UV Visible Spectroscopy, electronic transitions .pptx

UV Visible Spectroscopy, electronic transitions, instrumentation and applications

1. Subject Name: Instrumental Methods of Analysis Unit Name: UV Visible Spectroscopy Topic Name( s): UV Visible Spectroscopy Lecture No : 01-07

2. What is Light? According to Maxwell: light is an electromagnetic field characterized by a frequency f , velocity v, and wavelength λ. Light obeys the relationship f = v / λ.

3. Electromagnetic Radiation Electromagnetic radiation consists of discrete packets of energy which are called as photons. A photon consists of an oscillating electric field (E) & an oscillating magnetic field (M) which are perpendicular to each other.

4. Electromagnetic Radiation Frequency ( ν ): It is defined as the number of times electrical field radiation oscillates in one second. The unit for frequency is Hertz (Hz). 1 Hz = 1 cycle per second Wavelength ( λ ): It is the distance between two nearest parts of the wave in the same phase i.e. distance between two nearest crest or troughs. The relationship between wavelength & frequency can be written as: c = ν λ As photon is subjected to energy, so E = h ν = h c / λ

5. The Electromagnetic Spectrum n = c / l E = h n

6. Electromagnetic Radiation Violet 400 - 420 nm Yellow 570 - 585 nm Indigo 420 - 440 nm Orange 585 - 620 nm Blue 440 - 490 nm Red 620 - 780 nm Green 490 - 570 nm

7. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 7 Principles of Spectroscopy

8. History of Spectroscopy Spectroscopy began with Isaac Newton's optics experiments (1666–1672). Newton applied the word "spectrum" to describe the rainbow of colors . During the early 1800s, Joseph von Fraunhofer made experimental advances with dispersive  spectrometers  that enabled spectroscopy to become a more precise and quantitative scientific technique. Since then, spectroscopy has played and continues to play a significant role in chemistry, physics and astronomy.

9. Principles of Spectroscopy The principle is based on the measurement of spectrum of a sample containing atoms/ molecules. Spectrum is a graph of intensity of absorbed or emitted radiation by sample verses frequency ( ν ) or wavelength ( λ ). Spectrometer is an instrument design to measure the spectrum of a compound.

10. Absorption Spectroscopy: An analytical technique which concerns with the measurement of absorption of electromagnetic radiation. e.g. UV (185 - 400 nm) / Visible (400 - 800 nm) Spectroscopy, IR Spectroscopy (0.76 - 15 μ m) Emission Spectroscopy: An analytical technique in which emission (of a particle or radiation) is dispersed according to some property of the emission & the amount of dispersion is measured. e.g. Mass Spectroscopy Principles of Spectroscopy

11. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 11 Interaction of EMR with Matter

12. Interaction of EMR with Matter 1.Electronic Energy Levels: At room temperature the molecules are in the lowest energy levels E . When the molecules absorb UV-visible light from EMR, one of the outermost bond / lone pair electron is promoted to higher energy state such as E 1 , E 2 , …E n , etc is called as electronic transition and the difference is as: ∆E = h ν = E n - E where (n = 1, 2, 3, … etc) ∆E = 35 to 71 kcal/mole

13. Interaction of EMR with Matter 2.Vibrational Energy Levels: These are less energy level than electronic energy levels. The spacing between energy levels are relatively small i.e. 0.01 to 10 kcal/mole. e.g. when IR radiation is absorbed, molecules are excited from one vibrational level to another or it vibrates with higher amplitude. 3. Rotational Energy Levels: These energy levels are quantized & discrete. The spacing between energy levels are even smaller than vibrational energy levels. ∆ E rotational < ∆ E vibrational < ∆ E electronic

14. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 14 Electronic Transitions

15. The possible electronic transitions are

16. σ electron from orbital is excited to corresponding anti-bonding orbital σ *. The energy required is large for this transition. e.g . Methane (CH 4 ) has C-H bond only and can undergo σ → σ * transition and shows absorbance maxima at 125 nm. σ → σ * transition 1

17. 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. π → π * transition 2

18. Saturated compounds containing atoms with lone pair of electrons like O, N, S and halogens are capable of n → σ * transition . These transitions usually requires less energy than σ → σ * transitions. The number of organic functional groups with n → σ * peaks in UV region is small (150 – 250 nm). n → σ * transition 3

19. 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. n → π * transition 4

20. These electronic transitions are forbidden transitions & are only theoretically possible. Thus , n → π * & π → π * electronic transitions show absorption in region above 200 nm which is accessible to UV-visible spectrophotometer. The UV spectrum is of only a few broad of absorption. σ → π * transition 5 π → σ * transition 6 &

21. The possible electronic transitions can graphically shown as:

22. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 22 Various terms used in UV / Visible Spectroscopy

23. Chromophore The part of a molecule responsible for imparting color, are called as chromospheres. or The functional groups containing multiple bonds capable of absorbing radiations above 200 nm due to n → π * & π → π * transitions.

24. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 24

25. Auxochrome The functional groups attached to a chromophore which modifies the ability of the chromophore to absorb light, altering the wavelength or intensity of absorption. or The functional group with non-bonding electrons that does not absorb radiation in near UV region but when attached to a chromophore alters the wavelength & intensity of absorption.

26. Auxochrome e.g. Benzene λ max = 255 nm Phenol λ max = 270 nm Aniline λ max = 280 nm

27. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 27 Absorption & Intensity Shifts

28. When absorption maxima ( λ max ) of a compound shifts to longer wavelength, it is known as bathochromic shift or red shift. The effect is due to presence of an auxochrome or by the change of solvent. e.g . An auxochrome group like –OH, -OCH 3 causes absorption of compound at longer wavelength. Bathochromic Shift ( Red Shift) 1

29. In alkaline medium, p-nitrophenol shows red shift. Because negatively charged oxygen delocalizes more effectively than the unshared pair of electron. p-nitrophenol λ max = 255 nm λ max = 265 nm Bathochromic Shift ( Red Shift) 1

30. When absorption maxima ( λ max ) of a compound shifts to shorter wavelength, it is known as hypsochromic shift or blue shift. The effect is due to presence of an group causes removal of conjugation or by the change of solvent. Hypsochromic Shift (Blue Shift) 2

31. Aniline shows blue shift in acidic medium, it loses conjugation. Aniline λ max = 280 nm λ max = 265 nm Hypsochromic Shift ( Blue Shift) 2

32. When absorption intensity ( ε ) of a compound is increased, it is known as hyperchromic shift. If auxochrome introduces to the compound, the intensity of absorption increases. Pyridine 2methylpyridine λ max = 257 nm λ max = 260 nm ε = 2750 ε = 3560 Hyperchromic Effect 3

33. When absorption intensity ( ε ) of a compound is decreased, it is known as hypochromic shift. Naphthalene 2-methyl naphthalene ε = 19000 ε = 10250 Hypochromic Effect 4

34. Wavelength ( λ ) Absorbance ( A ) Shifts and Effects Hyperchromic Shift Hypochromic Shift Red Shift Blue Shift λ max

35. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 36 Beer-Lambert’s Law

36. The Bouguer-Lambert Law Lambert’s law states that the rate of decrease of intensity of monochromatic light with the thickness of the medium is directly proportional to the intensity of incident light.

37. Beer’s Law Concentration According to this law, when a beam of monochromatic radiation is passed through a solution of absorbing species, the intensity of beam of monochromatic light decreases exponentially with increase in concentration of absorbing species.

38. The Beer- Bouguer -Lambert Law

39. Beer Lambert Law As the cell thickness increases, the intensity of “I” (transmitted intensity of light) decreases.

40. Lambert’s law: “The intensity of beam of parallel monochromatic radiation decreases exponentially as it passes through a medium of homogenous thickness” (absorbance is proportional to the thickness of the solution). log I / I T = K 1 b Where K 1 – proportionality constant b – Thickness A = log I / I T = log (1/T) = -log T = 2 – log (%T)   Beer’s law: “The intensity of beam of parallel monochromatic radiation decreases exponentially with the number of absorbing molecules” (absorbance is proportional to concentration). log I / I T = K 11 c K 11 – proportionality constant C – concentration Beer – Lambert law: A combination of two laws yields the Beer – lambert law A = log I /I T = abc

41. A = € bc € - units are mol -1 cm -1 When € < 100  weakly absorbing € > 10,000  intensely absorbing Specific absorbance: A (1%, 1cm) The absorbance of a specified con. in a cell of specified path length. A = A 1% 1cm x bc where c is in gm/100ml & b is in cm A (1%, 1cm) units are dl g -1 cm -1 € = A 1% 1cm x M.Wt . 10 Molar Absorptivity :

42. 43 Deviations from Beer Lambert Law Deviations can be: Positive Deviation Negative Deviation There are 3 types of deviations usually observed: Applicable to Dilute Solution only Chemical Deviations Instrumental Deviations Applicable to Dilute Solution only: The real limitation of the law is that the beer’s law is successful in describing the absorption behaviour of dilute solutions only.

43. 44 Deviations from Beer Lambert Law Chemical Deviations : Association of molecules : This can be explained by taking the examples of methylene blue at small concentration(10 ‾ ⁵ molar) and at concentration above 10 ‾ ⁵molar . Dissociation of molecules: This can be explained by the fact that dichromate ions posses their maximum absorbance at 450nm which is orange in colour . But upon dilution, it will be dissociated to chromate ions having maximum absorbance at 410nm which is yellow in colour. This law is not valid in case if the absorbing material is coagulated into a small number of large units .

44. This law shows deviation if the absorbing material at the required wavelength contains presence of impurities. This law is not applicable in case of suspension.

45. 46 Deviations from Beer Lambert Law C. Instrumental Deviations: Strict adherence of an absorbing system to this law is observed only when the radiation used is monochromatic. Stray radiation, slit width also causes deviation. Hence, the reasons for the deviation depends on environment such as temperature, pressure, solvent, refractive index of the sample Poly Chromatic Light Stray Light

46. Principles of UV – Visible Spectroscopy 11/5/2022 Unit Number: 1, Lecture Number: 1-8 47

47. Principle The UV radiation region extends from 10 nm to 400 nm and the visible radiation region extends from 400 nm to 800 nm. Near UV Region: 200 nm to 400 nm Far UV Region: below 200 nm Far UV spectroscopy is studied under vacuum condition. The common solvent used for preparing sample to be analyzed is either ethyl alcohol or hexane.

48. Instrumentation Components of UV-Visible Spectrophotometer Source Filters & Monochromator Sample compartment Detector Recorder

49. Five Basic Optical Instrument Components 1) Source – A stable source of radiant energy at the desired wavelength (or range). 2) Wavelength Selector – A device that isolates a restricted region of the EM spectrum used for measurement (monochromators, prisms & filters). 3) Sample Container – A transparent container used to hold the sample (cells, cuvettes, etc). 4) Detector/Photoelectric Transducer – Converts the radiant energy into a useable signal (usually electrical). 5) Signal Processor & Readout – Amplifies or attenuates the transduced signal and sends it to a readout device as a meter, digital readout, chart recorder, computer, etc.

50. Schematic of a conventional Single-Beam Spectrophotometer

51. Optical system of a Double-Beam Spectrophotometer

52. Optical system of a Split-Beam Spectrophotometer

53. Light Sources Various UV radiation sources are as follows a. Deuterium lamp b. Hydrogen lamp c. Tungsten lamp  d. Xenon discharge lamp e. Mercury arc lamp Various Visible radiation sources are as follows a. Tungsten lamp b. Mercury vapour lamp c. Carbonone lamp

54. For Visible region Tungsten filament lamp Use for region 350nm to 2000nm. These measure most effectively in the visible region from 320 - 1100 nm Instruments that only use Tungsten halogen lamps as the light source will only measure in the visible region .

55.   For ultra violet region Hydrogen discharge lamp Consist of two electrode contain in Hydrogen filled silica envelop. Gives continuous spectrum in region 185-380nm. above 380nm emission is not continuous

56.   Deuterium Lamps: Deuterium arc lamps measure in the UV region 190 - 370 nm As Deuterium lamps operate at high temperatures, normal glass housings cannot be used for the casing. Instead, a fused quartz, UV glass, or magnesium fluoride envelope is used.  When run continuously typical lamp life for a Deuterium lamp is approximately 1000 hours Deuterium lamps are always used with a Tungsten halogen lamp to allow measurements to be performed in both the UV and visible regions.

57. TUNGSTEN LAMP DUETERIUM LAMP

58. Filters & Monochromators The Monochromator/Filter will select a narrow portion of the spectrum (the band pass) of a given source FILTERS ARE OF TWO TYPES: Absorption Filters Interference Filters MONOCHROMATORS ARE OF TWO TYPES: Refractive type PRISM TYPE Reflective type Diffraction type GRATING TYPE Transmission Type

59. Filters Absorption Filters Absorption filters, commonly manufactured from dyed glass or pigmented gelatin resins Band widths are extremely large {30 – 250 nm} Combining two absorbance filters of different λ max

60. The most common type of gelatin filter is constructed by sandwiching a thin layer of dyed gelatin of the desired colour between two thin glass plates.

61. INTERFERENCE FILTERS These are used to select wavelengths more accurately by providing a narrow band pass typically of around 10nm These filters rely on optical interference (destructive wave addition) to provide narrow bands of radiation.

62. Interference filter consists of a dielectric spacer film made up of CaF 2, MgF 2 between two parallel reflecting films . INTERFERENCE FILTERS

63. As light passes from one medium to the other the direction and wavelength of light can be changed based on the index of refraction of both mediums involved and the angle of the incident and exiting light. INTERFERENCE FILTERS

64. Due to this behaviour, constructive and destructive interference can be controlled by varying the thickness (d) of a transparent dielectric material between two semi-reflective sheets and the angle the light is shined upon the surface. As light hits the first semi-reflective sheet, a portion is reflected, while the rest travels through the dielectric to be bent and reflected by the second semi-reflective sheet . INTERFERENCE FILTERS

65. If the conditions are correct, the reflected light and the initial incident light will be in phase and constructive interference occurs for only a particular wavelength. INTERFERENCE FILTERS

66. MONOCHROMATORS PRISM MONOCHROMATOR:

67. Refractive type prism Monochromator : It consists of entrance slit, collimator lens, Prism, Focusing lens, Exit slit.

68. Reflective type prism Monochromator or Littrow type mounting :

69. GRATING MONOCHROMATOR Gratings are rulings made on glass, Quartz or alkyl halides Depending upon the instrument no. of rulings per mm defers If it is UV-Visible no. of gratings per mm are more than 3600.

70. DIFRACTION TYPE GRATING The mechanism is that diffraction produces reinforcement. The rays which are incident on the grating gets reinforced with the reflected rays and hence resulting radiation has wavelength governed by equation m λ = b(sin I + sin r) m – order λ – desired wavelength b – grating spacing i – angle of incidence r - angle of diffraction

71. TRANSMISSION TYPE GRATING: It is similar to diffraction grating, but refraction produces instead of reflection Refraction produces reinforcement The wavelength of radiation produced by transmission grating can be expressed by d sin Φ λ = d = 1/lines per cm m

72. SAMPLE COMPARTMENT Spectroscopy requires all materials in the beam path other than the analyte should be as transparent to the radiation as possible. The geometries of all components in the system should be such as to maximize the signal and minimize the scattered light. The material from which a sample cuvette is fabricated controls the optical window that can be used. Some typical materials are: Optical Glass - 335 - 2500 nm Special Optical Glass – 320 - 2500 nm Quartz (Infrared) – 220 - 3800 nm Quartz (Far-UV) – 170 - 2700 nm

73. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 75

74. (a) Open-topped rectangular standard cell (b) Apertured cell for limited sample volume Cell Type I Cell Type II Micro cell for very small volumes flow-through cell for automated applications

75. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 77

76. DETECTORS After the light has passed through the sample, we want to be able to detect and measure the resulting light. These types of detectors come in the form of transducers that are able to take energy from light and convert it into an electrical signal that can be recorded, and if necessary, amplified. Three common types of detectors are used Barrier Layer Cells Photo Emissive Cell Detector Photomultiplier Tubes Silicon Photodiode Array

77. It constitutes   The steel support plate 'A' layer of metallic selenium 'B', which is a few hundredths of a millimetre in thickness. 'C' is a thin transparent electrically-conductive layer, applied by cathodic sputtering. It consists of a metallic base plate like iron or aluminium which acts as one electrode. Barrier Layer Cell or Photo Voltic Cell

78. On its surface, a thin layer of a semiconductor metal like selenium is deposited. Then the surface of selenium is covered by a very thin layer of silver or gold which acts as a second collector tube. When the radiation is incident upon the surface of selenium, electrons are generated at the selenium- silver surface and the electrons are collected by the silver. This accumulation at the silver surface creates an electric voltage difference between the silver surface and the basis of the cell.

79. Photo Emissive Cells Detector Phototubes  are also known as photo emissive cells. A phototube consists of an evacuated glass bulb. There is light sensitive cathode inside it. The inner surface of cathode is coated with light sensitive layer such as potassium oxide and silver oxide. When radiation is incident upon a cathode, photoelectrons are emitted. These are collected by an anode. Then these are returned via external circuit. And by this process current is amplified and recorded .

80. The Photomultiplier Tube The Photomultiplier T ube  is a commonly used detector in UV Spectroscopy. It consists of a photo emissive cathode (a cathode which emits electrons when struck by photons of radiation), several dynodes (which emit several electrons for each electron striking them) and an anode.

81. The Photomultiplier Tube A photon of radiation entering the tube strikes the cathode, causing the emission of several electrons. These electrons are accelerated towards the first dynode (which is 90V more positive than the cathode). The electrons strike the first dynode, causing the emission of several electrons for each incident electron .

82. These electrons are then accelerated towards the second dynode, to produce more electrons which are accelerated towards dynode three and so on. Eventually, the electrons are collected at the anode. By this time, each original photon has produced 106 - 107 electrons. The Photomultiplier Tube

83. The resulting current is amplified and measured. Photomultipliers are very sensitive to UV and visible radiation. They have fast response times. Intense light damages photomultipliers; they are limited to measuring low power radiation. The Photomultiplier Tube

84. 86 Virtually all UV spectra are recorded solution-phase Cells can be made of plastic, glass or quartz Only quartz is transparent in the full 200-700 nm range; plastic and glass are only suitable for visible spectra Concentration is empirically determined Solvents must be transparent in the region to be observed; the wavelength where a solvent is no longer transparent is referred to as the cutoff Since spectra are only obtained up to 200 nm, solvents typically only need to lack conjugated p systems or carbonyls Sample Handling

85. 87 Additionally solvents must preserve the fine structure (where it is actually observed in UV!) where possible. H-bonding further complicates the effect of vibrational and rotational energy levels on electronic transitions Dipole-dipole interacts less so the more non-polar the solvent, the better (this is not always possible)

86. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 88 Woodward– Fieser Rules are named after Robert Burns Woodward and Louis Fieser ) are several sets of empirically derived rules which attempt to predict the wavelength of the absorption maximum ( λmax ) in an ultraviolet–visible spectrum of a given compound. Inputs used in the calculation are the type of chromophores present, the auxochromes (substituents on the chromophores, and solvent. Examples are conjugated carbonyl compounds, conjugated dienes, and polyenes. Woodward– Fieser Rules

87. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 89 a) Homoannular Diene:- Cyclic diene having conjugated double bonds in same ring.         b) Heteroannular Diene:- Cyclic diene having conjugated double bonds in different rings.             c) Endocyclic double bond:- Double bond present in a ring.            d) Exocyclic double bond: - Double bond in which one of the doubly bonded atoms is a part of a ring system. Here Ring A has one exocyclic and endocyclic double bond. Ring B has only one endocyclic double bond. Woodward– Fieser Rules

88. APPLICATIONS OF UV VISIBLE SPECTROPHOTOMETRY

89. Practical application of UV spectroscopy UV was the first organic spectral method, however, it is rarely used as a primary method for structure determination It is most useful in combination with NMR and IR data to elucidate unique electronic features that may be ambiguous in those methods It can be used to assay by the proper irradiation wavelengths for photochemical experiments, or the design of UV resistant paints and coatings The most ubiquitous use of UV is as a detection device for HPLC; since UV is utilized for solution phase samples vs. a reference solvent this is easily incorporated into LC design It is used for characterizing aromatic compounds and conjugated olefins. It can be used to find out molar concentration of the solute under study. UV is to HPLC what mass spectrometry (MS) will be to GC

90. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 92 The process of determining the quantity of a sample by adding measured increments of a titrant until the end-point, at which essentially all of the sample has reacted, is reached. The titration is followed by measuring the absorbance of radiation in the range ultraviolet to near-infrared by the sample. Examples of spectrophotometric titration curves: Spectrophotometric Titration

91. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 93 only the titrate absorbs; only the titrant absorbs; only the product of the titration reaction absorbs; both the titrate and the titrant absorb; both the titration reaction’s product and the titrant absorb; only the indicator absorbs. The red arrows indicate the end points for each titration curve. Spectrophotometric Titration

92. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 94 STEPS FOR ASSAY Step1: Select the Solvent Step2: Prepare the series of known dilutions. Step3: Set λ max in spectrophotometer. Step4: Measure absorbance. Step5: Plot calibration curve. Methods of calculating concentration in single component analysis: By using the relationship: A = E b c Where, A= Absorbance E= Molar Extinction Coefficient b= Path length of the sample (Cuvette) C= Concentration of the compound in solution Single Component Analysis

93. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 95 By using the formula: Cu = [Au/As]× Cs×d ; Where, Cu= Concentration of unknown, Cs= Concentration of standard, Au= Absorbance of Unknown As= Absorbance of standard d= Dilution factor By using the equations through Beer’s curve: Y = mX + C Where, M= gradient of the line C= y-intercept X and Y are Axis of Graph Single Component Analysis

94. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 96 Combination of drug products occupy a important role in therapeutics. When rationally formulated, fixed-combination drugs may produce greater convenience, lower cost, and sometimes greater efficacy and safety. Multi Component Analysis

95. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 97 Analysis of samples with numerous components presents a major challenge in modern analysis. Different analytical techniques can be applied for multicomponent analysis including spectrophotometry, chromatography and electrophoresis. Multi Component Analysis

96. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 98 Simultaneous Equation Method ( Vierotd’s Method) If a sample contains two absorbing drugs (X and Y) each of which absorbs at the λ-max of the other (λ1 and λ2), it may be possible to determine both the drugs by the simultaneous equations method. The λ max of two component should be reasonably dissimilar. The two component should not interact chemically. Criteria for obtaining maximum precision is between the range of 0.1 – 2.0 .

97. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 99 Simultaneous Equation Method ( Vierotd’s Method) ( A2/A1) / (aX2/aX1) and (aY2/aY1) / (A2/A1) The information required is The absorptivities of X at λ1 and λ2, aX1 and aX2 The absorptivities of Y at λ1 and λ2, aY1 and aY2 The absorbances of the diluted sample at λ1 and λ2, A1 and A2

98. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 100 Absorbance Ratio Method (Q-Absorbance method) The absorbance ratio method is a modification of the simultaneous equations method. It depends on the property that, for a substance, which obeys Beer’s law at all wavelength. The ratio of absorbances at any two wavelengths is a constant value independent of concentration or path length .

99. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 101 In the quantitative assay of two components in a mixture by the absorbance ratio method, absorbance are measured at two wavelengths: one being the λ-max one of the components (λ2) and other being a wavelength of equal absorptivity of two components ( λ1) i.e. an iso-absorptive point.

100. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 102 It can be determine by the following equation Cx = ( Qm-Qy ). A1 / ( Qx-Qy ). ax1 Cy = ( Qm-Qx ). A1 / ( Qy-Qx ). ay1 Where: Qm = A2/ A1 Qx = ax2/ ax1 Qy = ay2/ ay1 A2 =Absorbance at λ2 ; A1 =Absorbance at λ1 ax1= Absorptivity of Drug X at λ1 ay1= Absorptivity of Drug Y at λ1 ax2= Absorptivity of Drug X at λ2 ay2= Absorptivity of Drug Y at λ2 Absorbance Ratio Method (Q-Absorbance method)

101. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 103 Derivative Spectrophotometric Method Derivative spectroscopy involves the conversion of a normal spectrum to it’s first, second or higher derivative spectrum. The normal spectrum is known as fundamental, zero order or D spectra. The first derivative spectrum (D 1 ) is a plot of the rate of change of absorbance with wavelength against wavelength. i.e plot of ΔA/ Δλ vs. λ The second derivative spectrum is a plot of Δ 2 A/ Δλ 2 vs. λ Zeroth (a) First(b), Second (c) derivative spectra

102. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 104 For the quantitative estimation of binary mixtures by the derivative spectroscopy First of all we have to find out the Zero Crossing Points (ZCP) for both the components (A and B). Now select ZCP for A and B so that at that particular ZCP other component shows remarkable absorbance. Now prepare calibration curve of A at the ZCP of B and of B at the ZCP of A Derivative Spectrophotometric Method

103. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 105 Multiwavelength UV-Spectrophotometry Its determine the composition of a binary mixture with overlapping spectra without determining molar absorptivities . This method is very simple it requires only three measurements, the absorbance of a standard solution for each component and the unknown mixture itself. The standard solutions of drugs in the ratio of 1:1 μg /mL were prepared in specific solvent .

104. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 106 Multiwavelength UV-Spectrophotometry All the standard solutions were scanned over the range of 200- 400nm, in the multicomponent mode, using two sampling wavelength. The overlay spectra of mix standard solution drawn. The data from these scans were used to determine the concentrations of two drugs in tablet sample solution.

105. 11/5/2022 Unit Number: 1, Lecture Number: 1-8 107 Dual Wavelength Method In dual wavelength method, two wavelengths were selected for each drug in a way so that the difference in absorbance is zero for another drug. Dual wavelength spectroscopy offers an efficient method for analyzing a component in presence of an interfering component. For elimination of interferences, dual analytical wavelengths were selected in a way to make the absorbance difference zero for one drug in order to analyse the other drug.

106. References Elementary Organic Spectroscopy by Y. R. Sharma Chatwal , G.R., Anand , S.K. Instrumental Methods Of Chemical Analysis, 5th Ed., Himalaya Publishing House Pvt. Ltd., Mumbai. Skoog , D.A., Holler, F.J., Nieman . Principles of Instrumental Analysis, 5th Ed. Brooks/Cole, A division of Thomsan Learning, Inc., New York, 2006. http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/UV-Vis/spectrum.htm http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab1.htm