UV Vis Spectro & Fluorimetry BP701T_IMA_I PDF

Dr. Hemant R. Tawale, Asso. Prof. & HOD, KGRDCP & RI. M.Pharm., Ph.D.(Pharma. Analysis.), PDF (USA)., FIAST., PGDIPL., IEA., LSS., LSSGB. B Pharm 7th Semester BP701T. INSTRUMENTAL METHODS OF ANALYSI...

Dr. Hemant R. Tawale, Asso. Prof. & HOD, KGRDCP & RI. M.Pharm., Ph.D.(Pharma. Analysis.), PDF (USA)., FIAST., PGDIPL., IEA., LSS., LSSGB. B Pharm 7th Semester BP701T. INSTRUMENTAL METHODS OF ANALYSIS This subject deals with the application of instrumental methods in qualitative and quantitative analysis of drugs. This subject is designed to impart a fundamental knowledge on the principles and instrumentation of spectroscopic and chromatographic technique. This also emphasizes on theoretical and practical knowledge on modern analytical instruments that are used for drug testing. UNIT -I UV Visible spectroscopy Electronic transitions, chromophores, auxochromes, spectral shifts, solvent effect on absorption spectra, Beer and Lambert’s law, Derivation and deviations. Instrumentation - Sources of radiation, wavelength selectors, sample cells, detectors- Photo tube, Photomultiplier tube, Photo voltaic cell, Silicon Photodiode. Applications - Spectrophotometric titrations, Single component and multi component Analysis Fluorimetry Theory, Concepts of singlet, doublet and triplet electronic states, internal and external conversions, factors affecting fluorescence, quenching, instrumentation and applications P a g e 1 | 33  UV VISIBLE SPECTROSCOPY Introduction to Electro Magnetic Radiation (EMR): Light travels in a straight line, but phenomenon like interference, refraction, diffraction, etc. could not explain this. To explain these phenomena, light is supposed to travel in waves. Light or EMR is a form of energy that is transmitted through space at a constant velocity of 3 × 108 𝑚𝑒𝑡𝑒𝑟/𝑠𝑒𝑐𝑜𝑛𝑑. These radiations are said to have dual nature exhibiting both:  Wave character  Particle character or corpuscular theory According to wave theory, light travels in the form of waves. This wave motion consists of oscillating electric (E) & magnetic (H) fields (vectors) directed perpendicular to each & perpendicular to the direction of the propagation of wave (Fig. 1). Fig. 1: Wave nature of light Quantum theory of EMR:  Quantum theory describes the EMR as consisting of a stream of energy packets, called Photons or Quanta, which travel in the direction of propagation of the beam with the velocity of light.  Thus, during emission or absorption of light by chemical species, the energy changes take place only discretely always as integral multiples of small units of energy i.e. photon.  The energy of the photon is proportional to the frequency of radiation, i.e. E α ν, or, E = hν , Where, h = Plank’s constant = 6.626 x 10-27 erg.sec. The energy of a photon is called quantum of energy & this depends only on the frequency but not on the intensity of radiation.  The waves are characterized by their wavelengths or frequencies or wave numbers.  The energy carried by an EMR is directly proportional to the frequency.  All types of radiations travel with the same velocity & no medium is required for their propagation. They can travel through vacuum also.  When visible light (a group of EMR) is passed through a prism, it is split up into seven colours which correspond to definite wavelengths. This phenomenon is called ‘dispersion’. P a g e 2 | 33 Spectroscopy:  The word spectroscopy is derived from spectrum which means a band of different colours formed due to difference in wavelength and skopin means examination or evaluation. Thus, spectroscopy is the branch of science that deals with the examination or evaluation of spectrum. It is defined as the interaction between the matter & EMR. It deals with emission as well as absorption spectra.  It is used to measure the energy difference between various molecular energy levels & to determine the atomic & molecular structures. The instruments used in such studies are called spectrophotometer.  If EMR (of certain wavelength range) are passed through the substance under analysis for sometimes, then radiations of certain wavelengths are absorbed by the substance. The wavelengths which are absorbed characterize some practical functional groups present in the compound or the compound itself. This dark pattern of lines which corresponds to the wavelengths absorbs is called Absorption spectrum. After absorption, the transmitted light is analyzed by the spectrometer relative to the incident light of a given frequency. The absorbed energy may heat up the sample or is re-emitted.  An emission spectrum is produced by the emission of radiant energy by an excited atom. The excitation of atoms can be brought about thermally (by heating the substance strongly) or electrically (by passing electric discharge through the vapours of the substance at a very low pressure). When an electric discharge is passed through the vapours of the substance, energy is absorbed & electrons in the ground state are promoted to Meta-stable states. When electrons from the Meta-stable state jump to the lower energy state, then some energy of definite frequency is released as radiation. If this radiation emitted is analyzed with the help of a spectroscope, an emission spectrum is observed. Characteristics/Units of wave (Fig.2): a. Wavelength: It is the distance between the adjacent crests or troughs in a particular wave. It is denoted by ‘λ’ (lambda). It can be expressed in Angstrom (0A) or nanometer (nm) or millimicrons (mμ) or centimeter (cm) or micrometer (μm). 1 nm = 10-9 m = 10-3 μm = 10 0A = 10-7 cm = 1 mμ Nanometer is frequently used in UV-Visible technique. b. Wave Number: It is the reciprocal of wavelength & it is expressed in per centimeter; or it is defined as the total number of waves which can pass through a space of 1 cm. It is expressed as ‘ū (nu bar)’. It is frequently used in IR technique. c. Frequency: it is defined as the number of waves which can pass through a point in one second. It is expressed as ν (nu) in cycles per second or in Hertz (Hz). 1 Hz = 1 cycle sec-1 1 Frequency α i.e., greater the wavelength, smaller is the frequency. wavelengt c Frequency, ν = Where, c = velocity of EMR (light) = 2.998 x 10-8 cm/sec h d. Energy: Energy of a particular wave is calculated as hc E = hν = = hcū Where, h = Plank’s constant = 6.626 x 10-27 erg. sec. h P a g e 3 | 33  Fig. 2: Characteristics of wave Electromagnetic spectrum:  The electromagnetic spectrum, for most spectroscopic purposes, is considered to be consisting of region of radiant energy ranging from wavelengths of 10 m to 1 x12-12 cm.  When a molecule absorbs EMR, it can undergo various types of excitation. This excitation may be  Electronic excitation,  Rotation excitation,  Excitation leading to a change in nuclear spin,  Excitation resulting in bond deformation & so on.  If the energy available approaches the ionization potential of the molecule, an electron may be ejected & ionization may occur.  Since each mode of excitation requires a specific quantity of energy, the different absorptions appear in different regions of the electromagnetic spectrum.  The various regions of electromagnetic spectrum are set out (Table:1 & Fig.3) & are labeled either according to the wavelength/ wave no. range used, or according to the type of the molecular energy levels involved, e.g. UV (electronic) spectra, IR (vibrational) spectra or RF (NMR) spectra. Table-1: Various regions of electromagnetic spectrum Type of Wavelength Wave no. Type of molecular spectrum Radiation RF > 100 mm < 3 x 109 Hz NMR (Spin orientation) Microwave 1 – 100 mm 10 – 0.1 cm-1 Rotational Far IR 50 μm – 1mm 200 – 10cm-1 Vibrational fundamental or rotational Mid IR 2.5 μm – 50 μm 4000 – 667 cm-1 Vibrational fundamental Near IR 780nm – 2.5 μm (13 – 4) x 103 cm-1 Vibrational (overtones) Visible 380nm – 780nm (2.6 – 1.3) x 104 cm-1 Electronic (valence orbital) Near UV 200nm – 380nm (5 – 2.6) x 104 cm-1 Electronic (valence orbital) Vacuum UV 10nm – 200nm (102 – 5) x 104 cm-1 Electronic (valence orbital) X-rays 10pm – 10nm 109 - 106 cm-1 Electronic (core orbitals) Gamma rays 10-10 cm 1010cm-1 Mossbauer effect (Nuclear transitions) Cosmic rays 10-12 cm 1012cm-1 excited states of nuclei P a g e 4 | 33 Photons of electromagnetic radiation of different energies (frequencies or wavelengths) interact with molecules in a variety of ways: 1. X-rays can excite and eject inner shell electrons, causing ionization and bond fragmentation. 2. Ultraviolet (UV) radiation causes high energy electronic transitions and visible radiation induces low energy electronic transitions in molecules. The resulting excited states may relax via bond breakage or various radiative and non-radiative pathways. 3. Infrared (IR) radiation causes vibrations in molecular bonds, such as bond stretching, bond bending etc. Therefore, IR spectroscopy is often called vibrational spectroscopy. 4. Microwaves can cause the molecules to rotate in the gas phase. This is the subject matter of microwave or rotational spectroscopy. 5. Radio waves can induce resonance of atomic nuclei rotation in a strong magnetic field. This is the subject of nuclear magnetic resonance (NMR) spectroscopy. Fig.3: Electromagnetic radiation of different frequencies or wavelengths P a g e 5 | 33 Absorption of EMR by organic molecules:  When a molecule absorbs radiation, its energy increases in proportion to the energy of the photon as E = hν  Since the energy absorbed by a molecule is quantized, there will not be continuous absorption by a molecule throughout a particular spectral range; instead, the molecule absorbs those frequencies which will provide it with the exact quantity of energy (quantum theory) necessary to raise its normal energy level to a higher level or levels.  Thus, when light radiations are passed through an organic compound or, when an organic molecule interacts with EMR, then electrons of the component atoms are excited. It may change its energy from E1 to E2 by absorption of radiation of frequency ‘ν’ so that, E2 - E1 = ∆E = nhν, when, n = an integer.  The lowest state of energy of an atom or molecule is called ground state. By absorbing one quantum of energy ‘hν’, the molecule is promoted to the next higher level & is said to be in the ‘b’. Similarly, absorption of more energy in integral multiples of hν, will result in further excitation to next higher energy levels i.e. excited state.  Study of absorbed radiations from a continuous source that are utilized in raising the internal energy of a molecule constitutes absorption spectroscopy.  In general, an absorption spectrum curve will consist of a series of peaks, each peak coinciding with a value of ‘ν’ which satisfies the relation (E2 - E1 = nhν).  After absorption of energy, the excited species returns to the ground state by emitting the energies radiations. The study of this emitted radiation constitutes emission spectroscopy.  The portions of EMR which do not satisfy relation may either simply pass through the matter or undergo scattering or reflection with or without change of wavelength. When molecules absorb energy and get excited, then either of the following energy changes occurs. a. Transition of an electron to high energy level, b. Change in the intermolecular vibrations of the molecule, c. Change of the moment of inertia of the molecule around its center of gravity, d. Transitions between electronic levels are found in the UV & visible regions, e. Transitions between vibrational levels, but within same electronic level in mid & near IR region, f. Transitions between neighboring rotational levels in far IR & microwave regions. P a g e 6 | 33  UV-Visible Spectroscopy UV wavelength region  200nm – 380nm, Visible wavelength region 380nm – 780nm  It is also known as Electronic spectroscopy since it involves the promotion of electrons (σ, π, n- electrons) from the ground state to the higher energy state.  It very useful to measure the number of conjugated double bonds & also aromatic conjugation within the various molecules.  It distinguishes between conjugated & non-conjugated systems, α, β- unsaturated carbonyl compounds from β, γ-analogues, homo annular & hetero annular conjugated dienes, etc. Principle: Since the energy levels of a molecule are quantized, the energy required to bring about the excitation is a fixed quantity. Thus, the EMR with only a particular value of frequency will be able to cause excitation. If the substance is exposed to radiation of some different value of frequency, energy will not be absorbed & thus, light radiation will not suffer any loss in intensity.  When UV or Visible radiation is passed through a substance, absorption of energy results in the promotion of electron from the ground electronic state to the excited electronic state. The amount of absorption of energy depends upon wavelength of the radiation & the structure of compound.  During the process of absorption, a large number of photon-molecule collisions are possible but only those collisions will cause absorption of energy in which the energy of the photon matches the energy difference between the ground & the excited electronic state of the molecule. The absorption of energy is quantized.  The wavelength at which maximum absorption of radiation takes place is called λ max. This λmax is characteristic or unique for every substance & this is a qualitative aspect, useful in identifying the substance (Fig.4).  λmax is not usually affected by concentration of the substance. The absorbance of a solution increases with concentration of a substance, but there is no change in λmax when concentration changes. Absorbance Fig.4: UV Spectrum of a chemical (10, 15, 20, 30, 40, 50 µg/ml) P a g e 7 | 33 Purpose to measure UV/VIS spectra: There are five main reasons to measure UV/VIS spectra:  UV/VIS spectra allow components present in the sample solution to be identified. More precisely, the position and, to some extent, the profile of the absorption peaks allow specific compounds to be identified. For example, organic compounds can be identified by their spectra, or solvent purity can be easily checked by UV/VIS spectroscopy.  Absorption peaks can be used to quantify the investigated sample. For example, the sample concentration can be calculated from the absorbance value of the peak: Fig. 4: A higher concentration leads to higher absorbance value  Based on the relationship between absorbance and sample concentration, UV/VIS spectroscopy is applied as a quantitative analytical technique in market segments such as e.g. Water Testing, Food and Beverages, Pharmaceutical, Chemical and Biotech Industry.  The position of the peaks in the spectrum reveals information about the molecular structure of the sample. For example, specific functional groups of a molecular structure, such as carbon-oxygen, C=O, or carbon-carbon double bonds, C=C, absorb at specific characteristic wavelengths.  The spectrum may reveal specific physical properties of the sample molecules. For instance, from the UV/VIS spectrum it is possible to: – calculate the extinction coefficient of the sample – calculate the melting point of proteins and nucleic acids by measuring the UV/VIS spectra at different temperatures – determine the rate of a reaction by monitoring the absorption spectra as a function of time (also known as kinetic measurements).  Finally, position and profile of the peaks in the spectrum can give information about the microscopic environment of the sample molecules. As an example, the presence of impurities or other solvents in the sample solution has an effect on position and of the profile of the peaks. In other words, the peaks may be broader or have shifted due to impurities. P a g e 8 | 33 Electronic transitions:  Theory  When the molecule absorbs UV or visible light, its electron gets promoted from the ground state to the higher energy state.  In the ground state, the spins of the electrons in each molecular orbital are essentially paired.  In the higher energy state, if the spins of the electrons are opposite and unpaired, then it is called as an excited singlet state.  On the other hand, if spins of the electrons in the excited state are parallel and unpaired, it is called as an excited triplet state. The triplet state is always lower in energy than the corresponding excited singlet state. Therefore, triplet state is more stable as compared to the excited singlet state.  In the triplet excited state, electrons are farther apart in space & thus, electron-electron repulsion is minimized.  Normally the absorption of UV or visible light results in singlet ground state to excited singlet state transition, i.e. excitation proceeds with the retention of spins.  An excited singlet state is converted to excited triplet state with the emission of energy as light. The transition from the singlet ground state to excited triplet state is symmetryforbidden.  The higher energy states are designated as high energy molecular orbitals & also called as anti-bonding orbitals.  The higher probable transition due to absorption of quantized energy involves the promotion of one electron from the highest occupied molecular orbital (HOMO) to the lowest available unfilled molecular orbital (LUMO).  The higher is the energy gap, the lower is the wavelength of the light absorbed.  Types of Electronic Transitions (Fig.5) According to the molecular orbital theory, when a molecule is excited by the absorption of energy (UV or visible light), its electrons are promoted from a bonding orbital to an anti-bonding orbital. There are several types of electronic transitions available to a molecule including:  The anti-bonding orbital which is associated with the excitation of σ-electron is called σ*- anti-bonding orbital. σ→ σ* transition takes place when α-electron promoted to anti-bonding (σ *) orbital.  When a non-bonding electron (η) gets promoted to an anti-bonding sigma orbital (σ *), then it represents η→ σ * transition.  Similarly, π→ π* transition represents the promotion of π-electrons to an anti-bonding π- orbital, (π*-orbital)  Similarly, when a η-electron (non-bonding) is promoted to an anti-bonding π-orbital, it represents η→ π* transition. The energy required for various transitions obey the following order: σ → σ * > η→ σ * > π→ π* > η→ π* P a g e 9 | 33  Fig. 5: Types of electronic transitions. Let us consider various transitions involved in UV spectroscopy: a. σ → σ * transitions: (Fig. 6a)  It is a high energy process since α-bonds are very strong.  It is observed with saturated compounds (especially hydrocarbons), in which all the valence shells electrons are involved in the formation of sigma bonds do not show absorption in the normal UV region, i.e. 120nm – 180nm. e.g. methane, ethane, propane, cyclopropane, etc.  It requires radiation of very short wavelength. To study such high energy transitions, the entire path length must be evacuated, since oxygen begins to absorb strongly above 200nm. b. η → σ* transitions: (Fig. 6b)  It occurs in saturated compounds containing one hetero atom with unshared pair of electrons (η-electrons).  E.g. saturated halides, alcohols, ethers, amine, etc.  It requires comparatively less energy than that required for σ → σ* transitions.  In saturated alkyl halides, the energy required for such a transition decreases with the increase in size of the halogen atom (or decrease in the electro-negativity of the atom). c. π → π* transitions: (Fig. 6c)  This type transitions occur in the unsaturated centers of the molecule; i.e. in compounds containing double or triple bonds & also in aromatics.  Absorption usually occurs within the region of ordinary UV-spectrophotometer.  The excitation of π-electron requires smaller energy & hence, transition of this type occurs at longer wavelength.  A π-electron of a double bond is excited to π*-orbital. E.g. alkenes, alkynes, carbonyl compounds, cyanides, azo compounds, etc.  This transition requires still lesser energy as compared to η→σ* transition. d. η → π* transitions: (Fig. 6d)  In this type, an electron of unshared electron pair on hetero atom gets excited to π*-anti- bonding orbital.  This type of transition requires least amount of energy out of all the transitions, & hence occurs at longer wavelength.  Absorption occurring at lower wavelength is usually intense. In simple cases, it is quite easy to tell whether the transition is η→π* or π→π* since the excitation coefficient for the former is quite low as compared to that of later. The exact electronic structure of the molecules in the excited state is not known but the electronic transition involves the redistribution of electrons within the molecule. E.g. aldehydes, ketones, nitro compounds, etc. contain both η→π* & π→π* transitions. P a g e 10 | 33  Fig. 6a Fig. 6b Fig. 6c Fig. 6d The Chromophore Concept:  All those compounds which absorb light of wavelength between 400-800nm appear colored to the human eye. Exact color depends upon the wavelength of light absorbed by the compound.  Originally, a chromophore was considered any system or unsaturated atoms/groups, which is responsible for imparting color to the compound. E.g. Nitro group gives yellow color, aryl conjugated azo group gives azo dyes color. Chromophore/ chromogenic agent:  Chromophore is defined as any isolated covalently bonded group that shows a characteristic absorption in the ultraviolet or the visible region, OR, Chromophore is defined as any group which exhibits absorption of electromagnetic radiations in the visible or ultraviolet region.  The absorption occurs irrespective of the fact whether color is produced or not. Some important chromophores are  Ethylenic, acetylenic, carbonyls, acids, esters, nitrile groups, etc.  A carbonyl isolated group does not produce any color in the UV spectroscopy by absorbing light.  Chromophores -  in which the group contains π-electrons & they undergo π→π* transitions, e.g. ethylenes, acetylenes, etc.  which contain both π-electrons & η-electrons, such chromophores undergo two types of transitions i.e. π→π* & η→π*; e.g. carbonyls, nitriles, azo compounds, nitro compound, etc. Types of chromophore: Conjugated π-bond systems (resonating system) Metal complexes P a g e 11 | 33   Conjugated π-bond system chromophores  In the conjugated chromophores, the electrons jump between energy levels that are extended π orbitals, created by a series of alternating single and double bonds, often in aromatic systems.  E.g., azo compounds, pH indicators (Fig.7.), lycopene, β-carotene, and anthocyanins.  Lengthening or extending a conjugated system with more unsaturated bonds in a molecule will tend to shift absorption to longer wavelengths Fig.7: Phenolphthalein changes structure & color in different medium  Metal complex chromophores  The metal complex chromophores arise from the splitting of d-orbitals by binding of a transition metal to ligands.  Examples of such chromophores can be seen in chlorophyll, hemoglobin, hemocyanin, and colorful minerals such as malachite. Auxochrome:  An auxochrome can be defined as any group which does not itself acts as a chromophore but whose presence brings about a shift of the absorption band towards the red end of the spectrum (longer wavelength).  These are covalently saturated groups with lone pair of electrons.  The absorption at longer wavelength is due to the combination of a chromophore & an auxochrome to give rise to another chromophore. An auxochromic group is called as color enhancing group.  Auxochromic group do not show characteristic absorption above 200 nm. Some common auxochromic groups are –OH, -OR, _NH2, -NHR, -NR2, -SH, etc.  The effect of the auxochrome is due to its ability to extend the conjugation of a chromophore by the sharing of non-bonding electrons. Thus, a new chromophore results which has a different value of the absorption maximum as well as the extinction coefficient. E.g. In case of aniline absorption maximum takes place at 280nm because the pair of electrons on nitrogen atom is in conjugation with the π bond system of the benzene ring. P a g e 12 | 33  In acidic solutions, a blue shift is caused and absorption takes place at short wavelength 225nm. => λmax = 225nm, εmax = 203 NH2 => λmax = 280nm, εmax = 1430 Mechanism: Hence, (-NH2) amino group is an auxochrome. It involves the shift of absorption maximum towards shorter wavelength and may be caused by removal of conjugation in a system or by change of solvent.  All auxochromic groups contain non-bonding electrons. Due to this, there is extension of conjugation of the chromophore by sharing the non-bonding electrons. Spectral shifts (Fig.8): Changes in chemical structure or the environment lead to changes in the absorption spectrum of molecules and materials. There are several terms that are commonly used to describe these shifts,  Bathochromic effect,  Hypsochromic effect,  Hyperchromic effect,  Hypochromic effect Fig. 8: Spectral shifts in UV-Visible range. a. Bathochromic shift:  The absorptions of two or more chromophores which are separated by more than one bond are usually additive, but when chromophores are conjugated, i.e. separated by a single bond, pronounced effects are produced. The maximum absorption is shifted to longer wavelengths, thus bringing it into the working range of spectrophotometers.  The effect, by virtue of which the absorption maximum is shifted towards longer wavelength due to the presence of an auxochrome or by the change of solvent, is called as Bathochromic shift or Red shift. b. Hypsochromic shift:  It is an effect by virtue of which the absorption maximum is shifted towards shorter wavelength. The absorption shifted towards shorter wavelength is called Blue shift or Hypsochromic shift.  It may be caused by the removal of conjugation & also by changing the polarity of the solvent. E.g. aniline has λmax 280nm, because the pair of electrons on nitrogen atom is in conjugation P a g e 13 | 33  with the π-bond system of the benzene ring. But in acidic medium, aniline behaves as C 6H5- NH3+ (anilinium ion) as the electron pair is no longer present & hence conjugation is removed, which causes blue shift & absorption occurs at shorter wavelength (λmax = 203nm). c. Hyperchromic shift:  It is an effect due to which the intensity of absorption maximum increases, i.e. εmax increases. E.g. Pyridine => λmax = 257 nm, εmax = 2750 2-methyl pyridine => λmax = 262 nm, εmax = 3560  The introduction of an auxochrome usually increases intensity of absorption. d. Hypochromic shift:  It is an effect due to which the intensity of absorption maximum decreases i.e. extinction coefficient, εmax decreases.  The introduction of group which distorts the geometry of the molecule causes hypochromic effect. E.g. Biphenyl => λmax = 250 nm, εmax = 19000 2-methyl biphenyl => λmax = 237 nm, εmax = 10250  It is due to the distortion caused by the methyl group in 2-methyl biphenyl. Isoabsorptive points or Isobestic point (Fig. 9a & 9b):  The wavelength of equal absorptivity of the two species (A & B), or same substance in two different mediums, that wavelength is known as isobestic point. Fig. 9a: Spectra of p-nitrophenol in acidic and basic solution P a g e 14 | 33  Fig. 9b: Spectra of the indicator bromothymol blue as a function of pH Choice of Solvent used and effect of solvent on λmax:  A solvent is a liquid that dissolves another solid, liquid, or gaseous solute, resulting in a solution at specified temperature.  The solvent use should be high purity, generally referred to as ‘spectrograde’. Care should be taken to keep lint & dust from contaminating the final solutions.  A good solvent should be transparent over the desired range of wavelengths. Usually solvents which do not contain conjugated system are most suitable for running the UV spectrum. E.g. commonly used solvents are water, 95% ethanol, n-hexane, cyclohexane.  A solvent should be chosen so that it does not react chemically with the sample.  Solvents can be broadly classified into two categories:  Polar  Non-Polar.  A drug may show varied spectrum at particular wavelength in one particular condition but shall absorb partially at the same wavelength in another conditions.  These appeared changes in the spectrum are exclusively due to various characteristic features namely- 1. Nature of solvent 2.Nature of absorption band 3.Nature of the solute Solvent effects:  The solvent exerts a profound influence on the quality and shape of spectrum.  The absorption spectrum of pharmaceutical substance depends practically upon the solvent that has been employed to solubilize the substance.  A drug may absorb a maximum radiation energy at particular wavelength in one solvent but shall absorb partially at the same wavelength in another solvent.  Polarity plays an important role in the position and intensity of absorption maximum of a particular chromophore.  E.g. In case of non-polar solvents e.g. Iodine solution (purple color) the absorption maxima occur at almost the same wavelength as in iodine vapour (518 nm), whereas in case of polar P a g e 15 | 33  solvents, a brownish color is obtained instead of purple color, because the absorption occurs at shorter wavelengths.  Purified and certified solvents for spectroscopy should be used as we are looking for the “smooth” absorbance curve of solvent.  Absorption bands of many substances are relatively sharper and may also exhibit fine structure when measured in solvents of low dipole moment.  By increasing the polarity of the solvent, compounds like dienes & conjugated hydrocarbons do not experience any appreciable shift.  A suitable solvent for UV-spectroscopy should meet the following requirements:  It should not itself absorb radiations in the region under investigation.  It should be less polar so that it has minimum interaction with the solute molecules.  The absorption maximum for the polar compounds is usually shifted with the change in the polarity of the solvents.  If the chromophore involved in the transition is more polar in its ground state than in its excited state, then the ground state is more stabilized than the excited state by a more polar solvent due to solvation. Chromophores with n→π * or n→σ * transitions exhibit such behavior.  The solvent molecules are oriented around the solute (chromophore) molecules to fit with the ground state charge distribution of the solute molecules. Hydrogen bonding or polar solvents interact more strongly with unshared electron pairs of the ground state molecule.  On excitation, the charge distribution in such systems changes markedly and therefore, the solvent molecules would not have position and orientation to interact with the excited state charge distribution.  Thus, the ground state of such solute molecules is more stabilized than the excited state. This widens the energy gap between the ground and excited states with increasing polarity of the solvents (Fig.9).  Therefore, more energy is required for the n→π*kind of electronic transition with increasing solvent polarity. This results in the shift of spectral peak positions towards shorter wavelength.  On the other hand, if the excited state of the chromophore is more polar with respect to the ground state, then the excited state will be more solvated and more stabilized by a more polar solvent. This kind of property is observed in the case chromophores with π →π* transitions.  The π electron density is equally distributed in the ground state and the C nuclei are shielded whereas in the π* excited state the C nuclei become electron deficient due to the electron promotion.  This favors stronger interaction of the excited state molecule with more polar or hydrogen bonding solvents and thereby stabilizing the excited state more than the ground state.  This decreases the energy gap between the excited and the ground states with increasing solvent polarity (Fig.9) which results in shift of absorption peak positions towards longer wavelengths.  We may recall that a shift of the absorption peak position (λ max ) towards shorter wavelengths is called a blue shift or hypsochromic effect.  On the other hand, a shift of the λ max towards longer wavelength is termed as the red shift or bathochromic effect. When there is an increase in the absorption intensity, (i.e., absorbance) the effect is termed as hyperchromic effect. If there is a decrease in the absorption intensity, the effect is termed as hypochromic effect. P a g e 16 | 33  Fig.9: Effect of solvent polarity on n→π* and π →π* transitions.  In short, π* orbitals are more stabilized by hydrogen bonding with polar solvents like water & alcohol. It is due to greater polarity of π* orbitals compared to π-orbital. Thus, small energy will be required for such a transition & absorption shows a red shift.  If the group (carbonyl) is more polar in ground state than in the excited state, then increasing polarity of the solvent stabilizes the non-bonding electron in the ground state due to hydrogen bonding. Thus, absorption is shifted to shorter wavelength.  If the group is more polar in excited state, then absorption is shifted to longer wavelength with increase in polarity of the solvent which helps in stabilizing the non-bonding electrons in the excited state.  Increase in polarity of solvents shifts η→ π* & η→ σ* to shorter wavelength,  Increase in polarity of solvents shifts π → π* to longer wavelength. Example: Effect of solvent polarity on spectrum of mesityl oxide (Table.2): Table.2: P a g e 17 | 33 Table.3: λmax of different solvents in UV region Solvent λ of max. absorption Water 191 nm Ether 215 nm Methanol 203 nm Ethanol 204 nm Chloroform 237 nm Carbon tetrachloride 265 nm Benzene 280 nm Tetrahydrofuran 220 nm Laws governing absorption of radiation: Experimental measurements are usually made in terms of transmittance (T), which is defined as: I 𝑇 = t, where It is the light intensity after it passes through the sample and I0 is the initial light I0 intensity. 1 It The relation between A and T is: 𝐴 = log = − log 𝑇 = − log T I0 Absorption of light by a sample: There are two laws which govern the absorption of light by the molecules. These are: 1. Beer’s law, 2. Lambert’s Law 1. Beer’s Law: This law states that, “when a beam of monochromatic radiation is passed through a solution of an absorbing substance, the rate of decrease of intensity of radiation with concentration of the absorbing solution is proportional to the intensity of incident radiation as well as theconcentration of the solution.” dI i. e, − αI dC dI or, − = K. I (where K = proportionality constant) dC dI or, − = K. 𝑑𝐶 --------- equn. 1 I Let, when concentration = 0, then I = I0 (intensity of incident light) & when concentration = c, then I = It (intensity of transmitted light) I dI c By integrating the equn.1, ∫ t − = ∫ K. 𝑑𝐶 I0 I 0 P a g e 18 | 33  or, −[ln 𝐼]It = K [𝐶]c + 𝑏 -------- equn. 2 I0 0 When ‘c’ = 0, intercept ‘b’ = 0 Thus, -[ln It – ln I0] = K [c-0] + 0 or, -ln It + ln I0 = Kc or, ln I0 -ln It = Kc I0 or, ln = 𝐾𝑐 It or, I0 = 𝑒Kc , Or, I0 = It eKc---------- equn. 3 It or, It = 𝑒–Kc , I0 or, It = I0 e-Kc 2. Lambert’s Law: It states that, “when a beam of monochromatic radiation passes through a homogenous absorbing medium, the rate of decrease of intensity of radiation with thickness (path length) of the absorbing solution is proportional to the intensity of incident radiation.” dI i. e, − αI dt dI or, − = K. I (where K = proportionality constant) dt dI or, − = K. 𝑑𝑡 -------- equn. 4 I Let, when pathlength = 0, then, I = I0 (intensity of incident light) & when pathlength = t, then, I = It (intensity of transmitted light) I dI c By integrating the equn.4, ∫ t − = ∫ K. 𝑑𝑡 I0 I 0 or, −[ln 𝐼]It = K [𝑡]t + 𝑏 -------equn. 5 I0 0 When ‘t’ = 0, intercept ‘b’ = 0 Thus, -[ln It – ln I0] = K [t-0] + 0 or, -ln It + ln I0 = Kt or, ln I0 -ln It = Kt I0 or, ln = 𝐾𝑡 It or, I0 = 𝑒Kt , Or, I0 = It eKt ----------equn. 6 It or, It = 𝑒–Kt , I0 or, It = I0 e-Kt by combining equn. 3 & 6, we get I0 = It eKct --------- equn. 7 The above equn. can also be written by changing the natural logarithm to the base 10. I0 = It 10act, where ‘a’ = extinction coefficient = K/2.303 P a g e 19 | 33  or, I0 = 10act It I or, 𝑙𝑜𝑔 0 = 𝑎𝑐𝑡 It It We know, transmittance (T) is expressed in terms of %T, 𝑇= & I0 𝟏 absorbance 𝑨 = 𝒍𝒐𝒈 = − 𝐥𝐨𝐠 𝑻 = − 𝐥𝐨𝐠 ( 𝑰𝒕 ) = 𝐥𝐨𝐠 ( 𝑰 ) = 𝐚𝐜𝐭 = 𝛆 𝐜𝐭 𝟎 𝑻 𝑰𝟎 𝑰𝒕 Where ε = molecular extinction coefficient or molar absorptivity, c = concentration of solution in moles/liter, t = path length of the sample medium (usually 1cm) A or, 𝛆= ct 1% Molecualr weigh or, 𝛆 = E1cm × 10 Where, E1% = absorbance of 1% w/v solution using a path length 1 cm, which is constant for each substance. VISIBLE SPECTROSCOPY & COLORIMETRY:  It is concerned with the study of absorption of visible radiation whose wavelength ranges from 380nm – 780nm.  All coloured substances absorb in this wavelength region in different manner.  Colourless solutions are converted to coloured solution by reacting with chemicals called as ‘chromogenic reagent’ and the involved reaction is called as ‘chromogenic reaction’.  The absorbing capacity of a coloured substance is directly proportional to the amount of desired constituent. Properties of colored system:  Sensitivity: Solution should be intensely colored & hence an easily detectable change in intensity can be obtained by small changes in the concentration.  Stability: Intensity of color should remain constant for a long time.  Specificity: Only desired constituent should develop a color.  Conformity of Beer’s law: The measurement may be facilitated for a single or poly-component system, if Beer’s law is obeyed by the colored solution. Instrumentation (Table.4): UV or visible spectrophotometer / colorimeter consist of following apparatus as per requirement / maker.  Source of light/ radiation  Filter / monochromator (converts polychromatic light to monochromatic light).  Monochromator (Entrance slit, collimator, Prism/Grating, collimator, exit slit)  Sample cell  Detector Source of Light  Tungsten filament lamps and Hydrogen-Deuterium lamps are most widely used and suitable light source as they cover the whole UV region.   Tungsten filament lamps are rich in red radiations; more specifically they emit the radiations P a g e 20 | 33  of 375 nm, while the intensity of Hydrogen-Deuterium lamps falls below 375 nm.   Monochromator  Monochromators generally is composed of prisms and slits.   Most of the spectrophotometers are double beam spectrophotometers. P a g e 21 | 33   The radiation emitted from the primary source is dispersed with the help of rotating prisms.   The various wavelengths of the light source which are separated by the prism are then selected by the slits such the rotation of the prism results in a series of continuously increasing wavelength to pass through the slits for recording purpose.  The beam selected by the slit is monochromatic and further divided into two beams with the help of another prism.  Sample and reference cells  One of the two divided beams is passed through the sample solution and second beam is passé through the reference solution.   Both sample and reference solution are contained in the cells.   These cells are made of either silica or quartz. Glass can’t be used for the cells as it also absorbs light in the UV region. Detector  Generally, two photocells serve the purpose of detector in UV spectroscopy.   One of the photocell receives the beam from sample cell and second detector receives the beam from the reference.   The intensity of the radiation from the reference cell is stronger than the beam of sample cell. This results in the generation of pulsating or alternating currents in the photocells.  Amplifier  The alternating current generated in the photocells is transferred to the amplifier.  The amplifier is coupled to a small servometer.  Generally current generated in the photocells is of very low intensity, the main purpose of amplifier is to amplify the signals many times so we can get clear and recordable signals.  Recording devices  Most of the time amplifier is coupled to a pen recorder which is connected to the computer.  Computer stores all the data generated and produces the spectrum of the desired compound.  Table.4: Instruments used in UV-Visible Spectrophotometer Visible Spectrophotometer UV Apparatus Colorimeter Hydrogen lamp, deuterium Source of Tungsten lamp, Tungsten lamp, lamp, xenon lamp, mercury arc radiation carbon arc lamp carbon arc lamp lamp Prism type monochromator Filters/ Absorption filter, Grating type monochromator (Dispersive / Refractive, Littrow / monochromators interference filter (Diffraction, Transmission) Reflective type) Cylindrical Sample cell Rectangular glass type Rectangular quartz type Glass/plastic type Phototubes / photo emissive cells, Phototubes / photo emissive Barrier layer cell / Detector Photomultiplier tubes, Silicon cells, Photomultiplier tubes, photo voltaic cell Photodiode Silicon Photodiode P a g e 22 | 33  Fig.10: Single beam colorimeter Fig.11: Single beam spectrophotometer Fig.12: Double beam spectrophotometer Fig. 13: Cuvette-based single-beam array spectrophotometer P a g e 23 | 33 Deviation of Beer’s law (Fig.14):  A system is said to obey Beer’s law, when a plot of absorbance Vs concentration gives a straight line. The straight line is obtained by using line of best fit or method of least squares or by joining the maximum no. of points in such a way that positive & negative errors are balanced or minimized.  When a straight line is not obtained i.e. non-linear curve is obtained in a plot of concentration Vs absorbance i.e. called as Beer’s deviation; that may be positive or negative deviation.  Positive deviation => when a small change in concentration produces greater change in absorbance.  Negative deviation => when a large change in concentration produces small change in absorbance. 2.000 Calibration curve +ve deviation 1.500 Linearity Absorbance 1.000 -ve deviation 0.500 0.000 12 16 20 Concentration (μg/ml) Fig.14: Calibration curve of a chemical in different solution Factors involved for deviation: ii. Instrumental - a. Stray radiation b. Improper slit width c. Fluctuation in single beam d. Use of non-monochromatic light iii. Physiochemical changes in solution –  Association => Methylene blue at concentration 10-5 present as monomer form and absorbs at λmax 660nm, but above concentration 10-4 present as dimer & trimer form and absorbs at λmax 600nm.  Dissociation => K2Cr2O7 in higher concentration solution orange in color & shows λ max 450nm where as in lower concentration yellow solution & absorbs at λmax 410nm. Cr2O72- + H2O ↔ 2HCrO42- ↔ 2H+ + 2CrO42- Orange yellow  Ionization (change in pH)  Faulty development of colour (incompletion of reaction) shows instability in color. E.g. determination of Fe using thioglycolic acid before completion of reaction.  Refractive index at higher concentration. P a g e 24 | 33 Applications of UV-Visible spectroscopy:  Detection of functional groups: To detect the presence or absence of chromophore. The absence of a band at particular wavelength may be regarded as an evidence for the absence of a particular group in the compound. If the spectrum is transparent above 200nm, it shows the absence of (i) conjugation, (ii) a carbonyl group (aldehyde & ketones), (iii) benzene or aromatic compounds, (iv) bromo or iodo atoms.  Extent of conjugation: The extent of conjugation in polynes R-(CH=CH)n-R can be estimated. Addition in un-saturation with the increase in the number of double bonds (increase in the value of n) shifts the absorption to longer wavelength.  Qualitative analysis / Identification of an unknown compound: An unknown compound can be identified by comparing its spectrum with the known spectra. If the two spectra coincide, the two compounds must be identical. If two spectra do not coincide, then the expected structure is different from the known compound.  UV/VIS spectroscopy is used as a tool to identify if the analyte is pure and did not undergo decomposition. For example, this technique is used for quality control of incoming raw material, and for the purity check of biologically relevant compounds such as the nucleic acids, DNA and RNA. Additionally, the melting point of DNA can be determined by recording its UV/VIS spectrum at different temperatures. Finally, by means of UV/VIS spectroscopy it is possible to differentiate between saturated and unsaturated fatty acids present in olive oil, and thus to monitor its quality.  In food & beverage industry: To monitor and improve product quality and consistency. The influence of packing material and stabilizers as well as chemical deterioration and degradation processes can also be observed with this method. A typical application in this market segment is the check for the purity of olive oil, which enables the product to be classified as “Extra Virgin”, “Virgin”, or simply “Olive Oil”. Standards are in place for the evaluation of olive oil based on the absorbance characteristics of certain molecules in the UV/VIS spectrum. Olive oil contains about 98% triglycerides. Unsaturated fatty acids in the oil are susceptible to breakdown and oxidation. The oxidation of free fatty acids causes the formation of peroxides. This leads to rancidity and degradation of the olive oil over time. Beside other parameters, this effect is evaluated by the conjugated di-enes and tri-enes of unsaturated fatty acids (conjugated C=C double bonds) which absorb in the range of 230 to 270 nm.  In chemical industry: For the determination of the purity of organic solutions. Additional peaks appearing at specific wavelengths can be observed due to impurities in the sample. An example in the chemical industry is the purity control in alcohol. Alcohol can be contaminated by benzene, which absorbs light at 280 nm, whereas alcohol absorbs at 210 nm. The measurement of the UV/VIS spectrum can easily tell if the sample is contaminated if an extra peak is present at 280 nm.  Distinction in conjugated & non-conjugated compounds: It also distinguishes between a conjugated & a non-conjugated compound.  Structure elucidation of organic compounds.  Determination of pKa value of indicators: pKa = pH - [log (ionized/unionized)]. The value of log (ionized/unionized) can be determined spectrophotometrically i.e. concentration Vs. absorbance at different pH & from the equation pKa can be calculated.  Quantitative analysis:  Using εmax values (E1%1cm)  Raw material  Direct comparison method P a g e 25 | 33   Calibration curve method  Based on the Lambert-Beer Law, the concentration of a compound in a solution can be determined quantitatively by UV/VIS spectroscopy. To perform that, a calibration line is first determined by measuring the absorption of several standard solutions of known concentration. In this way, the concentration of samples such as, DNA, RNA, proteins, carbohydrates or organic compounds can be determined.  The linear relationship between absorbance and concentration of a sample opens the door for a variety of quantitative analyses.  To determine an unknown concentration of a sample solution by UV/VIS spectroscopy, a calibration line must first be created. This is done by measuring the light absorption of several standard solutions of different, known concentrations at a predefined, fixed wavelength. In the following example, 5 standard solutions of increasing concentrations were measured at a predefined wavelength: Fig. : The calibration step and the standard list to determine the calibration line are indicated on the instrument display  A calibration line was obtained by plotting the absorbance values as a function of the concentration. Finally, a linear regression of the measured values gives the calibration line: b s o r b a n c e Concentration (mg/L)  Using the calibration line, an unknown sample can now be determined from its absorbance.  Determination of molecular weight  Chemical kinetics: Zero order, 1st order, 2nd order reaction  Assay of pharmaceutical substances  Keto-enol tautomerism. P a g e 26 | 33 FLUORIMETRY / FLUORESCENCE SPECTROSCOPY Fluorescence: It is a phenomenon of emission of radiation when the molecules are excited by radiation at certain wavelength. Fluorimetry: It is measurement of fluorescence intensity at a particular wavelength with the help of a filter fluorimeter or a spectrofluorimeter. Principle:  Molecule contains σ electrons, π electrons and nonbonding (n) electron.  The electrons may be present in bonding molecular orbital. It is called as highest occupied molecular orbital (HOMO). It has lest energy and more stable.  When the molecules absorb radiant energy from a light source, the bonding electrons may be promoted to anti bonding molecular orbital (LUMO). It has more energy and hence less stable. The process of promotion of electrons from HOMO to LUMO with absorption of energy is called as excitation.  Singlet state: a state in which all the electrons in a molecule are paired   Doublet state: a state in which un paired electrons are present  or   Triplet state: a state in which unpaired electrons of same spin present    Singlet excited state: a state in which electrons are unpaired but of opposite spin like   (un paired and opposite spin) When light of appropriate wavelength is absorbed by a molecule the electrons are promoted from singlet ground state to singlet excited state. Once the molecule is in this excited state, relaxation can occur via several processes by emission of radiation. The processes can be the following 1) Collisional deactivation 2) Fluorescence 3) Phosphorescence Fig.15: Calibration curve of a chemical in different solution P a g e 27 | 33 Collisional deactivation: In which entire energy lost due to collision de activation and no radiation emitted. Fluorescence: excited singlet state is highly unstable. Relaxation of electrons from excited singlet to singlet ground state with emission of light. Phosphorescence: At favorable condition like low temperature and absence of oxygen there is transition from excited singlet state to triplet state which is called as inner system crossing. The emission of radiation when electrons undergo transition from triplet state to singlet ground state is called as phosphorescence. Internal conversion: Intermolecular process by which a molecule passes to a lower energy electronic state without emission of light. Overlap of vibrational energy levels in two electronic energy levels. External conversion: Deactivation of an excited electronic state by interaction and energy transfer between the excited molecule and solvent or other solutes. Intersystem crossing: Process in which spin of an excited electron is reversed and change in multiplicity results. Most common when vibrational manifold overlap exists and when the molecule has a heavy atom substituent (e.g., Br, I). Factors affecting fluorescence intensity: 1. Concentration 6. pH 2. Quantum yield of fluorescence 7. Temperature& viscosity 3. Intensity of incident light 8. Photodecomposition 4. Adsorption 9. Quenchers 5. Oxygen 10. Scatter 1. Concentration: Fluorescence intensity is proportional to concentration of substance only when the absorbance is less than 0.02 We know, Absorbance ‘A’ = log Io/It, Or, A= abc Io=intensity of incident light It=intensity of transmitted light a= absorptivity of constant b= Pathlength c= concentration 2. Quantum yield of fluorescence (ϕ):  (ϕ) = number of photons emitted/number of photons absorbed  It is always less than 1.0 since some energy is lost by radiation less pathways (Collisional, Intersystem Crossing, Vibrational Relaxation) 3. Intensity of incident light: Increase in the intensity of incident light on the sample fluorescence intensity also increases. 4. Adsorption: Adsorption of sample solution in the container may leads to a serious problem. 5. Oxygen: Oxidation of fluorescent species to a non-fluorescent species, quenches fluorescent substance. P a g e 28 | 33  29 6. pH: Alteration of pH of a solution will have significant effect on fluorescence. E.g., Aniline in alkali medium gives visible fluorescence but in acidic condition gives fluorescence in visible region. 7. Temperature and viscosity:  Temperature increases can increase the collisional de activation, and reduce fluorescent intensity.  If viscosity of solution is more the frequency of collisions are reduced and increase in fluorescent intensity. 8. Photochemical decomposition: Absorption of intense radiation leads to photochemical decomposition of a fluorescent substance to less fluorescent or non-fluorescent substance. 9. Quenchers:  Quenching is the reduction of fluorescence intensity by the presence of substance in the sample other than the fluorescent analyte.  Quenching is following types:  Inner fluorescent effect: Absorption of Incident (UV) light or emitted (fluorescent) light by primary and secondary filters leads to decrease in fluorescence intensity.  Self-quenching: At low concentration linearity is observed, at high concentration of the same substance increase in fluorescent intensity is observed. This phenomenon is called self-quenching.  Collisional quenching: Collisions between the fluorescent substance and halide ions leads to reduction in fluorescence intensity.  Static quenching: This occurs because of complex formation between the fluorescent molecule and other molecules. Ex: caffeine reduces fluorescence of riboflavin. 10. Scatter: Scatter is mainly due to colloidal particles in solution. Scattering of incident light after passing through the sample leads to decrease in fluorescence intensity. Instrumentation: 1) Source of light:  Mercury vapor lamp: Mercury vapor at high pressure give intense lines on continuous background above 350nm.low pressure mercury vapor gives an additional line at 254nm.it is used in filter fluorimeter.  Xenon arc lamp: It give more intense radiation than mercury vapor lamp. it is used in spectrofluorimeter.  Tungsten lamp: If excitation has to be done in visible region this can be used. It is used in low cost instruments. 2) Filters and Monochromators:  Filters: These are nothing but optical filters work on the principle of absorption of unwanted light and transmitting the required wavelength of light. In inexpensive instruments fluorimeter primary filter and secondary filter are present.  Primary filter: Absorbs visible radiation and transmit UV radiation.  Secondary filter: Absorbs UV radiation and transmit visible radiation.  Monochromators: They convert polychromatic light into monochromatic light. They can isolate a specific range of wavelength or a particular wavelength of radiation from a source.  Excitation monochromators: Provides suitable radiation for excitation of molecule.  Emission monochromators: Isolate only the radiation emitted by the fluorescent molecules. P a g e 29 | 33  30 3) Sample cells: These are meant for holding liquid samples. These are made up of quartz and can have various shapes ex: cylindrical or rectangular etc. 4) Detectors: Photometric detectors are used. They are  Barrier layer /photovoltaic cell:  It is employed in inexpensive instruments. For ex: Filter Fluorimeter.  It consists of a copper plate coated with a thin layer of cuprous oxide (Cu2O). A semi- transparent film of silver is laid on this plate to provide good contact.  When external light falls on the oxide layer, the electrons emitted from the oxide layer move into the copper plate.  Then oxide layer becomes positive and copper plate becomes negative.  Hence an emf develops between the oxide layer and copper plate and behaves like a voltaic cell. So, it is called photovoltaic cell.  A galvanometer is connected externally between silver film and copper plate and the deflection in the galvanometer shows the current flow through it.  The amount of current is found to be proportional to the intensity of incident light  Photomultiplier tubes (PMT):  These are incorporated in expensive instruments like spectrofluorimeter. Its sensitivity is high due to measuring weak intensity of light.  The principle employed in this detector is that, multiplication of photoelectrons by secondary emission of electrons.  This is achieved by using a photo cathode and a series of anodes (Dyanodes). Up to 10 dyanodes are used. Each dyanode is maintained at 75 - 100V higher than the preceding one.  At each stage, the electron emission is multiplied by a factor of 4 to 5 due to secondary emission of electrons and hence an overall factor of 106 is achieved.  PMT can detect very weak signals, even 200 times weaker than that could be done using photovoltaic cell. Hence it is useful in fluorescence measurements.  PMT should be shielded from stray light in order to have accurate results. Fig.16: Schematic diagram of Photo Multiplier Tube (PMT) Instruments: The most common types are: □ Single beam (filter) fluorimeter □ Double beam (filter) fluorimeter □ Spectrofluorimeter (double beam) P a g e 30 | 33  31  Single beam (filter) fluorimeter  It contains tungsten lamp as a source of light and has an optical system consists of primary filter.  The emitted radiations are measured at 900 by using a secondary filter and detector. Primary filter absorbs visible radiation and transmit UV radiation which excites the molecule present in sample cell.  Instead of 90 if we use 180 geometry as in colorimetry secondary filter has to be highly efficient otherwise both the unabsorbed UV radiation and fluorescent radiation will produce detector response and give false result.  Single beam instruments are simple in construction cheaper and easy to operate.  Double beam fluorimeter  It is similar to single beam except that the two incident beams from a single light source pass through primary filters separately and fall on another reference solution. Then the emitted radiations from the sample or reference sample pass separately through secondary filter and produce response combinedly on a detector. Fig.17: Double beam fluorimeter  Spectrofluorimeter: □ In this primary filter in double beam fluorimeter is replaced by excitation monochromator and the secondary filter is replaced by emission monochromator. □ Incident beam is split into sample and reference beam by using beam splitter. P a g e 31 | 33  32 Fig.18: Applications:  Fluorimetric methods are not useful in qualitative analysis and much used in quantitative analysis.  Determination of inorganic substances. Al3+, Li+, Zn2+  Determination of thiamine HCl.  Determination of phenytoin.  Determination of indoles, phenols, & phenothiazines  Determination of napthols, proteins, plant pigments and steroids.  Fluorimetry, nowadays can be used in detection of impurities in nanogram level better than absorbance spectrophotometer with special emphasis in determining components of sample at the end of chromatographic or capillary column.  Determination of ruthenium ions in presence of other platinum metals.  Determination of boron in steel, aluminum in alloys, manganese in steel.  Determination of boron in steel by complex formed with benzoin.  Estimation of cadmium with 2-(2 hydroxyphenyl) benzoxazole in presence of tartarate.  Respiratory tract infections. S.No Name of the compound Experimental Emission conditions/ PH Wavelength (nm) 1 Adrenaline 1 335 2 Cynacobalamine 7 305 3 Riboflavin 6 520 4 Morphine 7 350 5 Hydrocortisone Acidic 520 6 Pentobarbitone 13 440 7 Amylobarbitone 14 410 P a g e 32 | 33 References:  Skoog DA, Holler FJ, Crouch SR. Principles of Instrumental Analysis, 7th ed. Boston (USA): Cengage Learning Publishers;2016.  Beckett AH, Stenlake JB. Practical Pharmaceutical Chemistry, 4th ed. vol 2, University of London: The Athlone Press;2001.  Sharma BK. Instrumental Methods of Chemical Analysis. 25th ed. Meerut (India): Krishna Prakashan Media;2005.  Ravisankar S. Textbook of Pharmaceutical Analysis. 5th ed. India: RX Publisher;2018.  Sharma YR. Elementary Organic Spectroscopy, 5th rev. ed. New Delhi (India): S Chand Publishers;2013.  Vogel AI. Text book of Quantitative Chemical Analysis, 5 th ed. New York (USA): Longman Scientific & Technical Publisher;1989. P a g e 33 | 33