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  1. 1. Pharmaceutical Analysis-II Part I SPECtrOSCOPIC MEtHODS Uv-Visible spectrophotometer 1
  2. 2. Introduction to Spectroscopic Methods • This chapter presents a brief review of electromagnetic radiation and discusses how molecules and elements absorb and emit electromagnetic radiation. • Absorption and emission of electromagnetic radiation are the basis for identification and quantitative determinations in spectroscopic methods such as – UV spectrophotometry, – IR spectrophotometry, – NIR spectrophotometry, – atomic absorption spectrometry and – atomic emission spectrometry. • These methods are presented in subsequent chapters. Uv-Visible spectrophotometer 2
  3. 3. INTRODUCTION… INTERACTION B/N RADIATION AND MATTER • To understand how light interacts with matter, let’s see the following example • If beam of white light is passed through a beaker of water, it remains white. • If KMnO4 is added to the water---purple • KMnO4 allows blue and red color to pass and absorbs the other colors. • This is an example of the interaction b/n radiant energy and matter. • In this case, the radiant energy is visible and we can see the effect of absorption with our eyes. Uv-Visible spectrophotometer 3
  4. 4. INTRODUCTION… • However, absorption of radiation can take place over a wide range of radiant energy, most of which can not be seen. • Such absorption effects can be measured using suitable instruments E.g. UV-Visible and IR instruments Uv-Visible spectrophotometer 4
  5. 5. INTRODUCTION… vDefinition – Spectroscopy is the study of interaction between electromagnetic radiation (EMR) and matter. – Because these (uv-vis and IR) techniques use optical materials to disperse and focus the radiation, they often are identified as optical spectroscopies. – Despite the difference in instrumentation, all spectroscopic techniques share several common features. – Before we consider individual examples in greater detail, let’s take a moment to consider some of these similarities. Uv-Visible spectrophotometer 5
  6. 6. INTRODUCTION… vAll techniques use electromagnetic radiation as light source What is Electromagnetic Radiation (EMR)? • Electromagnetic radiation is a form of energy • It has properties of both waves and particles. – Its refraction when it passes from one medium to another shows its wave property Whereas – Absorption and emission properties are associated with its particulate property Uv-Visible spectrophotometer 6
  7. 7. INTRODUCTION… Now let’s discuss these important properties of EMR one by one a. Wave properties of EMR  EMR consists of oscillating electric and magnetic fields that propagate through space along a linear path and with a constant velocity Figure: Plane-polarized electromagnetic radiation showing the oscillating electric field in red and the oscillating magnetic field in blue. Uv-Visible spectrophotometer 7
  8. 8. INTRODUCTION… vIn a vacuum, electromagnetic radiation travels at the speed of light, c, which is 3x108m/s vEMR moves through a medium other than a vacuum with a velocity, v, less than that of the speed of light in a vacuum. vThis is a proof that EMR has wave property. Uv-Visible spectrophotometer 8
  9. 9. INTRODUCTION… • The interaction of electromagnetic radiation with matter can be explained using either the electric field or the magnetic field. Uv-Visible spectrophotometer 9
  10. 10. INTRODUCTION… v An electromagnetic wave, therefore, is characterized by several fundamental properties. The most important ones are discussed as follows A. Wavelength (l, lambda) v Is defined as the distance between successive maxima, or successive minima. v Different units of length are used to express wavelengths § E.g. Angstrom, centimeter, micro and nanometer § 1 m = 102 cm = 103 mm = 106 m = 109 nm = 1010 o. § 10 = 10-1 nm = 10-4  = 10-7 mm = 10-8 cm = 10‑ 10 m. v For UV-visible EMR the wavelength is usually expressed in nanometers , for IR radiation is given in microns (µm, 10–6 m). Uv-Visible spectrophotometer 10
  11. 11. INTRODUCTION… B. Amplitude (A) v Is the vertical distance from midline of a wave to the peak or trough. v It is Measured by units of distance C. Frequency (v,nu) Frequency is the number of waves that pass through a particular point in one second (Hz = 1 cycle/s) v Relationship b/n wavelength and frequency D. Ø This expresses the Number of waves per Centimeter (has a unit cm-1) Uv-Visible spectrophotometer 11
  12. 12. INTRODUCTION… Exercise • The wavelength of the sodium D line is 589 nm. What are the frequency and the wave number for this line? Uv-Visible spectrophotometer 12
  13. 13. INTRODUCTION… b. Particle properties of EMR vWhen matter absorbs electromagnetic radiation it undergoes a change in energy. vEMR consists of a beam of energetic particles called photons. vWhen a photon is absorbed by a sample it is “destroyed,” and its energy is acquired by the sample. Uv-Visible spectrophotometer 13
  14. 14. INTRODUCTION… • The energy of a photon, in joules, is related to its frequency, wavelength, or wavenumber by the following equations. • Where h is Planck’s constant, with a value of 6.626 X 10–34 J · s. • Example-- What is the energy per photon of the sodium D line ( λ= 589 nm)? Uv-Visible spectrophotometer 14
  15. 15. INTRODUCTION… 2. The Electromagnetic Spectrum vThe frequency and wavelength of EMR vary over many orders of magnitude. vFor convenience, EMR is divided into different regions based on the type of atomic or molecular transition that gives rise to the absorption or emission of photons (see the Figure below). – T h e s e re g i o n s co l l e c t i ve l y fo r m T h e Electromagnetic Spectrum Uv-Visible spectrophotometer 15
  16. 16.  The boundaries describing the electromagnetic spectrum are not rigid, and an overlap between spectral regions is possible. Figure :The electromagnetic spectrum showing the boundaries between different regions and the type of atomic or molecular transition responsible for the change in energy. Uv-Visible spectrophotometer 16
  17. 17. INTRODUCTION… 3. How does EMR interact with Matter? vMatter is in a continuous motion vMotion could be • rotational, • vibrational • electronic or • translational motion or combination of these. vEach motion is associated with different level of energy. Uv-Visible spectrophotometer 17
  18. 18. INTRODUCTION… 3. How does EMR interact with Matter?... vEach motion can be made to occur at a faster rate (at higher energy level) by applying an external energy. vThis can be achieved by applying one of the regions of the EMR , since each consists energetic particles called photons. vAfter absorbing energy, each type of motion are promoted from the lower energy level (Ground state) to higher energy level (Excited Level). Uv-Visible spectrophotometer 18
  19. 19. 3. How does EMR interact with Matter?... v The source of the energetic state depends on the photon’s energy. Example v Absorption of UV/Visible affects all types of motions, the major effect being transition of valence electrons v Absorption of IR radiation results in vibrational and rotational energy transitions Uv-Visible spectrophotometer 19
  20. 20. UV-Visible spectrophotometry Principles • Radiation in the wavelength range 200–800 nm is passed through a solution of a compound. • The electrons in the bonds within the molecule become excited so that they occupy a higher quantum state and in the process absorb some of the energy passing through the solution. • The more loosely held the electrons are within the bonds of the molecule, the longer the wavelength (lower the energy) of the radiation absorbed. Uv-Visible spectrophotometer 20
  21. 21. Applications in pharmaceutical analysis • A robust, workhorse method for the quantification of drugs in formulations where there is no interference from excipients. • Determination of the pKa values of some drugs. • Determination of partition coefficients and solubilities of drugs. • Used to determine the release of drugs from formulations with time, e.g. in dissolution testing. • Can be used to monitor the reaction kinetics of drug degradation. • The UV spectrum of a drug is often used as one of a number of pharmacopoeial identity checks. Uv-Visible spectrophotometer 21
  22. 22. Strengths • An easy-to-use, cheap and robust method offering good precision for making quantitative measurements of drugs in formulations. • Routine method for determining some of the physico- chemical properties of drugs, which need to be known for the purposes of formulation. • Some of the problems of the basic method can be solved by the use of derivative spectra. Limitations • Only moderately selective. The selectivity of the method depends on the chromophore of the individual drugs, e.g a coloured drug with an extended chromophore is more distinctive than a drug with a simple benzene ring chromophore. • Not readily applicable to the analysis of mixtures Uv-Visible spectrophotometer 22
  23. 23. Introduction § A spectroscopic technique which utilizes the UV/Visible region of the EMR is known as UV/visible spectroscopy /spectrophtometery/. § Near UV region 200 nm-400 nm § Visible region 400-800 nm § Absorption of light in these region mainly causes electronic transition. § The outer electrons in an organic molecule may occupy one of three different energy levels (- , - or n- energy level). Uv-Visible spectrophotometer 23
  24. 24. Accordingly there are three types of electrons. a) σ-electrons; • They are bonding electrons • They represent valence bonds and possess the lowest energy level ( the most stable) b) π-electrons; • They are bonding electrons, forming the pi-bonds (double bounds), and • possess higher energy than sigma-electrons. Uv-Visible spectrophotometer 24
  25. 25. c) n-electrons; • They are nonbonding electrons, • Present in atomic orbitals of hetero atoms (N, O, S or halogens). • They usually occupy the highest energy level of the ground state. Uv-Visible spectrophotometer 25
  26. 26. Electronic transitions of organic compounds § Electrons reside in orbitals. A molecule also posseses normally unoccupied orbitals called antibonding orbitals; these corresponds to excited state energy levels and are either * or *. Uv-Visible spectrophotometer 26
  27. 27. • In excited state the  -electrons occupy an anti- bonding energy level ( *) and the transition is termed  -  * transition. •  -electrons occupy an anti-bonding energy level ( *) and the transition is termed  -  * transition, • While the n-electrons may occupy  * or  * levels to give n-  * or n-  * transition. Uv-Visible spectrophotometer 27
  28. 28. Organic compounds containing -Electrons: vCompounds contain -electrons only are the saturated hydrocarbons, which absorb below 170 nm. vThey are transparent in the near UV region (200 - 400 nm) – And this make them ideal solvents for other compounds studied in this range. vThey are characterized by --* transition only. Uv-Visible spectrophotometer 28
  29. 29. Organic compounds containing n-Electrons : vSaturated organic compounds containing hetero atoms, possess n-electrons in addition to sigma- electrons. vThey are characterized by the  -* and n – * transitions. vn-electrons can also be transited to * when they exist in unsaturated compounds Uv-Visible spectrophotometer 29
  30. 30. Organic compounds containing -Electrons : vUnsaturated compounds containing no hetero atoms are characterized by the -* and -* transitions, such as ethylene (CH2=CH2). vWhen these compounds contain hetero atoms, they can undergo -*, -*, n-* and n-* transitions example: acetone (CH3-COCH3). Uv-Visible spectrophotometer 30
  31. 31. • Increasing order in absorption wavelength -* <n-* < -*< n-* Table: Electronic transitions involving n,  and  molecular orbitals Uv-Visible spectrophotometer 31
  32. 32. Of these transitions, the most important are the n-π* and π - π *, because they involve functional groups that are characteristic of the analyte and wavelengths that are easily accessible. The bonds and functional groups that give rise to the absorption of ultraviolet and visible radiation are called chromophores. Uv-Visible spectrophotometer 32
  33. 33. Factors governing absorption of radiation in the UV-Vis region vFactors leading to spectral changes are the following vConjugation vAttachment of auxochromes and chromophres vSolvent polarity vPH of the medium Uv-Visible spectrophotometer 33
  34. 34. vπ to π * transitions, when occurring in isolated groups in a molecule, give rise to absorptions of fairly low intensity. vHowever, conjugation of unsaturated groups in a molecule produces a remarkable effect upon the absorption spectrum. • The wavelength of maximum absorption moves to a longer wavelength and the absorption intensity may often increase. Uv-Visible spectrophotometer 34
  35. 35. Uv-Visible spectrophotometer 35
  36. 36. vThe same effect occurs when groups containing n electrons are conjugated with a π electron group; e.g., vThus, the characteristic energy of a transition and hence the wavelength of absorption is a property of a group of atoms rather than the electrons themselves. Uv-Visible spectrophotometer 36
  37. 37. • When such absorption occurs, two types of groups can influence the resulting absorption spectrum of the molecule: • chromophores and • auxochromes Chromophores v are functional groups, not conjugated with another group, which exhibit a characteristic absorption spectrum in the ultraviolet or visible region. Uv-Visible spectrophotometer 37
  38. 38. vSome of the more important chromophoric groups are: vIf any of the simple chromophores is conjugated with another (of the same type or different type) a multiple chromophore is formed having a new absorption band. Uv-Visible spectrophotometer 38
  39. 39. Auxochromes vThey Intensify the absorption of a molecule vAuxochromes do not absorb significantly in the 200- 800nm region, – but will affect the spectrum of the chromophore when attached to it. vThese include OH, NH2, CH3 , alkoxy and Halogens Uv-Visible spectrophotometer 39
  40. 40. Auxochromes cause two types of shifts • Bathochromic (Red) shift: shift of absorption to longer wavelength due to substitution and solvent effects.  Hypsochromic (Blue) shift: it is shift of absorption to shorter wavelength.  Hyperchromic & hypochromic effects: it is the increase and decrease in absorption intensity respectively. Uv-Visible spectrophotometer 40
  41. 41. Absorption characteristics of chromophores 1- Ethylenic chromophores: vTheir bands are difficult to observe in near UV region, so they are not Useful analytically. v However, substitution and certain structural features may cause red shift rendering the band observable in the near UV region. Examples: Alkyl substitution: cause red shift due to hyper-conjugation and stabilization of excited state. Attachement to auxochromes: cause red shift and increased absorption intensity due to extension of conjugation. Uv-Visible spectrophotometer 41
  42. 42. 2- Carbon-hetero atom chromophores: Such as -C=O, -C=N, -C=S, -N=O, ….etc. vThey exhibit some common characteristics; n-pi* band in the range of 275-300 nm., which is the most apparent band, has low intensity and long wavelength. v This band undergoes a blue shift on increasing the solvent polarity – due to increasing the energy of transition as a result of hydrogen bonding vAlkyl substitution; Cause red shift due to hyper-conjugation. Uv-Visible spectrophotometer 42
  43. 43. A] 1,3-Butadienes and conjugated enones Uv-Visible spectrophotometer 43 Separated chromophores (by two or more single bonds) CH2 = CH – CH2 – CH = CH2 have additive effect only because there is little or no electronic interaction between separated chromophores. CH2 = CH2 CH2 = CH – CH2 – CH = CH2 170-180 nm
  44. 44. • If the two chromophoric groups are present in a molecule and they are separated by only one single bond (a conjugated system), a large effect on the spectrum results, more than found by mere addition. CH2 = CH – CH = CH2 or CH2 = CH – CH = O Uv-Visible spectrophotometer 44 CH2 = CH – CH = CH2 170-180 205-215 nm
  45. 45. [B] Aromatic Systems I) Benzene ring : vBenzene has three maxima at 184 nm ( the most intense), 204 nm and at 254 nm. vThe first two bands have their origin in the ethylenic -* transition, while the longest B-band is a specific feature of benzenoid compounds. vThis band abbreviated B-band, is characterized by vibrational fine structures. vIn structure elucidation both the B-band and the 204-nm ethylenic band, termed E-band are useful while the far UV band (184 nm) is unsuitable for analytical purposes. Uv-Visible spectrophotometer 45
  46. 46. [B] Aromatic Systems… I) Benzene ring : Uv-Visible spectrophotometer 46 184 nm 204 nm 254 nm
  47. 47. [B] Aromatic Systems… II) Monosubstituted benzenes : vWhen the benzene ring is substituted with a single functional group a Red shift occurs for both the E- and B-bands with increase in the absorption intensity. vThis occurs whether the substituent is an electron donating or electron withdrawing group. vIn addition the B band loses most of its fine structure. Uv-Visible spectrophotometer 47
  48. 48. Effect of pH on absorption spectra vThe spectra of compounds containing acidic or basic groups are dependent on the pH of the medium e.g. phenols and amines. vUV-spectrum of phenol in acid medium (where the molecular form predominates) is completely different from its spectrum in alkaline medium (where the phenolate anion predominates). vSpectrum in alkaline medium exhibits bathochromic shift with hyperchromic effect. Uv-Visible spectrophotometer 48
  49. 49. Effect of pH on absorption spectra… vThe red shift is due to the participation of the pair of electrons in resonance with the  electrons of the benzene ring, vthus increasing the delocalization of the  electrons. Uv-Visible spectrophotometer 49 - + H in acid medium in alkaline medium O O OH OH (Phenol)lmax = 270 nm (phenate anion) lmax= 290 nm
  50. 50. v On the other hand, UV spectrum of aniline in acid medium show hypsochromic (blue) shift with hypochromic effect vThis blue shift is due to the protonation of the amino group, hence the pair of electrons is no longer available vand the spectrum in this case is similar to that of benzene (thus called benzenoid spectrum). Uv-Visible spectrophotometer 50 NH2 NH3 In alkaline medium in acid medium Aniline, lmax= 280 nm Anilinium ion lmax= 254 nm + + H+ - H+
  51. 51. Effect of Solvent on absorption spectra vThe solvent in which the absorbing species is dissolved also has an effect on the spectrum of the species. v Peaks resulting from n → π* transitions are shifted to shorter wavelengths (blue shift) with increasing solvent polarity. vThe ground state is more polar than the excited state vHydrogen bonding solvents with unshared electron pairs in the ground state molecule lowers the energy of the n-orbital Uv-Visible spectrophotometer 51
  52. 52. Effect of Solvent on absorption spectra… vOften the reverse (i.e. red shift) is seen for π → π* transitions. vThe ground state of the molecule is relatively non- polar, and the excited state is often more polar than the ground state. vAs a result, when a polar solvent is used, it interacts more strongly with the excited state than with the ground state, and the transition is shifted to longer wavelength. Uv-Visible spectrophotometer 52
  53. 53. Effect of Solvent on absorption spectra… vFor example, the figure below shows that the absorption maximum of acetone in hexane appears at 279 nm which in water is shifted to 264 nm, with a blue shift of 15 nm. Uv-Visible spectrophotometer 53
  54. 54. Calculation of λmax of an organic compound I. Woodward's rules: v Named after Robert Burns Woodward vThese rules are several sets of empirically derived rules vThey attempt to predict the wavelength of the absorption maximum ( λmax ) in an ultraviolet- visible spectrum of a given compound. Uv-Visible spectrophotometer 54
  55. 55. I. Woodward's rules… A. Rules for conjugated dienes v These rules specify a base value (214 nm) for the parent diene which is 1,3-butadiene. v The value is red shifted upon v alkyl substitution or v attachment of ring carbons or v ring residues or olefin Uv-Visible spectrophotometer 55 R2C=CR-CR=CR2
  56. 56. A. Rules for conjugated dienes… vIt is also affected by the presence of double bonds out side a ring (exocyclic), extra double bonds in conjugation, and auxochromes. Uv-Visible spectrophotometer 56
  57. 57. A. Rules for conjugated dienes… Examples Uv-Visible spectrophotometer 57
  58. 58. Examples… Uv-Visible spectrophotometer 58
  59. 59. B. Rules for enones Uv-Visible spectrophotometer 59
  60. 60. B. Rules for enones… • Examples Uv-Visible spectrophotometer 60
  61. 61. B. Rules for enones… • Examples Uv-Visible spectrophotometer 61
  62. 62. vα, β -unsaturated aldehydes, acids and esters follow the same general trends as enones, but have different base values. Uv-Visible spectrophotometer 62
  63. 63. C. Rules for Benzoyl Derivatives Uv-Visible spectrophotometer 63
  64. 64. C. Rules for Benzoyl Derivatives… • Example v The Woodward’s rules work well only for conjugated polyenes having four double bonds or less. v For conjugated polyenes with more than four double bonds the Kuhn rules are used. Uv-Visible spectrophotometer 64
  65. 65. II. Simplified Kuhn and Hausser rule v According to this rule λmax = 134(n)1/2 +31 • Where n is the number of conjugated double bonds Example • λmax =476 nm • λmax =476 nm • Uv-Visible spectrophotometer 65
  66. 66. II. Simplified Kuhn and Hausser rules… vThis rule is also useful for calculating number of double bonds from the observed λmax. Example, a compound with λmax of 433 nm will have 9 conjugated double bonds. Uv-Visible spectrophotometer 66
  67. 67. Information from UV VIS Data Qualitative or Quantitative ü UV-VIS data alone gives little structural information 1. Single band of low intensity with (ε 100 to 10,000) and λmax < 220 nm: Usually n → σ* transition possible and the groups present may be alcohols, ethers, amines, thiols, etc. 2. A single band of low intensity (ε= 10 to 100) λmax250- 360nm ,with no absorption in the shorter range 200-250nm indicates n--> π* transition of a simple or unconjugated chromophore. Egs.C=O, C=N, N=N, NO2,COOH, CONH2. Uv-Visible spectrophotometer 67
  68. 68. 3. Two bands of medium intensity with (ε 1,000 to 10,000) and both λmax > 200 nm: Usually is π → π* transition of aromatic system: Look for fine structure in longer wavelength band. 4 . Bands of high intensity with (ε 10,000 to 20,000) and λmax > 220 nm: Usually conjugated π system: Check dienes and α,β- unsaturated carbonyls 5. Band of low intensity with λmax > 300 nm (n → π*) and band of high intensity with λmax < 250 nm (π → π*): Show unconjugated ketones, esters, acids, etc. Uv-Visible spectrophotometer 68
  69. 69. Quantitative UV-Visible Spectrophotometer v The attenuation of EMR as it passes through a sample is described quantitatively by two separate, but related terms: transmittance and absorbance. v Transmittance is defined as the ratio of the EMR’s power exiting the sample, PT , to that incident on the sample from the source, P0, v Multiplying the transmittance by 100 gives the percent transmittance (%T), which varies between 100% (no absorption) and 0% (complete absorption). Uv-Visible spectrophotometer 69
  70. 70. Quantitative UV-Visible Spectrophotometer… vAn alternative method for expressing the attenuation of electromagnetic radiation is absorbance, A, which is defined as vAbsorbance is the more common because it is a linear function of the analyte’s concentration. Uv-Visible spectrophotometer 70
  71. 71. Quantitative UV-Visible Spectrophotometer… vBesides absorption by the analyte, several additional phenomena contribute to the net attenuation of radiation, including § reflection and absorption by the sample container, § absorption by components of the sample matrix and § the scattering of radiation. vTo compensate for this loss of the electromagnetic radiation’s power, we use a method called blank. Uv-Visible spectrophotometer 71
  72. 72. Absorbance and Concentration: Beer’s Law vBeer’s law states that, using a monochromatic wavelength, Absorbance is directly proportional to concentration. v Where vA is absorbance va is absorptivity where the concentration is expressed in gm/L v ɛ is molar absorptivity where the concnetration is expressed in mol/L vC is concentration v b is the path length of sample cell Uv-Visible spectrophotometer 72
  73. 73. Absorbance and Concentration: Beer’s Law… Examples: • A 5.00x10–4 M solution of an analyte is placed in a sample cell that has a pathlength of 1.00 cm. When measured at a wavelength of 490 nm, the absorbance of the solution is found to be 0.338. What is the analyte’s molar absorptivity at this wavelength? Ans(ɛ = 676 cm-1 M-1 ) v A sample has a percent transmittance of 50.0%. What is its absorbance? Ans (A= 0.301) v The molar absorptivity of a substance is 2.0 × 104 cm-1 mol- 1 L. Calculate the transmittance through a cuvette of path length 5.0 cm containing 2.0 × 10-6 mol L-1 solution of the substance. Ans (T= 0.63) Uv-Visible spectrophotometer 73
  74. 74. Limitations to Beer’s Law vDeviations from the direct proportionality between the measured absorbance and concentration when path length is constant may be encountered. vAssumptions of the absorption law: vThe incident beam is monochromatic vThe absorbers absorb independently of each other. vIncident radiation consists of parallel rays perpendicular to the surface of the absorbing medium. vPath length traversed is uniform over the cross section of the beam. vAbsorbing medium is homogenous and does not scatter the radiation. Uv-Visible spectrophotometer 74
  75. 75. Limitations to Beer’s Law… vDeviations from linearity are divided into three categories: vFundamental vChemical and vInstrumental Uv-Visible spectrophotometer 75
  76. 76. I. Fundamental Limitations: – Beer’s law is valid only for low concentrations/diluted solutions/ of analyte. – At higher concentrations the individual particles of analyte no longer behave independently of one another. – There will be reflection, Refraction and scattering – Positive deviations Uv-Visible spectrophotometer 76
  77. 77. II. Chemical Limitations vDeviations from Beer’s law also arise when an analyte associates, dissociates or reacts with a solvent to produce a product having a different absorption spectrum from the analyte. vDepending on the resulting products, it may result in positive or negative deviations. Uv-Visible spectrophotometer 77
  78. 78. III. Instrumental Limitations • Using polychromatic radiation always gives a negative deviation from Beer’s • Stray light causes negative deviations Uv-Visible spectrophotometer 78
  79. 79. UV-Visible spectrophotometer… INStrUMENtatION FOr UV-VIS SPECtrOMEtrY Uv-Visible spectrophotometer 79
  80. 80. INSTRUMENTATION v Today a wide range of instruments are available for making molecular absorption measurements in the UV-visible range. v These vary from simple and inexpensive machines for routine work to highly sophisticated devices. v However, the basic components of these instruments remain the same. v The five essential components of UV-VIS instruments are – A stable radiation source – Wavelength selector – Sample holder – Radiation detector or transducer , and – Signal processing and output device Uv-Visible spectrophotometer 80
  81. 81. Uv-Visible spectrophotometer 81 INSTRUMENTATION… vThe general layout of the essential components in a simple absorption instrument is
  82. 82. INSTRUMENTATION… 1. Radiation Sources – A deuterium discharge lamp for UV region (160-375 nm) – A tungsten filament lamp or tungsten-halogen lamp for Visible and NIR regions (350 - 2500 nm) – The instruments automatically swap lamps when scanning between the UV and VIS-NIR regions Uv-Visible spectrophotometer 82
  83. 83. INSTRUMENTATION… 2. Wavelength Selectors vIn spectrophotometric measurements we need to use a narrow band of wavelengths of light. vThis enhances the selectivity and sensitivity of the instrument and give a more linear relationship vThere are different types of wavelength selectors. vThese include Filters and moncochromators Uv-Visible spectrophotometer 83
  84. 84. INSTRUMENTATION… A. Filters v Either absorption or interference filters are used for wavelength selection: Absorption filters v Usually function via selective absorption of unwanted wavelengths and transmitting the complementary color. v The most common type consists of colored glass or a dye suspended in gelatin and sandwiched between two glass plates. v They are Inexpensive and widely used for band selection in the visible region. Uv-Visible spectrophotometer 84
  85. 85. INSTRUMENTATION… Absorption filters… • If a solution appears orange, this implies that orange light is not being absorbed. Uv-Visible spectrophotometer 85
  86. 86. INSTRUMENTATION… Interference filters vAs the name implies, an interference filter relies on optical interference to provide a relatively narrow band of radiation. vIt consists of a transparent material (calcium or magnesium fluoride) sandwiched between two semitransparent metallic films coated on the inside surface of two glass plates. vWhen it is subjected to a perpendicular beam of light, a fraction passes through the first metallic layer and the other is reflected. Uv-Visible spectrophotometer 86
  87. 87. INSTRUMENTATION… Interference filters… vFraction that is passed undergoes a similar partitioning upon passing through the second metallic film, thus narrower bandwidths are obtained. Uv-Visible spectrophotometer 87
  88. 88. INSTRUMENTATION… B. Monochromators vOne limitation of an absorption or interference filter is that they do not allow for a continuous selection of wavelength. vIf measurements need to be made at two wavelengths, then the filter must be changed in between measurements. vAnother limitation is that they do not give narrow band of wavelength. Uv-Visible spectrophotometer 88
  89. 89. INSTRUMENTATION… B. Monochromators… vAn alternative approach to wavelength selection, which provides for a continuous variation of wavelength, is the monochromator. vThese are of two types; vthe prism and vgrating monochromators. Uv-Visible spectrophotometer 89
  90. 90. INSTRUMENTATION… Prisms v The radiations of different colors having different wavelengths are refracted to different extent due to the difference in the refractive index of glass for different wavelengths. Uv-Visible spectrophotometer 90
  91. 91. INSTRUMENTATION… Prisms… v In a prism monochromator, shown below fine beam of the light from the source is obtained by passing through an entrance slit. This is then collimated on the prism with the help of a lens. v The refracted beams are then focused on an exit slit. The prism is then rotated in a predetermined way to provide the desired wavelength from the exit slit. Uv-Visible spectrophotometer 91
  92. 92. INSTRUMENTATION… Gratings v A grating is made by cutting or etching a series of closely spaced parallel grooves on the smooth reflective surface of a solid material as shown below v The surface is made reflective by making a thin film of aluminum on it and the etching is done with the help of a suitably shaped diamond tool. Uv-Visible spectrophotometer 92
  93. 93. INSTRUMENTATION… Gratings… vIn grating monochromator (Fig. above), a fine beam of the light from the source falls on a concave mirror through an entrance slit. vThis is then reflected on the grating which disperses it. vThe dispersed radiation is then directed to an exit slit. Uv-Visible spectrophotometer 93
  94. 94. INSTRUMENTATION… Gratings… vThe range of wavelengths isolated by the monochromator is determined by the extent of dispersion by the grating and the width of the exit slit. vRotation of the grating in a predetermined way can be used to obtain the desired wavelength from the exit slit. Uv-Visible spectrophotometer 94
  95. 95. INSTRUMENTATION… 3. Sample cells vThe UV-VIS absorption spectra are usually determined either in vapor phase or in solution. vSample containing the analyte is taken in a cell called a cuvette – which is transparent to the wavelength of light passing through it. vA variety of quartz cuvettes are available vThese are of varying path lengths and are equipped with inlet and outlets. Uv-Visible spectrophotometer 95
  96. 96. INSTRUMENTATION… 3. Sample cells… vFor measurements in the visible region the cuvettes made of glass can also be used. vHowever, since glass absorbs the ultraviolet radiations, these cannot be used in the UV region. vTherefore, most of the spectrophotometers employ quartz cuvettes (Fig below), as these can be used for both visible and UV region. Uv-Visible spectrophotometer 96
  97. 97. INSTRUMENTATION… 3. Sample cells… v Usually square cuvettes having internal path length 1.0 cm are used. v Though cuvettes of much smaller path lengths say of 0.1 mm or 0.05 mm are also available. Uv-Visible spectrophotometer 97 qThe faces of these cells t h r o u g h w h i c h t h e radiation passes are highly polished to keep reflection and scatter losses to a minimum.
  98. 98. INSTRUMENTATION… 3. Sample cells… vNow a days ‘spectral grade’ solvents are available which have – high purity and – negligible absorption in the region of absorption by the chromophore. vIn a typical measurement of absorption spectrum, the solution of the sample is taken in a suitable cuvette and the spectrum is run in the desired range of the wavelengths. Uv-Visible spectrophotometer 98
  99. 99. INSTRUMENTATION… 3. Sample cells… v The absorption by the solvent, if any, is compensated by – running the spectrum for the solvent alone in the same or identical cuvette and subtracting it from the spectrum of the solution. vThis gives the spectrum only due to the absorption of the species under investigation. vIn double beam spectrometers, the sample and the solvent are scanned simultaneously Uv-Visible spectrophotometer 99
  100. 100. INSTRUMENTATION… 4. Detectors vconvert a light signal to an electrical signal vThis is suitably measured and transformed into an output. vThey generate a signal, which is linear in transmittance i.e. they respond linearly to radiant power falling on them. vThe transmittance values can be changed logarithmically into absorbance units by an electrical or mechanical arrangement in the signal read out device. vThere are three types of detectors which are used in modern spectrophotometers. Uv-Visible spectrophotometer 100
  101. 101. INSTRUMENTATION… 4. Detectors… i. Phototube • A phototube consists of a photoemissive cathode and an anode in an evacuated tube with a quartz window. • These two electrodes are subjected to high voltage (about 100 V) difference. • When a photon enters the tube and strikes the cathode, an electron is ejected and is attracted to the anode resulting in a flow of current which can be amplified and measured. Uv-Visible spectrophotometer 101
  102. 102. INSTRUMENTATION… ii. Photomultiplier (PM) Tube vA photomultiplier tube consists of a series of electrodes, called dynodes. v The voltage of successive electrodes is maintained 50 to 90 volt more positive than the previous one. vWhen a radiation falls on the cathode an electron is emitted from it. vThis is accelerated towards the next photo emissive electrode by the potential difference between the two. Here, it releases many more secondary electrons. Uv-Visible spectrophotometer 102
  103. 103. INSTRUMENTATION… ii. Photomultiplier (PM) Tube… v These, in turn are accelerated to the next electrode where each secondary electron releases more electrons. v The process continues up to about 10 stages of amplification. The final output of the photomultiplier tube gives a much larger signal than the photocell. Uv-Visible spectrophotometer 103
  104. 104. INSTRUMENTATION… iii. Diode Array Detectors • Employ a large number of silicon diodes arranged side by side on a single chip. • When a UV-VIS radiation falls on the diode, its conductivity increases significantly. • This increase in conductivity is proportional to the intensity of the radiation and can be readily measured. Uv-Visible spectrophotometer 104
  105. 105. INSTRUMENTATION… iii. Diode Array Detector… • Since a large number of diodes can be arranged together, the intensity at a number of wavelengths can be measure simultaneously. Uv-Visible spectrophotometer 105
  106. 106. INSTRUMENTATION… 5. Signal Processing and Output Devices vThe electrical signal from the transducer is suitably amplified or processed before it is sent to the recorder to give an output. vThe subtraction of the solvent spectrum from that of the solution is done electronically. vThe output plot between the wavelength and the intensity of absorption is the resultant of the subtraction process and is characteristic of the absorbing species. Uv-Visible spectrophotometer 106
  107. 107. TYPES OF UV-VISIBLE SPECTROMETERS vBroadly speaking there are three types of spectrometers. 1. Single Beam Spectrometers vcontain a single beam of light. üThe same beam is used for reading the absorption of the sample as well as the reference. vThe radiation from the source is passed through a filter or a suitable monochromator to get a band on monochromatic radiation. Uv-Visible spectrophotometer 107
  108. 108. I. Single Beam Spectrometers… v It is then passed through the sample (or the reference) and the transmitted radiation is detected by the photodetector. v The signal so obtained is sent as a read out or is recorded. v Typically, two operations have to be performed – first, the cuvette is filled with the reference solution and the absorbance reading at a given wavelength or the spectrum over the desired range is recorded. Uv-Visible spectrophotometer 108
  109. 109. 1. Single Beam Spectrometers… Second, the cuvette is taken out and rinsed and filled with sample solution and the process is repeated. v The spectrum of the sample is obtained by subtracting the spectrum of the reference from that of the sample solution. Uv-Visible spectrophotometer 109
  110. 110. 2. Double Beam Spectrometers v In a double beam spectrometer, the radiation coming from the monochromator is split into two beams with the help of a beam splitter. v These are passed simultaneously through the reference and the sample cell. v The transmitted radiations are detected by the detectors and the difference in the signal at all the wavelengths is suitably amplified and sent for the output. Uv-Visible spectrophotometer 110
  111. 111. 3. Photodiode Array Spectrometer v In a photodiode array instrument, also called a multi- channel instrument, the radiation output from the source is focused directly on the sample. v This allows the radiations of all the wavelengths to simultaneously fall on the sample. v The radiation coming out of the sample after absorption (if any) is then made to fall on a reflection grating. Uv-Visible spectrophotometer 111
  112. 112. Uv-Visible spectrophotometer 112 3. Photodiode Array Spectrometer… vThe grating disperses all the wavelengths simultaneously. vThese then fall on the array of the photodiodes arranged side by side. vIn this way the intensities of all the radiations in the range of the spectrum are measured in one go. vThe advantage of such instruments is that a scan of the whole range can be accomplished in a short time.
  113. 113. UV-Visible spectrophotometer… aPPLICatIONS OF UV-VISIBLE SPECtrOPHOtOMEtEr Uv-Visible spectrophotometer 113
  114. 114. UV-Visible Spectrophotometer… Principles: radiation in the wavelength range 200-800nm is passed through a solution of a compound. Ø The electrons in the molecule become excited so that they occupy a higher quantum state and in process absorb some of the energy passing through the solution. Ø The wavelength at which the solution (analyte) absorbs and the Intensity of absorption is determined by the structure and the concentration of the analyte respectively. ØCan be used for qualitative and quantitative analysis if appropriate Instrument is used 114
  115. 115. Important advantages of spectrophotometric methods 1- Wide applicability; large number of organic and inorganic species absorb light in the UV-Visible ranges. 2- High sensitivity; analysis for concentrations in the range from 10- 4 to 10- 6 M are ordinary in the Spectrophotometric determinations. 3- Moderate to high selectivity; Due to selective reactions, selective measurements and different mathematical treatments. 4- Good accuracy; Relative errors in concentration measurement lie in the range of 0.1 to 2 %. 5- Ease and convenience; Easily and rapidly performed with modern instruments. 115
  116. 116. Qualitative Applications 1. Identification of chromophores 2. Confirmation of identity 3. Detection of some functional groups 4. Determination of approximate number of conjugated double bonds 5. Identification of the position and/or conformation of certain functional groups Uv-Visible spectrophotometer 116
  117. 117. 1- Identification of chromophores • Example, the presence of an absorbance band at a particular wavelength often is a good indicator of the presence of a chromophore. • Useful information about substance can be obtained via examination of its lmax and εmax, which could be correlated with the structural features (See the following table). Uv-Visible spectrophotometer 117
  118. 118. 1. Identification of chromophores… Uv-Visible spectrophotometer 118 Absorption characteristics of some common organic chromophores:
  119. 119. 2-Confirmation of identity • The spectrum is a physical constant, which along with melting & boiling points, refractive index and other properties may be used for characterization of compounds • Although UV-visible spectra do not enable absolute identification of an unknown, they frequently are used to confirm the identity of a substance: 119
  120. 120. 2-Confirmation of identity… 2.1 Through comparison of the measured spectrum with a reference spectrum. a) An absorption band at 254 nm with characteristic vibrational fine structures may be an evidence for existence of aromatic structure. b) Three characteristic bands at 278, 361 &550 nm with absorbance ratio of 2:3:1 is very characteristic for cyanocobalamin. 120
  121. 121. 2.2 Identification by using Absorbance ratio • Absorbance ratio of a given drug at two different wavelength is constant, provided that – beer’s Law is obeyed at the selected wavelengths – The same concentration of the sample is used for both wavelengths 121 2-Confirmation of identity…
  122. 122. 3- Detection of some functional groups a) An absorption band at about 280-290 nm, which is displaced toward shorter wavelength with increasing solvent polarity strongly, indicates the presence of aromatic carbonyl group. b) Confirmation of presence of aromatic amine or phenolic structure may be obtained by testing the pH effect on their spectra. 122 NH2 NH3 In alkaline medium in acid medium Aniline, lmax= 280 nm Anilinium ion lmax= 254 nm + + H+ - H+ - + H in acid medium in alkaline medium O O OH OH (Phenol)lmax = 270 nm (phenate anion) lmax= 290 nm
  123. 123. 4- Approximate determination of the number of double bonds: By using Simplified Kuhn and Hausser rule : lmax (nm) = 134 n + 31 where n is the number of conjugated double bonds. 5-Identification of the position and/or conformation of certain functional groups: d g b a C = C – C = C – C = O enones • a-Alkyl cause red shift about 10 nm & a-OH about 35 nm • b-Alkyl cause red shift about 12 nm & b-OH about 30 nm • g-Alkyl cause red shift about 18 nm & g-OH about 50 nm 123
  124. 124. II. Quantitative Analysis Scope - Applications of spectrophotometric methods are so numerous and touch every field in which quantitative chemical information are required. - In general, about 90% of all the quantitative determinations are performed by spectroscopic techniques. - In the field of health alone , 95 % of all quantitative determinations are performed by UV-Visible spectrophotometer and similar techniques. Uv-Visible spectrophotometer 124
  125. 125. II. Quantitative Analysis ... A. Assay of single components • The assay of an absorbing substance may be quickly carried out by preparing a solution in a transparent solvent and measuring its absorbance at a suitable wavelength. – Wavelength of maximum absorbance • The concentration of the absorbing species is then calculated from the measured absorbance using the following procedures. i. Use of standard absorptivity value- used for substances whose reference standards are expensive or difficult to obtain Uv-Visible spectrophotometer 125
  126. 126. Using Beer-Lambert Law A = abc a can be ɛ (molar absorptivity) when unit of concentration is in moles per liter or A (1%, 1cm) (specific absorbance) when unit of concentration is in g/L. A (1%, 1cm) is absorbance of a 1g/100ml(1% w/v) solution in 1cm cell. A simple easily derived equation allows interconversion of ɛ and A (1%, 1cm) Uv-Visible spectrophotometer 126 � = �1⊂� 1% 10 � ��������� ����ℎ�
  127. 127. A. Assay of single components… Example 1: Calculate the concentration of methyl testosterone in an ethanolic solution of which the absorbance in a 1 cm cell at its λmax, 241 nm, was found to be 0.890. The Specific Absorptivity in the B.P. is 540 at 241 nm. (0.00165 g/100ml) Example 2: calculate the concentration in µg/ml of a solution of tryptophan (M.wt = 204.2) in 0.1 M HCl, giving an absorbance at its λ max, 277 nm, of 0.613 in 4 cm cell. ( molar absorptivity is 5432). (5.76 µg/ml) Uv-Visible spectrophotometer 127
  128. 128. A. Assay of single components… Example-3: A 5.00x10–4 M solution of an analyte is placed in a sample cell that has a path length of 1.00 cm. When measured at a wavelength of 490 nm, the absorbance of the solution is found to be 0.338. What is the analyte’s molar absorptivity at this wavelength? Ans(Molar A. = 676 cm-1 M-1 ) Example -4: A sample has a percent transmittance of 50.0%. What is its absorbance? Ans (A= 0.301) Example-5: The molar absorptivity of a substance is 2.0 × 104 cm-1 mol-1 L. Calculate the transmittance through a cuvette of path length 5.0 cm containing 2.0 × 10-6 mol L-1 solution of the substance. Ans (T= 0.63) Uv-Visible spectrophotometer 128
  129. 129. A. Assay of single components… ii. Use of calibration graph , Y = ax + b Example: the absorbance values at 250 nm of 5 standard solutions, and sample solution of a drug are given below: Conc. (ug/ml) A 250 nm 10 0.168 20 0.329 30 0.508 40 0.660 50 0.846 Sample 0.661 Ø Calculate the concentration of the sample. (Y= 0.01679X-0.0008, C= 36.5 µg/ml) Uv-Visible spectrophotometer 129      -  2 2 ) ( X N Y) X)( ( - XY N X a       -  2 2 2 ) ( X N XY) ( X) ( - ) X )( Y ( X b
  130. 130. A. Assay of single components… iii. Single point standardization – this method involves the measurement of the absorbance of a sample solution and of a standard solution of the reference substance. – The standard and sample solution are prepared in similar manner. – The concentration of the substance in the sample is calculated from the proportional relationship that exists b/n absorbance and concentration. Uv-Visible spectrophotometer 130 std std Sample sample A xC A C 
  131. 131. iii. Single point standardization… • It is the procedure adopted in many spectrophotometric a s s a y s o f t h e B P a n d f o r t h e m a j o r i t y o f spectrophotometric assays of USP. • Occasionally, a linear but non proportional r/nship b/n concentration and absorbance occurs, which is indicated by a significant negative or positive intercept in a beer’s law plot….double point standardization Uv-Visible spectrophotometer 131
  132. 132. iv. Double point standardization • In the case mentioned before, a two –point bracketing standardization is therefore required to determine the concentration of the sample solution. • The concentration of one of the standard solution is greater than that of the sample while the other is with a` lower concentration than the sample. • The concentration of the test sample is given by: • Where, Std1is the more concentrated standard and Std2 is less concentrated standard Uv-Visible spectrophotometer 132 2 1 2 1 1 2 1 1 ) ( ) )( ( std std std std srd std std std Sample sample A A A A C C C A A C - -  - - 
  133. 133. B. Assay of Substances in multi component sample • The spectroscopic analysis of drugs rarely involves measurement of absorbance of samples containing only one absorbing component. • The pharmaceutical analyst frequently encounters the situation where the concentration of one or more substance is required in samples known to contain other absorbing substances which potentially interfere in the assay. Uv-Visible spectrophotometer 133
  134. 134. B. Assay of Substances in multicomponent sample… • The basis of all the spectrophotometric techniques for multicomponent samples is the property that all wavelengths: a) The absorbance of a solution is the sum of absorbances of the individual components or b) The measured absorbance is the difference b/n the total absorbance of the solution in the sample cell and that of the solution in the reference cell. Uv-Visible spectrophotometer 134
  135. 135. B. Assay of Substances in multicomponent sample… If the identity, concentration and absorptivity of the absorbing interferents are known: it is possible to calculate their contribution to the total absorbance of a mixture. Example: The max of ephedrine HCl and Chlorocresol are 257 nm and 279 nm respectively and the specific absorptivity values in 0.1M HCl solution are: • Ephedrine HCl at 257 nm = 9.0 Chlorocresol at 257 nm = 20 • Ephedrine HCl at 279 nm = 0 Chlorocresol at 279 nm = 105 Ø Calculate the concentration of ephedrine HCl and Chlorocresol in a batch of Ephedrine HCl injection, diluted 1 to 25 with water, giving the following absorbance values in 1 cm cells. (A279 (total) = 0.424, A257 (total) = 0.97) ü Ans. C. Ephedrine HCl in the injection = 2.475 gm/100ml C. Chlorocresol = 0. 1010 gm/100ml Uv-Visible spectrophotometer 135
  136. 136. B. Assay of Substances in multicomponent sample… Simultaneous equations method Note: Absorbance is additive Uv-Visible spectrophotometer 136
  137. 137. B. Assay of Substances in multicomponent sample… 2. Simultaneous equations method… Example: Palladium (II) and gold (III) can be analyzed simultaneously through reaction with methiomeprazine (C19H24N2S2). The absorption maximum for the Pd complex occurs at 480 nm, while that for the Au complex is at 635 nm. Molar absorptivity data at these wavelengths are A 2 5 . 0 - m L s a m p l e w a s t r e a t e d w i t h a n e x c e s s o f methiomeprazine and subsequently diluted to 50.0 mL. Calculate the molar concentrations of Pd(II), CPd, and Au(III), CAu, in the sample if the diluted solution had an absorbance of 0.533 at 480 nm and 0.590 at 635 nm when measured in a 1.00-cm cell. (CAu = 3. 60X10-5M, Cpd= 2.4X10-4M) Uv-Visible spectrophotometer 137
  138. 138. B. Assay of Substances in multicomponent… Difference Spectrophotometer the selectivity and accuracy of Spectrophotometric analysis of samples containing absorbing interferents may be markedly improved by the technique of difference spectrophotometer. Principle: a component in a mixture is analysed by carrying out a reaction which is selective for the analyte. • This could be simply bringing about a shift in wavelength through adjustment of pH of the solution in which the analyte is dissolved or a chemical reaction such as oxidation or reduction. • The measured value is the difference absorbance (∆A) b/n two equimolar solutions of the analyte in different chemical forms which exhibit different spectral characteristics. Uv-Visible spectrophotometer 138
  139. 139. B. Assay of Substances in multicomponent… Difference Spectrophotometer… ü The criteria for applying difference spectrophotometery to the assay of a substance in the presence of other absorbing substances are that: q Reproducible changes may be induced in the spectrum of the analyte by the addition of one or more reagents q The absorbance of the interfering substance is not altered by the reagents. ü The simplest and most commonly employed technique for altering the spectral characteristics of the analyte is the adjustment of the pH by means of aqueous solutions of acid, alkali or buffers. Uv-Visible spectrophotometer 139
  140. 140. B. Assay of Substances in multicomponent… Difference Spectrophotometer… ∆A = Aalk(total)-Aacid (total) = Aalk+Aint-(Aacid+Aint) = Aalk-Aacid ∆A = ∆ε .b. C • If the substance is not affected by pH, it can be quantitatively converted by means of a suitable reagent to a chemical species that has d/t spectral properties to its unreacted parent species. Uv-Visible spectrophotometer 140
  141. 141. B. Assay of Substances in multicomponent… Derivative spectroscopy • Derivative spectroscopy uses first or higher derivatives of absorbance with respect to wavelength for qualitative analysis and for quantification. • If a spectrum is expressed as absorbance, A, as a function of wavelength,, the derivative spectra are: Uv-Visible spectrophotometer 141
  142. 142. B. Assay of Substances in multicomponent… 4. Derivative spectroscopy… • A first-order derivative is the rate of change of absorbance with respect to wavelength. • It passes through zero at the same wavelength as λmax of the absorbance band. This is characteristic of all odd-order derivatives. • The most characteristic feature of a second-order derivative is a negative band with minimum at the same wavelength as the maximum on the zero-order band. • A fourth-order derivative shows a positive band. • A strong negative or positive band with minimum or maximum at the same wavelength as λ max of the absorbance band is characteristic of the even-order derivatives. Uv-Visible spectrophotometer 142
  143. 143. B. Assay of Substances in multicomponent… 4. Derivative spectroscopy… • Note that the number of bands observed is equal to the derivative order plus one. Advantages ü Derivative spectrum shows better resolution of overlapping bands the fundamental spectrum and may permit the accurate determination of the λ max of the individual bands. ü It permits discrimination against broad band interferences, arising from turbidity or non-specific matrix absorption. Ø Thus, the information content of a spectrum is presented in a potentially more useful form, offering a convenient solution to a number of analytical problems, such as resolution of multi- component systems, removal of sample turbidity, matrix background and enhancement of spectral details. Uv-Visible spectrophotometer 143
  144. 144. B. Assay of Substances in multicomponent… 4. Derivative spectroscopy… Background elimination Resolution Discrimination Uv-Visible spectrophotometer 144
  145. 145. B. Assay of Substances in multicomponent… 4. Derivative spectroscopy… • It is possible to measure the absolute value of the derivative at an abscissa value (wavelength) corresponding to a zero- crossing of one of the components in the mixture. • This is termed a zero-crossing measurement. • The zero-crossing derivative spectroscopic mode allows the resolution of binary mixtures of analytes by recording their derivative spectra at wavelengths at which one of the components exhibits no signal. • Zero-crossing measurements for each component of the mixture are therefore the sole function of the concentration of the others. Uv-Visible spectrophotometer 145
  146. 146. III. Other Applications • Monitoring drug degradation kinetics • Detection in Chromatography • Determination of Equilibrium Constants • Determination of complex stoichiometry • Spectrophotometeric titrations Uv-Visible spectrophotometer 146
  147. 147. III. Other Applications A. Monitoring drug degradation kinetics Ø Can be simply done when the product has a different absorption spectrum than that of un-degraded drug. Ø The rate of disappearance of the spectrum or appearance of other spectrum (as a function of time ) may be used to determine rate constant for hydrolysis or degradation. Ø Oxidation reactions and any other type of reactions that yield products whose spectra are different from the reactants , may be followed and their rate constant estimated. Uv-Visible spectrophotometer 147
  148. 148. B. Detection in Chromatography Ø Mainly used in HPLC and HPTLC. Ø They are the most widely used detectors, because: ØMost drugs absorb UV-Visible radiation. ØMore sensitive and more selective than the bulk property detectors Ø Some absorbance detectors have one or two fixed wavelengths (280 and/or 254 nm). Ø More modern HPLC instruments have variable wavelength detectors using the photodiodes Uv-Visible spectrophotometer 148
  149. 149. C. Determination of Equilibrium Constants Ø Acid dissociation constants and metal ion-ligand stability constants can be determined spectrophotometrically if the species involved have absorptivities which differ from one another. Example : Determination of the pKa of Methyl red indicator ; Acidic (HMR) and basic (MR-) forms of methyl red are shown below Uv-Visible spectrophotometer 149 CO2- (CH3)2N N NH + CO2- (CH3)2N N=N HO- H+ Acid form, pH= 4, (HMR) Red, 520 nm Basic form, pH= 6, (MR-) Yellow 430 nm
  150. 150. C. Determination of Equilibrium Constants… Ø The pKa of methyl red indicator is given by the equation: Ø Both HMR and MR- have strong absorption peaks in the visible portion of the spectrum. Uv-Visible spectrophotometer 150 A 430 nm 520 nm pH l 5.0 Measured at 520 nm Measured at 430 nm
  151. 151. Ø The color change interval from pH 4 to pH 6 can be obtained with acetate buffer system. Ø At pH = 4, the acid is completely unionized and at pH = 6, the acid is completely ionized Ø At intermediate pH values, the two species are present. Ø Plotting absorbance (A) against pH values at l1 and l2 gives two curves. Ø The pH at the point of intersection represents the pKa of the indicator. Uv-Visible spectrophotometer 151
  152. 152. D. Determination of complex stoichiometry Ø The stoichiometry for a metal–ligand complexation reaction has the following general form. Ø Can be determined by one of three methods: – the method of continuous variations – the mole-ratio method and – the slope-ratio method. Uv-Visible spectrophotometer 152
  153. 153. D. Determination of complex stoichiometry… i. Method of continuous variations (CVM) Ø Also called Job’s method and is the most popular. Ø In this method a series of solutions is prepared such that the total moles of metal and ligand, ntot, in each solution is the same. Ø Thus, if (nM)iand (nL)i are, respectively, the moles of metal and ligand in the i-th solution, then Uv-Visible spectrophotometer 153
  154. 154. i. Method of continuous variations… Ø The relative amount of ligand and metal in each solution is expressed as the mole fraction of ligand, (XL)i, and the mole fraction of metal, (XM)i, Ø Absorbance versus the mole fraction of ligand will be plotted. CVM Uv-Visible spectrophotometer 154 A L/M ratio 0.0 1.0 A [L]/[L]+[M]
  155. 155. i. Method of continuous variations… Ø The intersection of the two lines drawn from both sides occurs when stoichiometric mixing of metal and ligand is reached. Ø Mole fraction of ligand at this intersection is used to determine the value of y for the metal–ligand complex, MLy. Uv-Visible spectrophotometer 155
  156. 156. ii. Mole-ratio method Ø In the mole-ratio method the moles of one reactant, usually the metal, are held constant, while the moles of the other reactant are varied. Ø The absorbance is monitored at a wavelength at which the metal–ligand complex absorbs. Ø A plot of absorbance as a function of the ligand-to-metal mole ratio (nL/nM) has two linear branches that intersect at a mole ratio corresponding to the formula of the complex. Uv-Visible spectrophotometer 156
  157. 157. iii. slope-ratio method Ø In the slope-ratio method two sets of solutions are prepared. Ø The first set consists of a constant amount of metal and a variable amount of ligand, chosen such that the total concentration of metal, CM, is much greater than the total concentration of ligand, CL. Ø Under these conditions we may assume that essentially all the ligand is complexed. The concentration of a metal–ligand complex of the general form MxLy is Uv-Visible spectrophotometer 157
  158. 158. iii. slope-ratio method … Ø If absorbance is monitored at a wavelength where only MxLy absorbs, then and a plot of absorbance versus CL will be linear with a slope, sL, of Ø A second set of solutions is prepared with a fixed concentration of ligand that is much greater than the variable concentration of metal; thus Uv-Visible spectrophotometer 158
  159. 159. iii. slope-ratio method … Ø The mole ratio of ligand-to-metal is determined from the ratio of the two slopes. Uv-Visible spectrophotometer 159
  160. 160. E. Spectrophotometeric titrations Ø One or more of the reactants or products absorb radiation. Ø They are carried out in a vessel for which the light path is constant. Ø The absorbance is directly proportional to concentration. Titration Curves • Plot of absorbance as a function of titrant volume and consists of two straight-line regions with different slopes Uv-Visible spectrophotometer 160
  161. 161. E. Spectrophotometric titrations… Advantages § More accurate results than direct titrimetric analysis are obtained. § Can be used for the titration of very dilute solutions (Sensitive) § Do not need favorable equilibrium constants as those required for titration that depends upon observations near the end point. § Can be used for all types of reactions (Redox, acid- base, complexometric , pptmetry…etc). Uv-Visible spectrophotometer 161
  162. 162. Colorimetry Ø Is a technique which involves measurement of absorbance in the visible region is known as colorimetry. Ø Involves measurement of color intensity of compounds. Requirements for colorimetry ü the substance should be colored or ü The substance should be able to be derivatized in to colored product. ü While derivatizing § The reagent should be specific § The color produced should be stable enough until the analysis is completed § Color intensity should be directly proportional to the concentration of the analyte. Application- colored drugs and those drugs which can be derivatized. Uv-Visible spectrophotometer 162
  163. 163. THE END Uv-Visible spectrophotometer 163