Spectroscopy Techniques and Applications PDF

# Spectroscopy Techniques and Applications "The past, like the future, is indefinite and exists only as a spectrum of possibilities." -Stephan Hawking ## Chapter Outline * 3.1 Introduction * 3.2 Electromagnetic Spectrum * 3.3 Atomic and Molecular Spectroscopy * 3.3.1 Atomic Spectroscopy *...

# Spectroscopy Techniques and Applications "The past, like the future, is indefinite and exists only as a spectrum of possibilities." -Stephan Hawking ## Chapter Outline * 3.1 Introduction * 3.2 Electromagnetic Spectrum * 3.3 Atomic and Molecular Spectroscopy * 3.3.1 Atomic Spectroscopy * 3.3.2 Molecular Spectroscopy * 3.4 Ultraviolet and Visible Spectroscopy * 3.4.1 Electronic Transition * 3.4.2 Selection Rules * 3.4.3 Chromophores * 3.4.4 Auxochromes * 3.4.5 Factors Affecting λ_max and Intensity of Spectral Lines * 3.4.6 Franck-Condon Principle * 3.4.7 Bond Dissociation Energy * 3.4.8 Applications of Ultraviolet and Visible Spectroscopy * 3.4.9 Fluorescence and Phosphorescence * 3.4.10 Mechanism of Fluorescence and Phosphorescence * 3.4.11 Jablonski Diagram * 3.4.12 Applications of Fluorescence in Medicines ## 3.1 Introduction Spectroscopy is the branch of science which deals with transitions occurring in a molecule when it interacts with electromagnetic radiations. Our knowledge about molecular structure is derived indirectly from different spectroscopic techniques. It is not possible to see atoms and molecules even with the help of most powerful microscope because of their infinitesimal small dimensions. As atoms give line spectra and each atom gives its characteristic spectrum, so the molecular spectra depend upon the nature of atoms involved and also upon the arrangement of the atoms in the molecules. Atomic spectra is due to electronic transitions only i.e., electrons shift from one energy level to another. But in molecular spectra, rotational and vibrational transitions also take place in addition to electronic transitions and this spectra is therefore, much complicated than atomic spectra. The molecular spectra is of much help to a chemist in elucidating the structure of molecules. ## 3.2 Electromagnetic Spectrum The visible light represents only a small part of the entire electromagnetic spectrum, which extends from high energy cosmic rays to low energy radio or radar waves. The complete electromagnetic spectrum gives the arrangement of various types of electromagnetic radiations in order of their increasing (or decreasing) wavelengths or frequencies (Fig. 3.1). | Electromagnetic radiation | Frequency (v) | Wavelength (λ) | Spectrum | | :----------------------------------------- | :------------------------------------------------------- | :------------------------------------------------------------ | :---------------------------------------------------------------------------------- | | Radio frequency region | 3x 10⁶ - 3×10¹⁰ Hz | 10 m - 100 m | Nuclear magnetic resonance | | Microwave region | 3 × 10¹⁰-3 × 10¹² Hz | 100 m- 1 cm | Electron spin resonance | | | | 1 cm – 100 μm | Microwave (rotational) spectroscopy | | Infra-red region | 3 × 10¹²-3 × 10¹⁴ Hz | 100 mm – 1 μm | Infared (vibrational) spectroscopy | | Visible and Ultraviolet region | 3 x 10¹⁴-3 × 10¹⁶ Hz | 1 µm – 10 nm | Utra-violet visible (electronic) spectroscopy | | X-ray region | 3 x 10¹⁶-3 × 10¹⁸ Hz | 10 nm – 100 pm | Photoelectron spectroscopy | | y-ray region | 3 x 10¹⁸-3 × 10²⁰ Hz | 1 pm - 100 pm | Enegy changes involve the rearrangement of nuclear particles | 1 µm = 10⁴ cm; 1 nm = 10^-9 m = 10 Å ## 3.3 Atomic and Molecular Spectroscopy ### 3.3.1 Atomic Spectroscopy It is obtained by the interaction of electromagnetic spectrum with atoms. Each atom gives its own characteristic spectrum in the form of lines. It is, therefore referred to as line spectrum also. It can be obtained either by absorption or by emission of radiation. Depending upon the mode of formation, atomic spectrum is called absorption or emission spectroscopy. The characteristic line spectrum obtained in case of an atom is due to the electronic transitions between the energy levels. ### 3.3.2 Molecular Spectroscopy A molecule can absorb or emit energy as a result of transitions between different electronic energy levels. From Fig. 3.2, it is evident that each electronic level consists of a number of vibrational sub-levels represented by v = 0, 1, 2, 3 etc., and further each vibrational level consists of a number of rotational sub-levels represented by j = 0, 1, 2, 3 etc., v and j are called vibrational and rotational quantum numbers respectively. However, a molecule can absorb a quantum of energy and increase its vibrational energy and rotational energy also. The quanta associated with these three different types of energy levels namely, electronic, vibrational and rotational are of very different energy. The energies of the quanta that are absorbed or emitted by a molecule can be measured by spectroscopic means. The molecular spectra arise due to changes in rotational, vibrational and electronic energies possessed by molecules. The total energy of a molecule, according to Born-Oppenheimer approximation, is given by: E = E_tr + E_rot + E_vib + E_el where E_el >>> E_vib >> E_rot > E_tr where E_tr, E_rot, E_vib and E_el represent translational, rotational, vibrational and electronic energies respectively. The translational energy is not quantised, whereas all the other energies are quantised. Since the translational energy is negligibly small, we can write the above approximation as: E = E_rot + E_vib + E_el responding to the three types of energies possessed by molecules three quantum numbers namely, rotational quantum number (j), vibrational quantum number (v), and electronic quantum number have been assigned. A diagram illustrating the distribution of different energy levels is shown in Fig. 3.2. Two electronic levels n" and n' are shown. The lower level n" has a number of vibrational energy level denoted by the vibrational quantum number v″ = 0, 1, 2, and each vibrational level has its own rotational energy levels represented by the rotaional quantum number J" = 1, 2,..... In the same way, the upper electronic level n' has vibrational level v' = 0, 1, 2..... and vibrational level has rotational energy levels J' = 1, 2..... ## 3.4 Ultraviolet and Visible Spectroscopy To understand why some compounds are colored and some are not, and to determine the relationship of conjugation to color, we must make accurate measurements of light absorption at different wavelengths in and near the visible part of the spectrum. The visible region of the spectrum comprises photon energies of 36 to 72 kcal/mole, and the near ultraviolet region, out to 200 nm, extends this energy range to 143 kcal/mole. Ultraviolet radiation having wavelengths less than 200 nm is difficult to handle, and is seldom used as a routine tool for structural analysis. The energies noted above are sufficient to promote or excite a molecular electron to a higher energy orbital. Consequently, absorption spectroscopy carried out in this region is also called electronic spectroscopy. ### 3.4.1 Electronic Transition According to the Molecular orbital theory, when a molecule is excited by the absorption of energy (UV or visible light), its electron are promoted from a bonding to an antibonding orbitals. When a molecule is formed then valence electron taking part in bond formation exist in bonding orbital. Electrons forming sigma (σ) bond are called σ electrons and those which participate in a bond (π) are called π electrons. Unshared electrons in a molecule which do not take part in any bond formation exist as non bonding orbitals. Such electrons are called n-electrons, non bonding electrons. There are higher energy levels termed as antibonding orbitals which are associated with σ and π bond as σ* (sigma star) and π* (pi-star) orbitals. As n electrons do not take parts in bond formation thus is no antibonding orbital associated with it. ### 3.4.2 Selection Rules Transitions between vibrational levels within the same electronic level give rise to spectra which are called vibrational spectra. In the near infra-red, transitions between different rotational levels within the same vibrational level are responsible for rotational spectra in the far infra-red or microwave region. Infact a transition between two electronic level is usually accompanied by changes in the vibrational and rotational quantum numbers, so that electronic spectra are really electonic vibration-rotational spectra. In the same way, vibrational spectra are really vibrational-rotational and are normally given the name. However, very small changes are required for transitions between rotational level alone. Hence the spectra corresponding to the smallest changes in energy are thus called pure rotational spectra. The study of molecular spectra gives information about the structure and properties of molecules such as bond lengths, bond angles, bond strength, shapes, dipole moments etc. ### 3.4.3 Chromophores In Greek chromophores means colour carrier. It has been recognized since long that the colour of a molecule is due to one or more unsaturated groups present in it. These groups are called chromophores. They exhibit characteristic absorption in ultraviolet and visible regions. In simple words, chromophores are the functional groups which absorb electromagnetic radiations and may or may not impact colour to any compound. Typical examples of chromophores are C=C, CN, C=O, —N=N—, C=S, and -N=O in the order of increasing power of imparting colour. Simple chromophores like ethylene, acetylene undergo π→ π* transitions in the short wavelength region of ultraviolet radiations. Compound containing atoms as ——, —S, N shows absorption due to n → σ* transition. Some common chromophores along with the type of excitation and λ_max value have been summarized in the table below: | Chromophore | Example | Excitation | λ_max in nm | Molar Absorptivity | Solvent | | :----------- | :--------------- | :---------- | :----------------- | :---------------- | :------- | | C=C | Ethene | π- π* | 171 | 15,000 | hexane | | C=C | 1-Hexyne | π - π* | 180 | 10,000 | hexane | | C=O | Carbonyl | η – π* | 290 | 15 | hexane | | | | π - π* | 180 | 10,000 | hexane | | C-O | Nitromethane | η - π* | 275 | 17 | ethanol | | | | π - π* | 200 | 5,000 | ethanol | | -N=O | | η - π* | 200 | - | - | | C-X; X = Br | Methyl bromide | η - σ* | 205 | 200 | hexane | | X=I | Methyl Iodine | η - σ* | 255 | 360 | hexane | From the above chart it is clear that the only molecular species likely to absorb light in the 200 to 800 nm region are n-electron and hetero atoms having non-bonding electron pairs. Such light absorbing groups are referred to as chromophores. The non-bonding electrons on oxygen in alcohols and ethers do not give rise to absorotion above 160 nm. Consequently, pure alcohol and ether solvents may be used for spectroscopic studies. The presence of chromophores in a molecule is best documented by UV-visible spectroscopy. Fortunately, conjugation generally moves the absorptions maxima to longer wavelength, as in the case of isoprene, so conjugation becomes the major structural feature identified by this technique. ### 3.4.4 Auxochromes Those groups which do not impart colour to a compound by themselves, but increase the intensity of affect of a chromophore when attrached to them are called auxochromes. These groups show characteristic absorption below 2000 Å by themselves. When they are attrached to a chromophore, they usually cause a shift in the absorption towards longer wavelengths (Red end of the spectrum). Auxochromes also incease the intensity of absorption. Typical examples of auxochromes are -OH, -NH₂, -NR₂, -OR, -CH, and -Br. All auxochromes contain unshared pair of non-bonding electrons. The action of an auxochrome is due to the electronic transitions involving these non-bonding electrons, when conjugated with chromophores e.g., Nitrobenzene has only a tinge of colour but when -NH₂ group is attached at para position then conjugation leads to bathochromic shift i.e., towards longer wavelength and darkening of colour occur. ### 3.4.5 Factors Affecting λ_max and Intensity of Spectral Lines The effect of substitutents and solvent on π → π* transitions have been extensively studied. A most suitable solvent is one which does not itself absorb in the region under inventigation. Hexane and other hydrocarbons can be used as these are less polar and which have least interactions with the molecule under investigation. For ultraviolet spectroscopy, ethanol, water and cyclohexane serve the best purpose. The position and the intensity of absorption maximum is shifted for a particular chromophore by changing the polarity of the solvent. In general, the absorption maximum for the non-polar compounds is same in alcohol (polar) as well as in hexane (non-polar) solvent. The absorption maximum for the polar compounds ehanges with change in polarity of the solvent. The various effects are as follows: * **Bathochromic Shift:** It is an effect by virtue of which the absorption maximum is shifted towards longer wavelength due to the presence of an auxochrome or by change of solvent. If an electron donor group like OH, CH₃, NH₂ etc., is substituted in a compound, then the absorption band is shifted to longer wavelengths. Such shifts of absorption bands towards longer wavelengths due to substitution or solvent effect are known as bathochromic or red shifts. For example, the λ_maxfor CH and CH₃CH₃ are 254 mm and 261 mm. Thus, the substitution of hydrogen by CH₃ group in benzene causes bathochromic shift in the abosrption band. Conjugation of double bonds also cause bathochromic shift. For example: Ethylene (H₂C=CH₂) absorbs at λ_max = 175 nm in comparison to 1-butene (H₂C=CH-CH-CH₃) at λ_max= 185 nm or isobutene H₂C=C(CH₃)₂ at λ_max= 188 nm. The bathochromic shift is more pronounced as the number of alkyl group increases. * **Hypsochromic Shift:** It is an effect by virtue of which the absorption maximum is shifted towards shorter wavelength. If an electron acceptor group like acetyl group is substituted in a compound, then the absorption band is shifted to shorter wavelength. Such shifts of a compondand towards shorter wavelengths due to substitution of solvent effect is known as hypsochromic or blue shifts. If CH₃ group is introduced in an unsaturated chain of an organic compound, then conjugation is distroyed and absorption band of the compound is shifted to shorter wavelengths. Hydrogen bonding also produces Hypsochromic shift. | Compound | λ_max (nm) | Compound | λ_max (nm) | Compound | λ_max (nm) | | :------------- | :--------------- | :------------- | :--------------- | :------------- | :--------------- | | H-C=O | 290 | H-C=O | 235 | H-C=O | 205 | | | | | | | | | N | | Cl | | NH₂ | | Even change in solvent can also bring the Hypsochromic shift. For example: CH₃-C-CH₃ (acetone) absorbs at λ_max = 279 nm in hexane and at λ_max = 264.5 nm when dissolved in water. * **Hyperchromic Effect:** It is an effect due to which the intensity of absorption maximum increases i.e., ε_max increases. If a substituent group causes increase in the intensity of band, then the effect is called hyperchromic effect. The increase in the intensity of absorption band is due to the fact that the substituent group causes the increase in the molar extinction coefficient ε of the compound. For example, Benzene shows absorption at λ_max = 254 with ε_max = 200 whereas toluene shows absorption at λ_max = 261 with ε_max = 300, showing the hyperchromic effect. * **Hypochromic Effect:** If a substituent group causes the intensity of the band to decrease, then the effect is called hypochromic effect. As an example, ε_max for C₆H₆ and CH₃Cl are 200 and 170 respectively. Thus, substitution of Cl causes hypochromic effect. ### 3.4.6 Franck-Condon Principle The transitior between two electronic states take place according to this principle. The vibrational structure of electronic spectra can be investigated with the help of well known Frank-Condon principle. It states that an electronic transition takes place so rapidly that a vibrating molecule does not change its inter-nuclear distance to any appreciable extent during the transition because the electrons moves much faster than the nuclei hence the nuclei do not change their positions and velocities. This principle for the electronic transition of a atomic molecule has been explained in Fig. 3.7. The potential energy curve (Fig. 3.6) indicate that at equilibrium atoms of the diatomic molecule do vibrate i.e., their internuclear distance keep on changing. Two potential energy curves for the molecule in the ground state represented by E_o and in the first excited electronic state represented by E₁ are shown. The minimum in the potential energy curve E₁ occurs at slightly greater internuclear distance than the corresponding minimum in the ground state curve E_o. This is because the bonding in the excited state is weaker than in the ground state. The electronic transition is represented by a vertical line drawn between the energy levels of the two curves. From quantum mechanics it is known that the molecule is in the center of the ground vibrational level of the ground electronic state. When a photon falls on the molecule, the most probable electronic transition, according to Franck Condon principle, takes place from v" = 0 to v' = 2. The probability of other vibrational level transitions is low. As a result the relative intensities of these transitions are smaller than the intensity of the 0–2 transition as shown in Fig. 3.7. ### 3.4.7 Bond Dissociation Energy Let us consider a diatomic molecule which has a potential energy curve. E in the ground state and two potential energy curves E₁ and E₂ in the excited states. The curve E₂ does not have a minimum (Fig. 3.8) and the state is unstable for all inter nuclear separations. Since the bond is weaker in the excited state, the equilibrium internuclear distance is longer in E₁ than in E_o. According to Frank-Condon principle, the transitions are represented by arrows X i.e., (0 → 2) have maximum intensity. Two other possible transitions are represented by arrows Y and Z. The transition Y ends in the lowest vibrational level of the first excited electronic state, where the molecule is still held firmly. However, the transition Z promotes the molecule to a point where its energy is much higher than the dissociation energy D₂ of curve II is lesser than that of curve I i.e. D₂ < D₁. This is because of weaker bonding in exited state than in the ground state. The molecule will also dissociate if it is excited to a level above the point C where the potential energy curves for E₁ and E₂ cross. At an internuclear distance corresponding to point C, the molecule may change from one state to the other without energy change and continue to dissociate into two parts. ### 3.4.8 Applications of Ultraviolet and Visible Spectroscopy There are extensive applications of ultraviolet and visible spectroscopy in different fields of chemical interest. However, a few important applications are given here. * **Determination of dissociation energy:** The dissociation energy for a molecule can be determined with great accuracy from this spectrum. The dissociation energy is calculated from the wavelength that separates the discontinuous part of the spectrum from the continous part of the spectrum resulting from the dissociation of the molecule into two parts in varying amount of kinetic energy. * **Calculation of moment of inertia, vibrational frequency and interatomic distances of diatomic molecules:** This spectra also provide information regarding vibrational frequencies, moment of intertia, interatomic distance etc. In case of homo-nuclear diatomic molecules such as H₂ and N₂, the electronic transition takes place from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecule orbital (LUMO). This is in the short wave length side of the ultraviolet (UV) region. The behaviour of O₂ molecule is, however, exceptional, which shows a complex spectra. * **Identification of organic compounds:** It is also useful in establishing the identity of the new and unknown compounds. The spectrum of the new compound can be compared with the spectrum of the known compound. For instance, it helps in deciding whether a particular compound is cis or a trans geometrical isomer. The resemblance of the two spectra establishes the nature of the new compound. The absorption spectrum of an unknown compound is compared with those of a number of known compounds. If the absorption spectrum of the unknown compound tallies with a particular unknown compound, then the structure of the two will also be similar. The absorption spectra of synthetic (unknown) and natural vitamin A₂ are shown in Fig. 3.5. The similarity between the two spectra proved helpful in the identification and assignment of structure of synthetic vitamin A₂. The technique in which the spectrum of an unknown compound is tallied with those of known compounds, is known as finger printing. The points marked as A and C in the Fig. 3.8, where the value of λ_max is the same for two compounds are called isobestic points. * **Presence of impurities:** It can be used to detect the presence of impurities in a substance. The spectrum of the compound is compared with the spectrum of pure compound. The impurities will certainly cause extra absorption bands, characteristic to their structures. Ultraviolet and visible spectrophotometry is used for the control of purification of a compound. * **Determination of structure of organic compounds:** The fact that compounds having similar structures show similar absorption spectra that can be utilized in deciding the constitutions of compounds. For example, two possible structures for isatin have been suggested viz. Corresponding to the above two structures, two methyl ethers are known Comparison of the spectrum reveals that the absorption spectrum of isatin is similar to that of N-methyl ether, structure III. Thus, structure I is assigned to isatin. * **Confirmation of cis and trans hydrogen** Determination of Geometrical isomer i.e., whether compound is cis or trans is also established with the help of UV-visible spectroscopy. e.g., In case of cis isomer, due to steric repulsion, coplanarity is lost and thus conjuation is limited. It absorbs at shorter λ i.e., 268 nm as compared to trans which absorb at 272 nm. * **Determination of extrent of conjugation:** It is very useful technique to know the extent of multiple bonding in a molecule. It can also be used to distingush between conjugated diene and conjugated triene, conjugated and non-conjugated molecule, α, β unsaturated ketones and ẞ, y unsaturated ketones (these differ in the position of carbon-carbon double bond). The values of λ_max and ε_max are more for extended conjugated molecules as compared to those for an unconjugated alkenes. Bathochromic as well as hyperchromic effect are observed when the spectrum of conjugated triene is compared to that of conjugated diene (Fig. 3.9). * **Study of Kinetics:** If the λ_max values of reactants and products of a chemical reaction are quite different, then the change in the concentrations of reactants or products can be followed spectropho-tometrically and kinetics of the reaction can be studied. Spectrophotometric methods are particularly useful when the reactions are fast and the solutions are very dilute, e.g., photochemical transfor-mation of ergosterol to vitamin D₂ is well suited for spectrophotometric method. * **Other Physico-chemical Studies:** Apart from these, ultraviolet and visible spectrophotometry has been very useful in other physico-chemical studies, such as determination of dissociation constants of acids and bases, heat of formation of molecular addition compounds and complexes in solution, empirical formulae of complexes in solutions etc. ## 3.4.9 Fluorescence and Phosphorescence **Fluorescence:** Certain substance absorb photo-energy and get excited i. e., the electrons are raised from lower enrgy levels to higher energy level, but their excited molecule immediately or instantaneously re-radiates or emits part of the absorbed energy at a greater wavelength. Such substances which absorb photo-energy and emit a part of it immediately at a greater wavelength with a short time (10 sec.) are called Fluorescent Substances and this phenomenon is called Fluorescence. This process is instantaneous and starts immediately after the absorption of light and stops the moment the radiation is cut off. Examples of fluorescence are solutions of fluorescein and eosin. When their solution are placed in light, they show fluorescence from green to violet colour; uranyl sulphate, chloropyll (green colouring matter of plants) fluorospar (CaF₂) and petroleum also show this phenomenon. It may be pointed that different substances shows the phenomenon of fluorescence with different wavelengths. Thus, fluorospar show fluorescence with blue light, chlorophyll with red light, uranium glass with green light and so on. **Phosphorescence:** In this process the absorbed light energy raises the electron to a higher level and the excited electron revert back to the original state after some time lag and consequently absorbed light is emitted after some time. Thus, phosphorescence may be regarded as slow fluorescence. In this phenomenon, the emission of light of different wavelength continues even after the source of radiation has been cut off. The familiar examples of phosphorescence are BaS or SrS containing about 2.5% alkali chlorides (NaCl or KCl) and a trace of heavy metal sulphide. Such a mixture is generally used for painting watch dials, electric switches etc. Many dyestuffs which are fluorescent in aqueous solution become phosphorescent when dissolved in glycerol and cooled. ### 3.4.10 Mechanism of Fluorescence and Phosphorescence In fluorescence light absorbed by molecule is emitted almost instantaneously when or more steps. The emission of light ceases with the removal of source of light. The absorption of radiation takes the molecules to singlet excited state. As the excited molecule collides with the surrounding molecules, it gives up energy and steps down the ladder of vibrational levels of singlet excited state (radiationless decay). As the transition occurs from lowest vibrational energy level of singlet excited state to singlet ground state, radiation is emitted and fluorescence is observed. In phosphorescence light absorbed by molecule is given out slowly and even long after the removal of source of light. The absorption of radiation takes the molecule to singlet excited state. As excited molecule collides with the surrounding molecule it gives up energy and steps down the ladder of vibrational levels of singlet excited state. The initial steps are common in fluorescence and phosphorescence, till the vibrational energy levels of triplet excited state. At this stage, singlet and triplet excited states share a common point. At this point intersystem crossing occurs i.e., switch from singlet to triplet state. As the return from a triplet state to single state is spin forbidden, so triplet state acts as a slowly radiating reservoir. ### 3.4.11 Jablonski Diagram Fluorescence and Phosphorescence can be more conveniently shown with the help of Jablonski's diagram. In order to understand Jablonski diagram, we should know the term spin multiplicity. **Spin multiplicity:** Spin multiplicity of a state is given by (2S + 1) where S is the total electron spin. * If S = 0, then spin multiplicity = 1, Such an electronic state is called Singlet state. * If S = 1, then spin multiplicity = 3, Such an electronic state is called Triplet state. Consider an energy level, containing two electrons as shown in Fig. 3.7(a). If a radiation of suitable frequency falls on it, one of the paired electrons is promoted to upper energy level. If the spins of two electrons are parallel Fig. 3.7(b), then total spin S = S₁ + S₂ = 1/2 + 1/2 = 1 => spin multiplicity = (2S + 1) = 3 As spin multiplicity is 3, so the molecule is in Triplet excited state. If the spins of two electrons are antiparallel, then total spin S = S₁ + S₂ = 1/2 - 1/2 = 0 => Spin multiplicity = (2S + 1) = 1 As spin multiplicity is 1, so the molecule is in Singlet excited state. Since the electron can jump to any of the higher electronic states depending upon the energy of photon absorbed, we get a series of Singlet excited states represented as S₁, S₂, S₃, S₄..... and are known as first singlet excited state, second singlet excited state, third singlet dexcited states, and so on. Similarly T₁; T₂, T₃ are known as first triplet excited state, second triplet excited state, third triplet excited state and so on. For each singlet excited state there is corresponding triplet excited state and as the energy of singlet excited state is higher than the triplet state. Accordingly the energy sequence may be written as: E_s1 > E_t1; E_s2 > E_t2; E_s3 > E_t3 Fluorescence and phosphorescence are shown in Jablonski diagram in Fig. 3.12. The transitions which involve the return of excited states (S₃, S₂ or T₃, T₂) to the first excited state (S₁ or T₁) do not involve the emission of any radiations and are thus referred to as Non-radiative or radiationless transitions. When the energy of activated molecule is dissipated in the form of heat through molecular collisions, it is called **Internal conversion (IC)** it occurs in less than 10^-11 second. The molecule may lose energy by intersystem crossing (ISC). ISC involves the transition from singlet state to corresponding triplet state. For example, from S₂ to T₂ or S₁ to T₁. ISC like IC is also a non-radiative or radiationless transition. ISC is a spin forbidden process and hence occurs at slow rate. The transition from S₁ to S_o state is an allowed transition and occurs in 10^-8 second. The emission of radiation in this transition is called **Fluorescence**. The transition from the triplet state T₁ to the ground state S_o is slow and is spin forbidden. The emission of radiation in this transition is called **phosphorescence**. The life time of phosphorescence is larger and varies from 10^-3 second to 100 seconds. Both fluorescence and phosphorescence emit radiations of shorter frequencies or larger wavelength than the incident radiation (or exciting radiation) because some part of radiation absorbed by the molecule is dissipated in the form of heat during the non-radiative transitions. ## 3.4.12 Applications of Fluorescence in Medicines Clinically compatible time-resolved fluorescence spectroscopy and imaging systems developed by several research groups have shown that fluorescence can be employed to know about the diseased tissues in patients. It has also been investigated that the fluorescence lifetime contrast can be used in tissue characterization and diagnosis. Systematic studies of the fluorescence decay characteristics in patients are helpful for a complete and accurate clinical diagnosis. Some important applications in the field of medicine are listed below: * The analysis of the concentration of riboflavin (Vitamin B₂) in chloroform has been carried out. * Use of fluorescent microscopes and fluoroscope used in X-ray diagnosis help in testing the condition of food stuff and detecting ring worms etc. * Fluorescence is helpful in tissue characterization and diagnosis. * Applications of time resolved fluorescence to diagnosis of pathologic conditions in humans such as: * diagnosis of cancer of the gastrointestinal (GI) tract, bronchi/lung, skin, head and neck, and brain; * ophthalmic pathologies; and * atherosclerotic cardiovascular disease. * The potential role of autofluorescence is in the diagnosis of head and neck tumors including the cancer of the oral cavity through a variety of spectroscopic and imaging techniques. * Autofluorescence technique has been used in the treatment of primary brain tumors. * Several fluorescence techniques have been employed for the characterization of skin. Analysis of skin physiology, optical biopsy of skin, and detection of dermatological disorders including fungal infections, skin age, hair pigment, and cancer has been carried out by researchers in the field. * Time-resolved Fluorescence has been applied in the detection of eye diseases and research. ## Applications of fluorescence in other fields are as under: * For lighting purposes in fluorescent tubes: Mercury arc producing large proportion of ultra violet light is used in a tube coated with fluorescent salts which gives visible light. * By mixing fluorescent dyes with coloured paints, the fluorescence of the dye helps the light reflected by the paint to produce extra-ordinary brightness and luster. These materials are used for road signs. For example, Brilliant Sulpho Flavine FF and Rhodamine 6G dispersed in a special kind of plastic are used for this purpose. * In industry for testing and identifying materials, e.g. in rubber industry. * In television-the cathode stream of the photoelectric effect is made visible in the cathode ray tube by adding ZnS to which a little Ni is added to cut off phosphorescence which otherwise makes the picture blurred. * A fluorescent dye is used as whitener for washing cloth when mixed with washing powder. Both fluorescence and phosphorescence emit radiations of shorter frequencies or larger wavelength than the incident radiation (or exciting radiation) because some part of radiation absorbed by the molecule is dissipated in the form of heat during the non-radiative transitions.