UV- Visible Spectrophotometry: Introduction, Spectral Shifts, Instrumentation and Applications.pptx

This presentation covers the fundamental principles, instrumentation, and applications of this analytical technique. Learn how to measure light absorption by samples in the ultraviolet and visible regions of the electromagnetic spectrum. Understand Beer's Law and its significance in quantitative analysis. Explore various applications in chemistry, biochemistry, and environmental science. Whether you're a student or professional, this presentation will enhance your knowledge of UV-Vis spectroscopy.

1. UV-VISIBLE SPECTROSCOPY Presented by- Jaskiran Kaur Assistant Professor SCOP, Barabanki 1

2. CONTENTS Introduction The Nature Of Electronic Excitations Electronic Transitions Chromophores Auxochromes Spectral Shifts Solvent Effects On The Absorption Spectra Beer and Lambert’s Law Instrumentation Applications 2

3. INTRODUCTION Spectroscopy is the measurement and interpretation of Electro Magnetic Radiation (EMR) absorbed or emitted when the molecules or atoms or ions of a sample move from one energy state to another energy state. This change may be from Ground State to excited state or excited state to Ground state. At ground state, the energy of a molecule is the sum total of rotational, Vibrational and electronic energies. In other words, spectroscopy measures the changes in rotational, vibrational and /or electronic energies. 3

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6. THE NATURE OF ELECTRONIC EXCITATIONS When continuous radiation passes through a transparent material, a portion of the radiation may be absorbed. If that occurs, the residual radiation, when it is passed through a prism, yields a spectrum with gaps in it, called an absorption spectrum. As a result of energy absorption, atoms or molecules pass from a state of low energy (the initial, or ground, state) to a state of higher energy (the excited state). The electromagnetic radiation that is absorbed has energy exactly equal to the energy difference between the excited and ground states. In the case of ultraviolet and visible spectroscopy, the transitions that result in the absorption of electromagnetic radiation in this region of the spectrum are transitions between electronic energy levels. As a molecule absorbs energy, an electron is promoted from an occupied orbital to an unoccupied orbital of greater potential energy. 6

7. The Excitation Process 7

8. ELECTRONIC TRANSITIONS The electrons that contribute to the absorption characteristics of an organic molecule are: Bonding electrons: These electrons directly participate in bond formation. Non-bonding electrons: Unshared outer electrons that are localized about atoms such as O, N, Halogens (X), S. There are different types of electrons contributing electronic transition. Sigma ( σ ) electrons Pi ( π ) electrons Non-bonding (n) electrons Anti -Bonding (*) 8

9. Sigma ( σ ) electrons: Saturated bonds only contain sigma electrons which are held very tightly resulting in very high bond strengths. Compounds containing only σ -electrons are useful as solvents for near UV region. Example : Saturated hydrocarbons, alkanes like n-hexane are used as solvents in UV region. Pi ( π ) electrons: Pi electrons are present in unsaturated compounds like alkenes, alkynes and aromatic compounds. Examples include all alkenes, alkynes and aromatic compounds. Non-bonding (n) electrons: Non-bonding electrons are less fimly held unshared outer electrons localized on hetero atoms such as oxygen, nitrogen, sulphur or halogens. Sigma electron > Pi electron > Non-bonding electron. Anti-Bonding electrons: The unoccupied, or antibonding orbitals (π* and *), are the orbitals of highest energy. 9

10. Types of E lectronic Transitions σ → σ* transition π → π* transition n → σ* transition n → π* transition 10

11. 1. σ → σ* transition σ electron from orbital is excited to corresponding anti-bonding orbital σ*. The energy required is large for this transition. e.g. Methane (CH4) has C-H bond only and can undergo σ → σ* transition and shows absorbance maxima at 125 nm. 2. π → π* transition π electron in a bonding orbital is excited to corresponding anti-bonding orbital π*. Compounds containing multiple bonds like alkenes, alkynes, carbonyl, nitriles, aromatic compounds, etc undergo π → π* transitions. e.g. Alkenes generally absorb in the region 170 to 205 nm. 3. n → σ* transition Saturated compounds containing atoms with lone pair oaf electrons like O, N, S and halogens are capable of n → σ* transition.These transitions usually require less energy than σ → σ* transitions. The number of organic functional groups with n → σ* peaks in UV region is small (150 – 250 nm). 11

12. 4 . n → π* transition An electron from non-bonding orbital is promoted to anti-bonding π* orbital. Compounds containing double bond involving hetero atoms (C=O, C≡N, N=O) undergo such transitions. n → π* transitions require minimum energy and show absorption at longer wavelength around 300 nm. 12

13. CHROMOPHORES Absorption of light in UV-visible region gives rise to absorption spectra. This spectrum arises due to transitions among electronic energy levels ( Eo to E1) of certain groups present in the molecule. This group of atoms which is responsible for the absorption in UV-visible region is known as the chromophore. Mostly the unsaturated groups, heteroatoms or heteroatoms containing lone pair of electrons are the possible chromophores. These chromophores show absorption in certain region of spectrum (Say from 400 to 200 am), however at particular wavelength it shows maximum absorption (say at 254 nm) and that wavelength is called as “Wavelength of maximum absorption.” It is denoted by max. The nuclei determine the strength with which the electrons are bound and thus influence the energy spacing between ground and excited states. 13

14. Hence, the characteristic energy of a transition and the wavelength of radiation absorbed are properties of a group of atoms rather than of electrons themselves . The group of atoms producing such an absorption is called a chromophore. As structural changes occur in a chromophore, the exact energy and intensity of the absorption are expected to change accordingly . 14

15. AUXOCHROMES The attachment of substituent groups in place of hydrogen on a basic chromophore structure changes the position and intensity of an absorption band of the chromophore. The substituent groups may not give rise to the absorption of the ultraviolet radiation themselves, but their presence modifies the absorption of the principal chromophore . Substituents that increase the intensity of the absorption, and possibly the wavelength, are called auxochromes . These groups do not absorb the radiations in UV-visible region, however when they are attached to chromophore they shift the absorption bands cither towards higher wavelengths or lower wavelengths i.e. they bring out the changes in absorption spectum of chromophore when attached to it. These auxochromes generally have nonbonding valence electrons. Eg . –OH, - NH2, -Cl etc . 15

16. SPECTRAL SHIFTS There are four types of shifts observe in the UV – Visible Spectroscopy. Bathochromic shift ( Red shift ) Hypsochromic shift ( Blue shift ) Hyperchromic shift Hypochromic shift 16

17. BATHOCHROMIC SHIFT: (Red Shift-A shift to lower energy or longer wavelength) B athochromic shift is an effect by virtue of which the absorption maximum is shifted towards the longer wavelength due to the presence of an auxochrome or change in solvents. This shift can be caused by an increase in the conjugation of a molecule, a change in the solvent polarity, or interactions such as hydrogen bonding. Examples : Conjugation : In benzene, the absorption maximum occurs around 254 nm. When benzene is converted to aniline (with an amino group), the absorption maximum shifts to around 280 nm due to increased conjugation. Solvent Effect : For a compound like 4-nitrophenol, the absorption maximum shifts from 315 nm in water to 400 nm in ethanol due to the solvent polarity effect. 17

18. HYPSOCHROMIC SHIFT: ( Blue Shift-A shift to higher energy or shorter wavelength ) H ypsochromic shift is an effect by virtue of which absorption maximum is shifted towards the shorter wavelength. generally it is caused due to the removal of conjugation or by changing the polarity of the solvents. This shift can be caused by a decrease in conjugation, a change in the solvent polarity, or steric effects that disrupt the electronic environment . Examples : Conjugation : In the case of benzaldehyde, when it is converted to benzoic acid, the absorption maximum shifts from 248 nm to 230 nm due to the disruption of conjugation. Solvent Effect : For a compound like 4-nitroaniline, the absorption maximum shifts from 380 nm in ethanol to 350 nm in water due to the solvent polarity effect. 18

19. HYPERCHROMIC SHIFT Hyperchromic shift is an effect by virtue of which absorption maximum increases. the introduction of an auxochrome in the compound generally results in the hyperchromic effect. This shift can be caused by factors such as an increase in the number of absorbing species, a change in the molecular environment that enhances the transition probability, or structural modifications that increase the oscillator strength . Example: Aromatics in Solvents : The addition of a solvent that enhances the solubility of an aromatic compound can cause a hyperchromic shift due to better solvation and more efficient light absorption. For instance, dissolving an aromatic compound like naphthalene in ethanol instead of water can result in a hyperchromic effect 19

20. HYPOCHROMIC SHIFT A hypochromic shift is a decrease in the absorbance or intensity of the absorption band. Hypochromic effect is defined as the effect by virtue of intensity of absorption maximum decreases. Hypochromic effect occurs due to the distortion of the geometry of the molecule with an introduction of new group. This shift can be caused by factors such as a decrease in the number of absorbing species, changes in the molecular environment that reduce the transition probability, or structural modifications that decrease the oscillator strength . Example: Complex Formation : When a ligand binds to a metal ion to form a complex, it can result in a hypochromic shift. For example, the complexation of 2,2'-bipyridine with a metal ion like Fe(II) often results in a decrease in the intensity of the absorption band due to changes in the electronic environment of the ligand. 20

21. SOLVENT EFFECTS ON THE ABSORPTION SPECTRA The choice of the solvent to be used in ultraviolet spectroscopy is quite important, which can alter the electronic environment of the solute. The absorption spectrum of a pharmaceutical substance depends partially upon the solvent that has been employed to solubilize the substance. A drug may absorb a maximum of radiant energy at a particular wavelength in one solvent but shall absorb practically little at the same wavelength in another solvent. These apparent changes in spectrum are exclusively due to various characteristic features, namely: (a) Nature of the solvent, (b) Nature of the absorption band, and (c) Nature of the solute. 21

22. Some salient features of 'Solvent Effects' are enumerated below : Absorption bands of many substances are relatively sharper and may also exhibit fine structure when measured in solvents of low dipole moment Interactions of solvent-solute are found to be much stronger in such substances where strong dipole forces are involved , Solvent effects do help in reorganizing electronic transitions of the type n- π * that essentially involve the nonbonding electrons of nitrogen and oxygen , The nonbonding electrons of nitrogen and oxygen usually interact with polar solvents that ultimately give rise to a characteristic shift to shorter wavelengths. Example : The spectrum of lodine in a nonpolar solvent like CHCl3 , is found to be distinctly different (purple to the naked eye) when the same is compared in a polar solvent such as C2H5OH (brownish to the naked eye) A spectrum normally shows appreciable changes with varying pH when an ionizable moiety is present in the molecule and thereby constitutes part of the chromophore structure . 22

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24. 1 . Solvent Polarity : Bathochromic Shift (Red Shift) : In polar solvents, the absorption maximum can shift to longer wavelengths (lower energy). This occurs because polar solvents can stabilize the excited state more than the ground state of the solute, lowering the energy gap between the two states. Example : The absorption maximum of benzene shifts from around 204 nm in hexane (non-polar) to around 256 nm in ethanol (polar). 2. Solvent-Solute Interactions : Hydrogen Bonding : Solvents that can form hydrogen bonds with the solute can significantly impact the absorption spectrum. Hydrogen bonding can either stabilize or destabilize different electronic states, leading to shifts in the absorption band. Example : The absorption maximum of phenol shifts to longer wavelengths when it forms hydrogen bonds with solvents like water or ethanol. 24

25. 3. Solvent Effect on Molar Absorptivity (Intensity) : Hyperchromic Effect : Some solvents can enhance the intensity (molar absorptivity) of the absorption band by better solubilizing the solute or by increasing the transition probability. Example : Naphthalene in ethanol shows a higher intensity absorption band compared to in hexane. 4. Solvent Polarity and π-π* Transitions: Solvent polarity can have a pronounced effect on π-π* transitions, which involve the excitation of an electron from a bonding π orbital to an antibonding π* orbital. Polar solvents often cause a bathochromic shift for these transitions. Example : The absorption maximum for β-carotene shifts to a longer wavelength in more polar solvents . 5. Solvent Polarity and n-π Transitions*: For n-π* transitions, which involve the excitation of a non-bonding (n) electron to an antibonding π* orbital, polar solvents generally cause a hypsochromic shift. This is because polar solvents stabilize the non-bonding electrons more effectively than the antibonding π* orbitals. Example : The absorption maximum for acetone shifts to a shorter wavelength in more polar solvents. 25

26. BEER – LAMBERT’S LAW 26 The Beer-Lambert law states that: “For a given material sample path length (L) and concentration (C) of the sample are directly proportional to the absorbance of the light” The Beer-Lambert law is expressed as : A = ε Lc   where, A is Absorbance ε is molar extinction coefficient/ molar absorptivity L is the distance covered by the light through the solution c is the concentration of the absorbing species

27. Beer’s law was stated by August Beer which states that absorbance and concentration are directly proportional to each other . Johann Heinrich Lambert stated Lambert law. It states that absorbance and path length are directly proportional . 27 Monochromatic

28. 28 The Beer Lambert Equation is based on the hypothesis that the intensity (I) of the monochromatic light is decreased by dI ( i.e negative) as it passes through a thickness dx of a material and is directly proportional to the concentration (C) as well as the intensity (I). α C I = k C I = - k C dx If Iₒ represents the incident intensity of the radiation before it passes through a medium of thickness L that has an absorption coefficient k, the transmitted intensity I will be given by the integrated form of the above equation. = - k C dx = -k C [x] ln I – ln Iₒ = - k C (L-0) ln = - k C L ln = k C L 2.303 log = k C L  

29. log = Assuming as ε (Extinction coefficient/ Molar absorptivity) log = ε log = ε C L Here ε C L is Absorption/ Absorbance log = A Transmittance is the inverse of absorbed intensity log = A OR -log T= A   29

30. Deviations from Beer Lambert’s Law A system is said to obey Beer’s law, when a plot of Concentration Vs absorbance gives a straight line . The straight line is obtained by joining the maximum no of points in such a way that positive and negative errors are balanced or minimised . When a straight line is not obtained, that is a non-linear curve is obtained in a plot of concentration Vs absorbance, the system is said to undergo deviation from Beer’s Law. Such deviation can be positive deviation or negative deviation . Positive deviation results when a small change in concentration produces a greater change in absorbance. Negative deviation results when a large change in concentration produces smaller change in Absorbance . It is normally seen that several system obey Beer’s Law only in concentration range, above which may show deviation ( eg ) 10 - 50 g/ml, it may obey, but may exhibit deviation above 50 g/ml. 30

31. Several reasons for the observed deviations from Beer’s law are as follows: Instrumental deviations Factors like stray radiation, improper slit width, fluctuations in single beam and when monochromatic light is not used can influence the deviation. Physicochemical changes in solution Factors like Association, Dissociation, ionisation (Change in pH), faulty development of colour (incompletion of reaction), refractive index at high concentrations, can influence such deviations. 31

32. INSTRUMENTATION 32 A typical UV-Visible Spectrophotometer consists of the following components: Radiation source Wavelength selector Sample container Radiation transducer Signal processors Readout device Schematic diagram of instrumentation of UV Visible Spectrophotometer

33. 33 Source of Radiation: They must provide sufficient energy over the wavelength region (400 - 800pm) for easy detection & measurement . It must supply continuous radiation of adequate intensity, stable and free from fluctuation over the entire wavelength region in which it is used. The output power should be stable for reasonable period . The following are the example of lamps: Hydrogen Discharge lamp Deuterium lamp Tungsten lamp Xenon discharge lamp

34. Hydrogen and Deuterium Discharge lamp: A continuum spectrum in the UV region is produced by electrical excitation of hydrogen or deuterium . A continuum spectrum is obtained from 160-375nm . The energy of photon can vary continuously. Only quartz cuvettes are used, because glass absorb radiation of wavelength less than 350nm . Advantages: Radiation is stable . Intensity of radiation is emitted 3-5 times the intensity of hydrogen lamp . Disadvantages: Expensive 34

35. b . Tungsten Lamp Most common light source used in spectrophotometer. The lamp consist of a tungsten filament in a vacuum bulb it offers sufficient intensity . It has long life. Produces wavelength region 350-2500nm . In the visible region the energy output varies with power of operating voltage. The voltage control is essential for stable radiation. Tungsten halogen lamps contain small quantity of iodine within a quartz envelop that houses the filament . Quartz is required due to the high operating temperature. A dvantages : Have long half life,Stable , cheap, easy to use . Disadvantages: Intensity at lower wavelength region in very feeble . Need voltage control for stable radiation. 35

36. C. Xenon Discharge Lamp This lamp produces intense radiation by passage of current through an atmosphere xenon. The spectrum is continuous over the range between 200 - 600nm. Xenon gas is stored under pressure in the range of 10 - 30 atm. The excess xenon lamp possess two tungsten electrodes separated by about 8nm. When an intense arc is formed between two tungsten electrodes by applying a low voltage the UV light is produced . The intensity of UV radiation produced by xenon discharge lamp is greater than that of hydrogen lamp . Advantages: The intensity of UV radiation produced are greater than that of hydrogen lamp . Emit both UV and visible wave length. 36

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39. Collimating System : The radiation emitted by the source is collimated (made parallel) by lenses, mirrors and slits . 1. Lenses : Materials used for the lenses must be transparent to the radiation being used. Ordinary silicate glass transmits between 350 to 3000 nm. 2. Mirrors : These are used to reflect, focus or collimate light beams in spectrophotometer. To minimize the light loss, mirrors aluminized on their front surfaces . 3. Slits : Slit is an important device in resolving polychromatic radiation into monochromatic radiation . To achieve this, entrance slit and exit slit are used. The width of slit plays an important role in resolution of polychromatic radiation. 39

40. Wavelength Selector: Most of the spectroscopic analysis radiation that consist of a limited narrow continuous group of wave length is called as band . Ideally the output from a wavelength selector would be a radiation of single wavelength / frequency . A narrow band width represents better performance. Dispersive devices or wavelength selectors ae classified broadly into two categories: Filters Monochromators 1. Filters: Filters are the devices which allow selective transmission of light of specific wavelengths. Selection of filters is usually done on a compromise between peak transmittance and band pass width . Advantages : These are simpler in construction and cheaper in cost. Disadvantages : Accuracy is very low. Types of filters used in Spectroscopy 40

41. Types of filters used in Spectroscopy: Absorption filters Interference filters Absorption filters – Absorption filters are simple filters which Works by selective absorption of unwanted radiation and transmits the radiation which is required. Examples Glass and Gelatin filters . Interference filters- Works on the interference phenomenon, causes rejection of unwanted wavelength by selective reflection . It is constructed by using two parallel glass plates, which are silvered internally and seperated by thin film of dielectric material of different (CaF2, SiO , MgF2) refractive index. These filters have a band pass of 10- 15nm with peak transmittance of 40-60 %. 2. Monochromators: All monochromators contain the following component parts , An entrance slit , A collimating lens , A dispersing device (a prism or a grating ), A focusing lens, An exit slit , 41

42. Types of Monochromators 42

43. Prism Type: Prism is made from glass, Quartz or fused silica. Quartz or fused silica is the choice of material of UV spectrum. When white light is passed through glass prism, dispersion of polychromatic light in rainbow occurs. Now by rotation of the prism different wavelengths of the spectrum can be made to pass through in exit slit on the sample. The effective wavelength depends on the dispersive power of prism material and the optical angle of the prism. Dispersive Type: The source of light through entrance slit falls on a collimator . The parallel radiation from collimator are dispersed into different colours or wavelengths & by using another collimator, the images of entrance slit are reformed . The reformed ones will be either violet, indigo, blue, green, yellow, orange or red . The required radiation on exit slit are be selected by rotating the prism or by keeping the prism stationary & moving the exit slit. 43

44. 2. Littrow Type: The principle of working is similar to the refractive type except that, a reflective surface is present on one side of the prism . It has optical angle 30̊ and its one surface is aluminized with reflected light back to pass through prism and to emerge on the same side of the light source that is light doesn’t pass through the prism on other side. Hence the dispersed radiations gets reflected & can be collected on the source of light . 44

45. Grating Type: Are most effective one in converting a polychromatic light to monochromatic light. As a resolution of +/- 0.1 nm could be achieved by using gratings , they are commonly used in spectrophotometers . Diffraction type: More refined dispersion of light is obtained by means of diffraction ratings . These consist of large number of parallel lines (grooves) about 15000-30000/ inch is ruled on highly polished surface of aluminium . These gratings are replica made from master gratings by coating the original master grating with epoxy resin and are removed after setting . To make the surface reflective, a deposit of aluminium is made on the surface. In order to minimize to greater amounts of scattered radiation and appearance of unwanted radiation of other spectral orders, the gratings are blazed to concentrate the radiation into a single order. 45

46. 2. Transmission type: It is similar to diffraction grating but refraction takes place instead of reflection . Refraction produces reinforcement. This occurs when radiation transmitted through grating reinforces with the partially refracted radiation. 46 Transmission Type Diffraction Type

47. Sample cells/Holders: The cells or cuvettes are used for handling liquid samples. The cell may either be rectangular or cylindrical in nature. For study in UV region; the cells are prepared from quartz or fused silica. For study in Visible region ; Color corrected fused glass is used The surfaces of absorption cells must be kept scrupulously clean. No fingerprints or a touch should be present on cells. Cleaning is carried out washing with distilled water or with dialcohol , acetone . The thicknes s of the cell is generally 1cm, call maybe be rectangular in shape or cylindrical with flat ends. Two opposite faces of the cuvettes are slightly translucent and the other two faces are clear. 47

48. Detectors: Device which converts light energy into electrical signals, that are displayed on readout devices. The transmitted radiation falls on the detector which determines the intensity of radiation absorbed by sample. The following types of detectors are employed in instrumentation of absorption spectrophotometer: a)Photovoltaic cell b ) Phototubes/Photo emissive tube c ) Photomultiplier tube d) Silicon Photodiode 48

49. Photo Tubes/ Photo emissive Tubes: Photo tubes are also known as Photo emissive tubes. A photo tube consists of an evacuated glass tube. There is a light sensitive cathode inside it. The inner surface of the cathode is coated with a light sensitive layer such as potassium oxide or silver oxide. When radiation is incident upon the cathode, electrons are emitted. Electrons are emitted which are attracted to anode causing current to flow . And by this process current is amplified and recorded. 49

50. 2. Photomultiplier tube : The photomultiplier tube is a commonly used detector in UV spectroscopy. It consist of a cathode covered with a photo emissive material, which emits electrons when exposed to radiation . The tube contain additional electrodes called dynodes . Dynodes is maintained at a potential 90v more positive than the cathode and so the electrons emitted by the cathode are attracted towards it . Each photo electron striking the dynodes causes emission of several additional electrons . These electrons are then accelerated towards the second dynode, which is at 90v more than dynode 1. Again several electrons are emitted for each electron . Repetition of the process leads to formation of 10^6 to 10^7 electrons for each photon striking the cathode. Ideal for sensing weak light intensities. 50

51. 51 Cross sectional view of photomultiplier tube

52. 3. Photo Voltaic cell: The detector has a thin film metallic layer coated with silver or gold and acts as an electrode. It also has a metal base plate which acts as another electrode. These two layers are separated by a semiconductor layer of selenium . When light radiation falls on selenium layer, electrons become mobile and are taken up by transparent metal layer. This creates a potential difference between two electrodes and causes the flow of current. When it is connected to galvanometer, a flow of current observed which is proportional to the intensity and wavelength of light falling on it. 52

53. APPLICATIONS Chemical identification and quantification:  UV-Vis spectroscopy can confirm chemical identity and quantify the purity of drugs/drug ingredients . Quantification of impurities:  UV-Vis is commonly utilized in pharmaceutical monographs for quantifying impurities in drug ingredients and drug products . Dissolution testing:  UV-Vis spectroscopy has long been a method for analyzing the results of dissolution testing of solid oral dosage forms like tablets . Development of Active Pharmaceutical Ingredients (APIs):  From scans to stop-flow kinetics, UV-Vis is a common technique in drug discovery and development . Protein analysis and measurements:  Simple techniques enable quantification and qualification of biomaterials from protein to bacteria and nucleic acids . Structure elucidation of organic compounds: UV spectroscopy is useful in the structure elucidation of organic molecules, the presence or absence of unsaturation, the presence of hetero atoms. 53

54. 7. Quantitative Analysis: UV absorption spectroscopy can be used for the quantitative determination of compounds that absorb UV radiation. This determination is based on Beer’s law which is as follows . A = ε Lc 8. Qualitative Analysis: UV absorption spectroscopy can characterize those types of compounds which absorbs UV radiation. Identification is done by comparing the absorption spectrum with the spectra of known compounds . 9. Chemical Kinetics: Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is passed through the reaction cell and the absorbance changes can be observed. 10. Detection Of Functional Groups: This technique is used to detect the presence or absence of functional group in the compound. 11. As HPLC Detector: A UV/Vis spectrophotometer may be used as a detector for HPLC. 54

55. 12. Spectrophotometric titrations: Generally, in normal titrimetric methods, the equivalence point of a reaction is visually detected by the colour produced by an indicator (as in acid-base titrations etc.) or by the colour of the reactant (permanganate etc ). Such titrations give results within one per cent accuracy. In titrations, if the colour change is gradual or the colour change does not contrast sharply, accurate results are not obtained. All such problems can be overcome if photometric or spectrophotometric titrations are carried out . In spectrophotometric titrations, a spectrophotometer is used to determine the equivalence point. The method consists in placing the titration vessel directly in the path of the light of the instrument . The absorbance of the solution is then determined after adding titrant and a plot of absorbance as a function of the volume of the tritrant is made. 55

56. 13. Single component Analysis : Each molecule absorbs UV-Vis light at characteristic wavelengths. By measuring the absorbance of a solution at a specific wavelength, one can determine the concentration of the molecule of interest using Beer-Lambert law. Application: Used for quantifying the concentration of a specific molecule in a solution when no other molecules absorb light at the chosen wavelength . 14. Multi component Analysis : In mixtures with multiple absorbing components, each component contributes to the overall absorbance. Advanced techniques and mathematical treatments (like chemometrics ) can be used to deconvolve the spectrum and determine concentrations of individual components. Used for analyzing mixtures where multiple species are present and can absorb UV-Vis light simultaneously. 56

57. THANK YOU! 57