive. These features make fluorescence spectroscopy an attractive technique as compared to other forms of optical spectroscopy or other analytical techniques such as chromatography and electrophoresis. Fluorescence spectroscopy has been used widely as a tool for quantitative analysis, characterization, and quality control in the pharmaceutical, environmental, agricultural, nanotechnology and biomedical fields.

The emission of light from an excited electronic state of a molecular species is called luminescence. The discovery and characterization of luminescence begun from the 15th century. In 1506 Nicolas Monardes was the first to describe the bluish opalescence of the water infusion from the wood of a small Mexican tree. In 1612 Galileo described the emission of light (phosphorescence) from the famous Bolognian stone, which discovered by Vincenzo Casciarolo, a Bolognian shoemaker. Galileo wrote: "It must be explained how it happens that light is conceived into the stone, and is given back after some time, as is childbirth". Even though, some of the first scientific reports of luminescence appeared in the middle of the 18th century. In 1845 Sir J.F.W. Herschel reports on an experiment he did twenty years earlier. Herschel made the first observation of fluorescence from quinine sulfate (quinine: (*R*)-(6-methoxyquinolin-4-yl)-((2*S*, 4S, 8R)- 8-vinylquinuclidin-2-yl)methanol, C20H24N2O2, quinine absorbs in the UV region), he observed that an otherwise colorless solution of quinine in water emitted a blue color under certain circumstances. Herschel concludes that a species in the solution, "exert its peculiar power on the incident light" and disperses the blue light. The experiment can be repeated simply by observing glass of tonic water exposed to sunlight. Often a blue glow is visible at the surface (Rendell, 1987).

The phenomenon of fluorescence was known by the middle of the nineteenth century. British scientist Sir George G. Stokes first made the observation that the mineral fluorspar exhibits fluorescence when illuminated with ultraviolet light, and he coined the word "fluorescence". In 1852, Sir G.G. Stokes studied the same compound (quinine) that has been used by Herschel and found that the fluorescing (*emitted*) light has longer wavelengths than the excitation (*absorbed*) light, a phenomenon that has become to be known as the Stokes

Current Achievement and Future Potential of Fluorescence Spectroscopy 211

fluorescence energy transfer, and intersystem crossing may also depopulate the first excited state ; and 3. Return of the fluorophore to its ground state, accompanied by the emission of light. The light energy emitted is always of a longer wavelength (*lower energy*) than the light energy absorbed, due to the energy dissipation during the transient excited lifetime, as shown in Step 2. Consequently, the ratio of the number of fluorescence photons emitted (stage 3) to the number of photons absorbed (stage 1) represents the *fluorescence quantum* 

Fig. 1. The Jablonski diagram illustrates the three stages involved in the creation of an excited electronic singlet state by optical absorption and subsequent emission of

A fluorophore can repeatedly undergo the fluorescence process—in theory, indefinitely. This is extremely useful, because it means that one fluorophore molecule can generate a signal multiple times. This property makes fluorescence a very sensitive technique for visualizing microscopic samples—even a small amount of the stain can be detected. In reality, however, the fluorophore's structural instability during the excited lifetime makes it susceptible to degradation. High-intensity illumination can cause the fluorophore to change its structure so that it can no longer fluoresce—this is called photobleaching. When a fluorescent sample, such as a slide with mounted tissue, is photobleached, the fluorophores are no longer promoted to an excited state, even when the required light energy is supplied

Now that we've introduced the general process of fluorescence, let's take a look at the basic properties of the light spectrum and its importance in fluorescence. The visible spectrum (Figure 2) is composed of light with wavelengths ranging from approximately 380

Light waves with shorter wavelengths have higher frequency and higher energy. Light waves with longer wavelengths have lower frequency and lower energy. As we stated before, an excited fluorophore emits lower-energy light than the light it absorbed. Therefore, there is always a shift along the spectrum between the color of the light absorbed by the

*yield* (Lakowicz, 2006).

fluorescence

(Lakowicz, 2006).

nanometers to 750 nanometers.

fluorophore during excitation, and the color emitted.

shift. Stokes' paper demonstrated the fundamental property of fluorescence, which simply can be summarized as a photon of ultraviolet radiation collides with an electron in a simple atom, exciting and elevating the electron to a higher energy level. Subsequently, the excited electron relaxes to a lower level and emits light in the form of a lower-energy photon (*higher wavelength*) in the visible light region. In 1871 Adolph Von Baeyer, a German chemist, synthesized the fluorescent dye, fluorescein. In 1880 Edmund Bequerel showed that certain metal ion complexes emit radiation with a very long decay time (Rendell, 1987).

Jabłoński and others developed a modern theoretical understanding of Stokes observation some 70 years later. In the 1920s and 1930s Jabłoński investigated polarized light and fluorescence and was able to show that the transition moments in absorption and emission are two different things. Thus the foundation for the concept of anisotropy was laid. For that and other accomplishments Jabłoński has been referred to as the father of fluorescence and his work has had a major impact on the theoretical understanding of photophysics (Rendell, 1987; Lakowicz, 2006).

How and why do certain molecules known as fluorophores or fluorescent molecules (such as: dyes, polyaromatic hydrocarbon, or heterocyclic,…etc.) emit different colors of light?. Briefly the answer for this question is that some molecules are capable of being excited, via absorption of light energy, to a higher energy state, also called an excited state. The energy of the excited state, which cannot be sustained for long, "decays" or decreases, resulting in the emission of light energy. This process is called *fluorescence*. To "fluoresce" means to emit light via this process. A fluorophore is a molecule that is capable of fluorescing due to the presence of certain chromophores within a molecule. In its ground state, the fluorophore molecule is in a relatively low-energy, stable configuration, and it does not fluoresce. When light from an external source hits a fluorophore molecule, the molecule can absorb the light energy. If the energy absorbed is sufficient, there are multiple excited states or energy levels that the fluorophore can attain, depending on the wavelength and energy of the external light source. Since the fluorophore is unstable at high-energy configurations, it eventually adopts the lowest-energy excited state, which is semi-stable. The length of time that the fluorophore is in excited states is called the *excited lifetime*, and it lasts for a very short time, ranging from 10-15 to 10-9 seconds. Next, the fluorophore rearranges from the semi-stable excited state back to the ground state, and the excess energy is released and emitted as light. The emitted light is of lower energy, and thus longer wavelength, than the absorbed light. This means that the color of the light that is emitted is different from the color of the light that has been absorbed. Emission of light returns the fluorophore to its ground state. The fluorophore can absorb light energy again and go through the entire process repeatedly (Lakowicz, 2006).

The cyclical fluorescence process, shown in Figure 1, can be summarized as: 1. Excitation of a fluorophore through the absorption of light energy, the excitation wavelength is usually the same as the absorption wavelength of the fluorophore; 2. A transient excited lifetime with some loss of energy, during this period, the fluorophore undergoes conformational changes and is also subject to possible interactions with its molecular environment, with two important consequences: first, the energy of higher excited state is partially dissipated as a heat, yielding a relaxed lowest singlet excited state from which fluorescence emission originates, second, not all the molecules initially excited by absorption (stage 1) return to the ground state by fluorescence emission, as other processes such as collisional quenching,

shift. Stokes' paper demonstrated the fundamental property of fluorescence, which simply can be summarized as a photon of ultraviolet radiation collides with an electron in a simple atom, exciting and elevating the electron to a higher energy level. Subsequently, the excited electron relaxes to a lower level and emits light in the form of a lower-energy photon (*higher wavelength*) in the visible light region. In 1871 Adolph Von Baeyer, a German chemist, synthesized the fluorescent dye, fluorescein. In 1880 Edmund Bequerel showed that certain

Jabłoński and others developed a modern theoretical understanding of Stokes observation some 70 years later. In the 1920s and 1930s Jabłoński investigated polarized light and fluorescence and was able to show that the transition moments in absorption and emission are two different things. Thus the foundation for the concept of anisotropy was laid. For that and other accomplishments Jabłoński has been referred to as the father of fluorescence and his work has had a major impact on the theoretical understanding of photophysics (Rendell,

How and why do certain molecules known as fluorophores or fluorescent molecules (such as: dyes, polyaromatic hydrocarbon, or heterocyclic,…etc.) emit different colors of light?. Briefly the answer for this question is that some molecules are capable of being excited, via absorption of light energy, to a higher energy state, also called an excited state. The energy of the excited state, which cannot be sustained for long, "decays" or decreases, resulting in the emission of light energy. This process is called *fluorescence*. To "fluoresce" means to emit light via this process. A fluorophore is a molecule that is capable of fluorescing due to the presence of certain chromophores within a molecule. In its ground state, the fluorophore molecule is in a relatively low-energy, stable configuration, and it does not fluoresce. When light from an external source hits a fluorophore molecule, the molecule can absorb the light energy. If the energy absorbed is sufficient, there are multiple excited states or energy levels that the fluorophore can attain, depending on the wavelength and energy of the external light source. Since the fluorophore is unstable at high-energy configurations, it eventually adopts the lowest-energy excited state, which is semi-stable. The length of time that the fluorophore is in excited states is called the *excited lifetime*, and it lasts for a very short time, ranging from 10-15 to 10-9 seconds. Next, the fluorophore rearranges from the semi-stable excited state back to the ground state, and the excess energy is released and emitted as light. The emitted light is of lower energy, and thus longer wavelength, than the absorbed light. This means that the color of the light that is emitted is different from the color of the light that has been absorbed. Emission of light returns the fluorophore to its ground state. The fluorophore can absorb light energy again and go through the entire process repeatedly

The cyclical fluorescence process, shown in Figure 1, can be summarized as: 1. Excitation of a fluorophore through the absorption of light energy, the excitation wavelength is usually the same as the absorption wavelength of the fluorophore; 2. A transient excited lifetime with some loss of energy, during this period, the fluorophore undergoes conformational changes and is also subject to possible interactions with its molecular environment, with two important consequences: first, the energy of higher excited state is partially dissipated as a heat, yielding a relaxed lowest singlet excited state from which fluorescence emission originates, second, not all the molecules initially excited by absorption (stage 1) return to the ground state by fluorescence emission, as other processes such as collisional quenching,

metal ion complexes emit radiation with a very long decay time (Rendell, 1987).

1987; Lakowicz, 2006).

(Lakowicz, 2006).

fluorescence energy transfer, and intersystem crossing may also depopulate the first excited state ; and 3. Return of the fluorophore to its ground state, accompanied by the emission of light. The light energy emitted is always of a longer wavelength (*lower energy*) than the light energy absorbed, due to the energy dissipation during the transient excited lifetime, as shown in Step 2. Consequently, the ratio of the number of fluorescence photons emitted (stage 3) to the number of photons absorbed (stage 1) represents the *fluorescence quantum yield* (Lakowicz, 2006).

Fig. 1. The Jablonski diagram illustrates the three stages involved in the creation of an excited electronic singlet state by optical absorption and subsequent emission of fluorescence

A fluorophore can repeatedly undergo the fluorescence process—in theory, indefinitely. This is extremely useful, because it means that one fluorophore molecule can generate a signal multiple times. This property makes fluorescence a very sensitive technique for visualizing microscopic samples—even a small amount of the stain can be detected. In reality, however, the fluorophore's structural instability during the excited lifetime makes it susceptible to degradation. High-intensity illumination can cause the fluorophore to change its structure so that it can no longer fluoresce—this is called photobleaching. When a fluorescent sample, such as a slide with mounted tissue, is photobleached, the fluorophores are no longer promoted to an excited state, even when the required light energy is supplied (Lakowicz, 2006).

Now that we've introduced the general process of fluorescence, let's take a look at the basic properties of the light spectrum and its importance in fluorescence. The visible spectrum (Figure 2) is composed of light with wavelengths ranging from approximately 380 nanometers to 750 nanometers.

Light waves with shorter wavelengths have higher frequency and higher energy. Light waves with longer wavelengths have lower frequency and lower energy. As we stated before, an excited fluorophore emits lower-energy light than the light it absorbed. Therefore, there is always a shift along the spectrum between the color of the light absorbed by the fluorophore during excitation, and the color emitted.

Current Achievement and Future Potential of Fluorescence Spectroscopy 213

A fluorescent molecule absorbs light over a range of wavelengths—and every chemical molecule has a characteristic excitation range. However, some wavelengths within that range are more effective for excitation than other wavelengths. This range of wavelengths reflects the range of possible excited states that the fluorophore can achieve. Thus for each fluorescent molecule, there is a specific wavelength—the excitation maximum—that most effectively induces fluorescence. Now let's look at the light that is emitted by the fluorophore molecules when they are excited at the optimal excitation wavelength. Just as fluorophore molecules absorb a range of wavelengths, they also emit a range of wavelengths. There is a spectrum of energy changes associated with these emission events. When we excite the previously described dye solution at its excitation maximum, 550 nanometers, light is emitted over a range of wavelengths. A molecule may emit at a different wavelength with each excitation event because of changes that can occur during the excited lifetime, but each emission will be within the range. Although fluorophore molecules emit same intensity of light, the wavelengths and therefore the colors of the emitted light are not homogeneous. However, a larger population of molecules has most

Based on this distribution of emission wavelengths, we say that the emission maximum of this fluorophore is 570 nanometers. The range of wavelengths is represented by the

Fig. 4. Fluorescence Emission Spectrum of dye solution monitored at λex = 550 nm

The emission intensity is proportional to the amplitude of the fluorescence excitation spectrum at the excitation wavelength. Fluorescence emission intensity depends on the same

intensely fluorescence at 570 nanometers (Lakowicz, 2006).

Fluorescence Emission Spectrum, Figure 4.


Fig. 2. Visible light spectrum

For example, let's say that we have a tube that contains a particular fluorescent dye. If we shine 480 nanometer light at the dye solution, some of the fluorophore molecules will become excited. However, the majority of the molecules are not excited by this wavelength of light. As we increase the excitation wavelength, say to 520 nanometers, more molecules are excited. However, this is still not the wavelength at which the proportion of excited molecules is maximal. For this particular dye, 550 nanometers is the wavelength that excites more fluorophores than any other wavelength of light. At wavelengths longer than 550 nanometers, the fluorophore molecules still absorb energy and fluoresce, but again in smaller proportions. The range of excitation wavelengths can be represented in the form of a fluorescence excitation spectrum, which looks like the spectrum shown in Figure 3.

Fig. 3. Excitation spectrum of dye solution recorded at λem = 570 nm, the excitation wavelength maximum at 550 nm.

For example, let's say that we have a tube that contains a particular fluorescent dye. If we shine 480 nanometer light at the dye solution, some of the fluorophore molecules will become excited. However, the majority of the molecules are not excited by this wavelength of light. As we increase the excitation wavelength, say to 520 nanometers, more molecules are excited. However, this is still not the wavelength at which the proportion of excited molecules is maximal. For this particular dye, 550 nanometers is the wavelength that excites more fluorophores than any other wavelength of light. At wavelengths longer than 550 nanometers, the fluorophore molecules still absorb energy and fluoresce, but again in smaller proportions. The range of excitation wavelengths can be represented in the form of a

fluorescence excitation spectrum, which looks like the spectrum shown in Figure 3.

Fig. 3. Excitation spectrum of dye solution recorded at λem = 570 nm, the excitation

Fig. 2. Visible light spectrum

wavelength maximum at 550 nm.

A fluorescent molecule absorbs light over a range of wavelengths—and every chemical molecule has a characteristic excitation range. However, some wavelengths within that range are more effective for excitation than other wavelengths. This range of wavelengths reflects the range of possible excited states that the fluorophore can achieve. Thus for each fluorescent molecule, there is a specific wavelength—the excitation maximum—that most effectively induces fluorescence. Now let's look at the light that is emitted by the fluorophore molecules when they are excited at the optimal excitation wavelength. Just as fluorophore molecules absorb a range of wavelengths, they also emit a range of wavelengths. There is a spectrum of energy changes associated with these emission events. When we excite the previously described dye solution at its excitation maximum, 550 nanometers, light is emitted over a range of wavelengths. A molecule may emit at a different wavelength with each excitation event because of changes that can occur during the excited lifetime, but each emission will be within the range. Although fluorophore molecules emit same intensity of light, the wavelengths and therefore the colors of the emitted light are not homogeneous. However, a larger population of molecules has most intensely fluorescence at 570 nanometers (Lakowicz, 2006).

Based on this distribution of emission wavelengths, we say that the emission maximum of this fluorophore is 570 nanometers. The range of wavelengths is represented by the Fluorescence Emission Spectrum, Figure 4.

Fig. 4. Fluorescence Emission Spectrum of dye solution monitored at λex = 550 nm

The emission intensity is proportional to the amplitude of the fluorescence excitation spectrum at the excitation wavelength. Fluorescence emission intensity depends on the same

Current Achievement and Future Potential of Fluorescence Spectroscopy 215

Other intermolecular processes (e.g. quenching, energy transfer, solvent interaction etc.) are

Once a molecule is excited by absorption of light it can return to the ground state with emission of fluorescence, but many other pathways for de-excitation are also possible, these

Jablonski diagram (Fig. 7) explains the mechanism of light emission in most organic and inorganic luminophores. Upon absorption of the light by a molecule, the electron promoted from ground electronic state (S0) to an excited state that should possess the same spin multiplicity (such as, S1, S2,….) this process usually occurs within ∼10-15 s. This excludes the triplet excited state as the final state of electronic absorption because transitions between states of different multiplicities are improbable "*forbidden*" (e.g. T→S or S→T). According to the quantum mechanical selection rules for electronic transitions, spin state should be maintained upon excitation because it is harder for an electron to go from a singlet state to

omitted (Rendell, 1987; Lakowicz, 2006).

are summarized in Figure 6.

Fig. 5. Possible energy states according to their spin multiplicity

Fig. 6. All possible pathways for de-excitation processes

parameters as absorbance—defined by the Beer-Lambert law as the product of the molar extinction coefficient, optical path-length, and concentration—as well as on the fluorescence quantum yield, the intensity of the excitation source, and the efficiency of the instrument and, in dilute solutions, is linearly proportional to these parameters.

The summary points of this introduction to fluorescence are: 1. Fluorophores are molecules that, upon absorbing light energy, can reach an excited state, and then emit light energy. 2. The three-stage process of excitation, excited lifetime, and emission is called fluorescence. 3. Fluorophores absorb a range of wavelengths of light energy, and also emit a range of wavelengths. Within these ranges are the excitation maximum and the emission maximum. Because the excitation and emission wavelengths are different, the absorbed and emitted lights are detectable as different colors or areas on the visible spectrum.

The purpose of this chapter is to review the articles on the interior cited aspects published since 2000 about various aspects of application of fluorescence spectrophotometry in chemical analysis.
