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Uv visible spectroscopy- madan

uv and visible spectroscopy, instrumental analysis

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Uv visible spectroscopy- madan

  1. 1. INSTRUMENTAL TECHNIQUES OF ANALYSIS MADAN SIGDEL M. Pharm. (Pharmacology)
  2. 2. UNIT - ONE Visible and ultraviolet spectroscopy a. Introduction and elemental theory b. Instrumentation, measurement and sample handling c. Applications I. Chromophores- isolated functional group II. Quantitative studies concentration, rate measurements and acid/base dissociation
  3. 3. SPECTROSCOPY  It is the branch of science that deals with the study of interaction of matter with light. OR • It is the branch of science that deals with the study of interaction of electromagnetic radiation with matter.
  4. 4. ELECTROMAGNETIC RADIATION Electromagnetic radiation consist of discrete packages 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.
  5. 5. 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.
  6. 6. The relationship between wavelength & frequency can be written as: c=νλ As photon is subjected to energy, so E= hν= hc/λ
  7. 7. ELECTROMAGNETIC SPECTRUM
  8. 8.  a) when a group is more polar in ground state than exited state than increase polarity of the solvent stabilizes the non-bonding electrons in the ground state because H-bonding. Thus absorption shifted to lower wave length.  b) when the group is more polar in the exited state, then absorption get shifted to longer wave length with increased polarity of the solvent which helps in stabilizing the non-bonding electrons in the exited state.  The increased in polarity of the solvent generally shifts n-π* and n-σ * bands to shorter wav lengths and π-π* bands to longer wave lengths.
  9. 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. 10.  1. 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 μm)  2. 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
  11. 11. INTERACTION OF EMR WITH MATTER 1. Electronic Energy Levels:  At room temperature the molecules are in the lowest energy levels E0.  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 E1, E2, …En, etc is called as electronic transition and the difference is as: ∆E = h ν = En - E0 where (n = 1, 2, 3, … etc) ∆E = 35 to 71 kcal/mole
  12. 12. 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.
  13. 13. 3. Rotational Energy Levels: • These energy levels are quantized & discrete. • The spacing between energy levels are even smaller than vibrational energy levels. ∆Erotational < ∆Evibrational < ∆Eelectronic
  14. 14. THEORY INVOLVED • When a beam of light falls on a solution or homogenous media ,a portion of light is reflected ,from the surface of the media, a portion is absorbed within the medium and remaining is transmitted through the medium. • Thus if I0 is the intensity of radiation falling on the media • Ir is the amount of radiations reflected, • Ia is the amount of radiation absorbed & • It the amount of radiation transmitted then I0 = Ir + Ia + It
  15. 15. ABSORPTION LAWS  Lambert’s law  Beer’s law  Beer-lambert’s law
  16. 16. LAWS GOVERNING ABSORPTION OF RADIATION  The two laws related to the absorption of radiation are: Beer’s law ( related to concentration of absorbing species)  Lambert’s law (related to thickness/path length of absorbing species)  These two laws are applicable under the following condition: I = I a + I t I = Intensity of incident light I a = Intensity of absorbed light I t =Intensity of transmitted light and No reflection/scattering of light takes place
  17. 17. Beer’s law “The intensity of a beam of monochromatic light decreases exponentially with increase in the concentration of absorbing species. arithmetically Accordingly, - dI / dc α I (The decrease in the intensity of incident light (I) with concentration c is proportional to the intensity of incident light (I)) -dI / dc = kI (removing and introducing the constant of proportionality ‘k’) -dI / I = k dc (rearranging terms) -In I = kc + b ……Equation (1) (on integration , b is constant of integration) When concentration = 0, there is no absorbance. Hence I= Io Substituting in equation 1, -In Io = k*0 + b -In Io = b
  18. 18. Substituting the value of b, in equation 1, -In I = kc –InIo In Io – In I = kc In Io/I = kc (since log A-log B = log A/B) Io / I = e kc (removing natural logarithm) I / Io = e –kc (making inverse on both sides) I = Io e -kc ….Equation (2) (equation of Beer’s law)
  19. 19. Lambert’s law “The rate of decrease of intensity (monochromatic light) with the thickness of the medium is directly proportional to the intensity of incident light” i.e. –dI / dt α I This equation can be simplified similar to equation 2 to get the following equation (by replacing ‘c’ with ‘t’) I = Io e –kt ….. Equation (3) [equation of Lambert’s law]
  20. 20. BEER – LAMBERT,S LAW Equations (2) and (3) can be combined to get I= Io e –kct I = Io 10 –kct (converting natural algorithm to base 10) I / Io = 10 –kct (rearranging terms) Io / I = 10 kct (inverse on both side Log Io / I = kct (taking log on both sides) ….. Equation 4 It can be learnt that transmittance (T) = I / Io and Absorbance (A) = log 1 / T Hence A = log 1 / I/ Io A = log Io /I ……. Equation 5
  21. 21. Using Equation 4 & 5 , Since A= log Io /I and log Io /I = Kct we can infer that, A= Kct (instead of K, we can use ε) A= ε ct (Equation of beer – Lambert’s law) Where: A – Absorbance or optical density or extinction co- efficient. ε – Molecular extinction coefficient c – Concentration of the drug (mol/lit) t – Path length (normally 10mm or 1cm)
  22. 22.  Absorbance of a material is a logarithmic ratio of the amount of radiation falling upon a material to the amount of radiation transmitted through the material. A=log Io/I  transmittance is the fraction of incident light (electromagnetic radiation) at a specified wavelength that passes through a sample T=I/Io
  23. 23. DEVIATION FROM THE BEER’S LAW  Beer’s law: it states that if we plot absorbance A against concentration C a straight line passing through origin is obtained, but usually a deviation from a linear relationship between concentration and absorption and an apparent failure of beer’s law. There are two type of deviation :  POSITIVE DEVIATION : When a small change in concentration produces a greater change in absorbance.  NEGATIVE DEVIATION : When a large change in concentration produces a smaller change in absorbance.
  24. 24. REASONS FOR DEVATION FROM BEER’S LAMBERT LAW  Instrumental deviation  Physicochemical change in solution  Instrumental deviation: Factors like stray radiation, improper slit width, fluctuation in single beam and when monochromatic light is not used can influence the deviation.  Physicochemical change in solution: Factors like association, dissociation, ionization (change in pH), faulty development of color (incompletion of reaction), refractive index at high concentration, can influence such deviation.
  25. 25. References  Instrumental Methods of Chemical Analysis by Gurdeep R. Chatwal ( page no : 108 to 113 )  Instrumental Methods of Analysis by Scoog and West)  http://en.wikipedia.org/wiki .  Instrumental analysis by willard and merit ( page no : 160 to 163 )
  26. 26. PRINCIPLE OF UV-VISIBLE SPECTROSCOPY  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.
  27. 27. PRINCIPLES OF UV-VISIBLE SPECTROSCOPY  UV-visible spectroscopy measure the response of a sample to ultraviolet and visible range of electromagnetic radiation.  Molecules have either n, π or б Electrons.  These electrons absorb UV radiation & undergoes transitions from ground state to excited state.
  28. 28.  The absorption of uv radiation brings about the promotion of an electron from bonding to antibonding orbital.  The wavelength of radiation is slowly changed from minimum to maximum in the given region, and the absorbance at every wavelength is recorded.  Then a plot of energy absorbed Vs wavelength is called absorption spectrum. The significant features: λmax (wavelength at which there is a maximum absorption) єmax (The intensity of maximum absorption) The UV spectrum depends on  solvents  concentration of solution
  29. 29.  σ electrons  π electrons  n electrons  σ -electrons: these electrons are involved in saturated bonds such as those b/w carbon of hydrogen in paraffin. These bands are known as σ bonds. As amount of energy required to excite electrons in σ bonds in much more than that produced by UV-light.  Compounds containing σ bonds do not absorb UV radiations. Therefore paraffin compounds are very use full as solvents.
  30. 30.  π-electrons: these electrons are involved in unsaturated hydrocarbons. Typical compounds with π bonds are trienes and aromatic compounds.  n-electrons: these are electrons which are not involved in bonding b/w atoms in molecule. Eg: organic compounds containing N,O & halogen.  As n electrons can be excited by UV radiations, any compound that contain atoms like N,O,S, halogens or unsaturated hydrocarbons may absorb UV radiation.
  31. 31.  Electronic transitions
  32. 32. 1. σ → σ* transition  σ electron from orbital is excited to corresponding anti- bonding orbital σ*.  The energy required is large for this transition.  e.g. Methane (CH4) has C-H bond only and can undergo σ → σ* transition and shows absorbance maxima at 125 nm.
  33. 33. 2. π → π* transition • π electron in a bonding orbital is excited to corresponding anti-bonding orbital π*. • Compounds containing multiple bonds like alkenes, alkynes, carbonyl, nitriles, aromatic compounds, etc undergo π → π* transitions. • e.g. Alkenes generally absorb in the region 170 to 205 nm.
  34. 34. 3. n → σ* transition • 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).
  35. 35.  4 • n → π* transition • An electron from non-bonding orbital is promoted to anti-bonding π* orbital. • Compounds containing double bond involving hetero atoms (C=O, C≡N, N=O) undergo such transitions. • n → π* transitions require minimum energy and show absorption at longer wavelength around 300 nm.
  36. 36. 5 • σ → π* transition & 6• π → σ* transition  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 .How does a spectrophotometer work.mp4  The UV spectrum is of only a few broad of absorption.
  37. 37. TERMS USED IN UV/ VISIBLE SPECTROSCOPY 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. e.g. NO2, N=O, C=O, C=N, C≡N, C=C, C=S, etc
  38. 38. CHROMOPHORE To interpretate UV – visible spectrum following points should be noted: 1. Non-conjugated alkenes show an intense absorption below 200 nm & are therefore inaccessible to UV spectrophotometer. 2. Non-conjugated carbonyl group compound give a weak absorption band in the 200 - 300 nm region.
  39. 39. CHROMOPHORE e.g. Acetone which has λmax = 279 nm and that cyclohexane has λmax = 291 nm. When double bonds are conjugated in a compound λmax is shifted to longer wavelength. e.g. 1,5 - hexadiene has λmax = 178 nm 2,4 - hexadiene has λmax = 227 nm CH3 C CH3 O O CH2 CH2 CH3 CH3
  40. 40. CHROMOPHORE 3. Conjugation of C=C and carbonyl group shifts the λmax of both groups to longer wavelength. e.g. Ethylene has λmax = 171 nm Acetone has λmax = 279 nm Crotonaldehyde has λmax = 290 nm CH3 C CH3 O CH2 CH2 C CH3 O CH2
  41. 41. 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.
  42. 42. AUXOCHROME e.g. Benzene λmax = 255 nm Phenol λmax = 270 nm Aniline λmax = 280 nm OH NH2
  43. 43. ABSORPTION AND INTENSITY SHIFTS
  44. 44. • When absorption maxima (λmax) 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, -OCH3 causes absorption of compound at longer wavelength. • Bathochromic Shift (Red Shift)1
  45. 45. • In alkaline medium, p-nitrophenol shows red shift. p-nitrophenol λmax = 255 nm λmax = 265 nm • Bathochromic Shift (Red Shift)1 OH N + O - O OH - Alkaline medium O - N + O - O
  46. 46. • When absorption maxima (λmax) 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
  47. 47. • Aniline shows blue shift in acidic medium, it loses conjugation. Aniline λmax = 280 nm λmax = 265 nm • Hypsochromic Shift (Blue Shift)2 NH2 H + Acidic medium NH3 + Cl -
  48. 48. • 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 2-methyl pyridine λmax = 257 nm λmax = 260 nm ε = 2750 ε = 3560 • Hyperchromic Effect3 N N CH3
  49. 49. • When absorption intensity (ε) of a compound is decreased, it is known as hypochromic shift. Naphthalene 2-methyl naphthalene ε = 19000 ε = 10250 CH3 • Hypochromic Effect4
  50. 50. Wavelength ( λ ) Absorbance(A) SHIFTS AND EFFECTS Hyperchromic shift Hypochromic shift Red shift Blue shift λmax
  51. 51. WOODWARD-FEISER RULE  Woodward (1941) : gave certain rules for correlating max with molecular structure  Scott-Feiser (1959): modified rule with more experimental data, the modified rule is known as Woodward-Feiser rule  used to calculate the position of max for a given structure by relating the position and degree of substitution of chromophore.
  52. 52. 1. HOMOANNULAR DIENE: CYCLIC DIENE HAVING CONJUGATED DOUBLE BONDS IN THE SAME RING. 2. Heteroannular diene: cyclic diene having conjugated double bonds in different ring
  53. 53. 2. Endocyclic double bond: double bond present in a ring 3. Exocyclic double bond: double bond in which one of the doubly bonded atoms is a part of a ring system Ring A Ring B Ring A has one exocyclic and endocyclic double bond. Ring B has only one endocyclic double bond
  54. 54. WOODWARD-FEISER RULE FOR CONJUGATED DIENES, TRIENES, POLYENES  Each type of diene or triene system is having a certain fixed value at which absorption takes place; this constitutes the BASIC VALUE or PARENT VALUE  The contribution made by various alkyl substituents or ring residue, double bonds extending conjugation and polar groups such as –Cl, -Br are added to the basic value to obtain max for a particular compound
  55. 55. PARENT VALUES AND INCRIMENTS FOR DIFFERENT SUBSTITUENT/GROUPS a) Parent value i. Acyclic conjugated diene and : 215nm heteroannular conjugated diene ii. Homoannular conjugated diene : 253nm iii. Acyclic triene : 245nm
  56. 56. b) INCREMENTS i. Each alkyl substituents or ring residue : 5 nm ii. Exocyclic double bond : 5 nm iii. Double bonds extending conjugation : 30nm c) Auxochrome : -OR : 6 nm -SR : 30 nm -Cl, -Br : 5 nm -NR2 : 60nm -OCOCH3 : 0 nm
  57. 57. CALCULATE MAX FOR 1,4- DIMETHYLCYCLOHEX-1,3-DIENE CH3 CH3 CH3 CH3 Parent value for homoannular ring : 253 nm Two alkyl substituents : 2 * 5= 10 nm Two ring residue : 2 * 5= 10 nm calculated value : =273 nm observed value : = 263 nm
  58. 58. CALCULATE MAX  Parent value for heteroannular diene : 215 nm Four ring residue : 4 * 5 = 20 nm calculated value : 235 nm observed value : 236 nm
  59. 59. CALCULATE MAX  Parent value for heteroannular diene : = 215 nm  Three ring residue : 3 * 5 = 15 nm  One exocyclic double bond : = 5 nm  Calculated value : = 235 nm  Observed value : = 235 nm
  60. 60. WOODWARD-FEISER RULES FOR ,- UNSATURATED CARBONYL COMPOUNDS a) Parent values i. ,-unsaturated acyclic or six membered ring : 215 nm ketone ii. ,-unsaturated five – membered ring ketone : 202nm iii. ,-unsaturated aldehyde : 207 nm b) Increments i. Each alkyl substituent or ring residue at  position : 10 nm at  position : 12 nm at  position : 18 nm
  61. 61. ii. EACH EXOCYCLIC DOUBLE BOND : 5 NM iii. Double bond extending conjugation : 30 nm iv. Homoannular conjugated diene : 39 nm v. Auxochromes position    -OH 35 30 50 -OR 35 30 17 -SR - 85 - -OCOCH3 6 6 6 -Cl 15 12 - -NR2 - 95 -
  62. 62. CALCULATE MAX CH3-C(O)-C(CH3)=CH2 O CH3-C-C= CH2 CH3  Parent value for ,-unsaturated acyclic : 215 nm ketone  one alkyl substituent in  position : 10 nm  calculated value = 225 nm  observed value = 220 nm
  63. 63. CALCULATE MAX  Parent value for ,-unsaturated 6 : 215 nm membered cyclic ketone One ring residue at  position : 10nm Two ring residue at  position : 2* 12 = 24 nm Double bond exocyclic to two ring : 2* 5 = 10nm calculated value : 259nm observed value : 256nm
  64. 64. CALCULATE MAX CH3 C=CH-C-CH3 CH3 C-CH=CH-CH3 CH3  Parent ,-unsaturated acyclic ketone 215  2  alkyl substitute 24  Calculated value 239nm  Parent acyclic conjugated diene 215 2 alkyl subst. 10 2 ring residue 10 Exocyclic double bond 5  Calculated value 240
  65. 65. SOLVENT EFFECTS  The most suitable solvent is one which does not it self absorb in the region under investigation.  A dilute sample solutions is preferred for analysis  Most commonly used solvent is 95% ethanol. It is best solvent as it is cheap, transparent down to 210µm.
  66. 66.  Commercial ethanol should not be used, because it has benzene ring which strongly absorb UV region.  Some other solvents are used which are transparent above 210µm are n-hexane, methyl alcohol, cyclohexane, acetonitrile, diethylether.  Hexane & other hydrocarbons can be used because these are less polar and have least interactions with the molecular under investigations.
  67. 67.  For UV spectroscopy, ethanol, water and cyclohexane serve the purpose best.  The position as well as the intensity of absorption maximum get shifted for a particular chromophore by changing the polarity of the solvent.  By increasing the polarity of the solvent, compounds such as dienes and conjugated hydrocarbons do not experience any appreciable shift.  Hence, in general the absorption maximum for the non-polar compounds is usually shifted with the change in polarity of the solvents. α,β-unsaturated carbonyl compounds show two different shifts.
  68. 68. INSTRUMENTATION Various components of UV spectrometers are as follows:  Radiation source:  Monochromators  Sample cells  Detectors  Readout device
  69. 69. 80 AMPLIFIER DETECTOR RECORDER Radiation source Sample holder Monochromator BASIC COMPONENTS IN UV-VISIBLE SPECTROPHOTOMETERS analysisHow does a spectrophotometer work.mp4
  70. 70. SOURCE OF RADIANT ENERGY: REQUIREMENTS OF AN IDEAL SOURCE  It should be stable and should not allow fluctuations.  It should emit light of continuous spectrum of high and uniform intensity over the entire wavelength region in which it’s used.  It should provide incident light of sufficient intensity for the transmitted energy to be detected at the end of optic path.  It should not show fatigue on continued use.
  71. 71. FOR VISIBLE RADIATION TUNGSTEN HALOGEN LAMP  Its construction is similar to a house hold lamp.  The bulb contains a filament of Tungsten fixed in evacuated condition and then filled with inert gas.  The filament can be heated up to 3000 k, beyond this Tungsten starts sublimating .
  72. 72.  To prevent this along with inert gas some amount of halogen is introduced (usually Iodine).  Sublimated form of tungsten reacts with Iodine to form Tungsten –Iodine complex.  Which migrates back to the hot filament where it decomposes and Tungsten get deposited. DEMERIT: It emits the major portion of its radiant energy in near IR region of the spectrum.
  73. 73. SOURCE FOR UV RADIATION: I.HYDROGEN DISCHARGE LAMP:  In Hydrogen discharge lamp pair of electrodes is enclosed in a glass tube (provided with silica or quartz window for UV radiation to pass trough) filled with hydrogen gas.  When current is passed trough these electrodes maintained at high voltage, discharge of electrons occurs which excites hydrogen molecules which in turn cause emission of UV radiation.
  74. 74. II.DEUTERIUM LAMP:  It’s similar to Hydrogen discharge lamp but instead of Hydrogen gas, Deuterium gas is used. MERIT:  Intensity of radiation is more as compare to Hydrogen discharge lamp. DEMERIT:  Expensive.
  75. 75. III. XENON DISCHARGE LAMP:  It possesses two tungsten electrodes separated by some distance.  These are enclosed in a glass tube with quartz or fused silica and xenon gas is filled under pressure.  An intense arc is formed between electrodes by applying high voltage. This is a good source of continuous plus additional intense radiation. DEMERIT:  The lamp since operates at high voltage becomes very hot during operation and hence needs thermal insulation.
  76. 76. MERCURY ARC LAMP:  In mercury arc lamp, mercury vapour is stored under high pressure and excitation of mercury atoms is done by electric discharge. DEMERIT: Not suitable for continuous spectral studies, because it doesn’t give continuous radiations.
  77. 77. COLLIMATING SYSTEM The radiation emitted by the source is collimated (made parallel) by lenses, mirrors and slits. LENSES:  Materials used for the lenses must be transparent to the radiation being used.  Ordinary silicate glass transmits between 350 to 3000 nm and is suitable for visible and near IR region.  Quartz or fused silica is used as a material for lenses to work below 300nm.
  78. 78. MIRRORS  These are used to reflect, focus or collimate light beams in spectrophotometer.  To minimize the light loss, mirrors are aluminized on their front surfaces.
  79. 79. SLITS:  Slit is an important device in resolving polychromatic radiation into monochromatic radiation.  To achieve this, entrance slit and exit slit are used.  The width of slit plays an important role in resolution of polychromatic radiation.
  80. 80. MONOCHRMATORS : It’s a device used to isolate the radiation of the desired wavelength from wavelength of the continuous spectra. Following types of monochromatic devices are used:
  81. 81. A. FILTERS:  Selection of filters is usually done on a compromise between peak transmittance and band pass width; the former should be as high as possible and latter as narrow as possible. 1. Absorption filters 2. Interference filter
  82. 82. I) Absorption filters: Absorption filters works by selective absorption of unwanted radiation and transmits the radiation which is required.
  83. 83. Selection of absorption filter is done according to the following procedure:  Draw a filter wheel.  Write the color VIBGYOR in clockwise or anticlockwise manner, omitting Indigo.
  84. 84.  If solution to be analyzed is BLUE in color a filter having a complimentary color ORANGE is used in the analysis.  Similarly, we can select the required filter in colorimeter, based upon the color of the solution.
  85. 85.  An Absorption glass filter is made of solid sheet of glass that has been colored by pigments which is dissolved or dispersed in the glass.  The color in the glass filters are produced by incorporating metal oxides like ( Cr, Mn, Fe, Ni, Co, Cu etc.).
  86. 86.  Gelatin filter is an example of absorption filter prepared by adding organic pigments; here instead of solid glass sheets thin gelatin sheets are used. Gelatin filters are not use now days.  It tends to deteriorate with time and gets affected by the heat and moisture. The color of the dye gets bleached.
  87. 87. MERITS:-  Simple in construction  Cheaper  Selection of the filter is easy DEMERITS:-  Intensity of radiation becomes less due to absorption by filters.  Band pass (bandwidth) is more (±20-30nm) i.e. if we have to measure at 400nm; we get radiation from 370-430nm. Hence less accurate results are obtained.
  88. 88. II) Interference filter  Works on the interference phenomenon, causes rejection of unwanted wavelength by selective reflection.  It’s constructed by using two parallel glass plates, which are silvered internally and separated by thin film of dielectric material of different (CaF2, Sio, MgF2) refractive index. These filters have a band pass of 10-15nm with peak transmittance of 40- 60%.
  89. 89. MERITS:-  Provide greater transmittance and narrower band pass (10-15nm) as compare to absorption filter.  Inexpensive  Additional filters can be used to cut off undesired wavelength.
  90. 90. b) PRISM:-  Prism is made from glass, Quartz or fused silica.  Quartz or fused silica is the choice of material of UV spectrum.  When white light is passed through glass prism, dispersion of polychromatic light in rainbow occurs. Now by rotation of the prism different wavelengths of the spectrum can be made to pass through in exit slit on the sample.
  91. 91.  The effective wavelength depends on the dispersive power of prism material and the optical angle of the prism.  There are two types of mounting in an instrument one is called ‘Cornu type’ and its adjusted such that on rotation the emerging light is allowed to fall on exit slit.
  92. 92.  The other type is called “Littrow type”, in which one surface is aluminized with reflected light back to pass through prism and to emerge on the same side of the light source i.e. light doesn’t pass through the prism on other side.
  93. 93. NICHOL PRISM ROCHON PRISM
  94. 94. c) DIFFRACTION GRATINGS:  More refined dispersion of light is obtained by means of diffraction gratings.  These consist of large number of parallel lines ( grooves) about 1500-3000/ inch is ruled on highly polished surface of aluminum.
  95. 95.  These acts as scattering centers for light beam impinging on it. Because of constructive interference, the separation of desired wavelength is accomplished.  The resolved power of grating depends on the number of lines. Generally resolving power of grating is better than that of prism and hence grating is used and is preferred.
  96. 96. Comparison Prism Grating Made of Glass-: Visible Quartz/fused silica-: UV Alkali halide:-IR Grooved on highly polished surface like alumina. Working Principle Angle of Incident Law of diffraction nλ= d (sini±sinθ) Merits/demerits Prisms give non- liner dispersion hence no overlap of spectral order Grating gives liner dispersion hence overlap of spectral order.
  97. 97. Merits/ demerits Prisms are not moisture resistant Prisms are not sturdy long lasting. Expensive Moisture resistant Grating are sturdy and long lasting. Economical.
  98. 98.  SAMPLE HOLDERS/CUVETTES:-  The cells or cuvettes are used for handling liquid samples. The cell may either be rectangular or cylindrical in nature. The cells that may hold the sample must be made of substances which are transparent in the spectral region of interest.  For study in UV region; the cells are prepared from quartz or fused silica.  The internal diameter of the cells is 0.5cm, 1cm, 2cm, or 4cm.
  99. 99.  The cuvettes with lid are used for handling volatile type solvents and solutions.  The surfaces of absorption cells must be kept scrupulously clean. No fingerprints or blotches should be present on cells. Cleaning of the cells is carried out washing with distilled water or with dilute alcohol, acetone.
  100. 100. 5. DETECTORS:-  The light or the intensity of transmitted radiation by a sample is collected on a detector device. Most modern detectors generate an electrical current after receiving the radiation. The generated currents are often amplified and pass on to a meter (a galvanometer or recorder). Requirements of an ideal detector:-  It should give quantitative response.  It should have high sensitivity and low noise level.  It should have a short response time.  It should provide signal or response quantitative in wide spectrum of radiation received.  It should generate sufficient signal or electrical current, which can be measured or easily amplified for detection by meter.
  101. 101. The following types of detectors are employed in instrumentation of absorption spectrophotometer:- (a)Barrier layer cell/Photovoltaic cell (b)Phototubes/Photoemessive tube (c)Photomultiplier tubes
  102. 102. a)Barrier layer cell/Photovoltaic cell CONSTUCTION: It consist of a metallic base plate (A) of Iron or Aluminum (act as one of the electrodes) over which a thin layer of Selenium (B) is deposited (act as semi conducting surface), upon that a fine layer of silver or gold (D) is spread (act as another electrodes) upper layer of which act as collecting ring (E), between the layer of Selenium (B) and Silver (D) is hypothetical barrier layer(C).
  103. 103. WORKING: When a radiant energy is made incident on the selenium layer (B) it results in the excitation of electrons from the same, which passes through hypothetical barrier layer and are collected on collector ring (E), this causes a potential difference between electrodes and if external circuit is complete the current flows, which is the measure of radiant energy falling on the Selenium layer. The current can be amplified and measured.
  104. 104.  (b)Photo Tubes/Photoemessive Tubes: CONSTRUCTION: This consist of spherical shaped vacuum bulb photoemessive cathode and an anode .The inner surface of semi-cylindrical cathode mounted inside the bulb is coated with a photosensitive material like Cesium oxide, Potassium oxide and silver oxide, which emits electrons when irradiated with radiant energy. High potential is maintain across electrodes.
  105. 105. WORKING:  When transmitted radiant energy is made incident upon cathode it causes generation of electrons which flows towards anode via external circuit and photo current results. The number of electrons ejected from the surfaces is directly proportional to the amount of radiant energy striking the anode surface.
  106. 106. (c)Photomultiplier Tube: CONSTRUCTION: In a vacuum tube a photocathode is fixed which receives radiant energy transmitted from the samples. Some 8 to 10 dynodes are fixed each with increasing potential of about 90 volts. Near the last dynode is fixed an anode or an electron collector electrode.
  107. 107.  The transmitted radiant energy strikes the cathode causes generation of electron which get attracted towards dynodes maintained at higher potentials which further causes the generation of electrons more than the previous ones, these steps is continued for 8 to 10 times depending upon the number of dynodes used.  The electron generated by the last dynode gets collected over anode which result in potential imbalance and hence current flows which can be amplified and measured.
  108. 108. 119 SINGLE BEAM SPECTROPHOTOMETER
  109. 109. Radiation Source Entrance Slit Exit Slit Monochromator Sample Cell Detector Galvanometer Readout Device
  110. 110.  In a single beam UV-Visible spectrophotometer, light from the radiation source after passing through a monochromator enters the sample cell containing the sample solution.  A part of the incident light (Io) is absorbed by the sample and remaining gets transmitted (It). 121
  111. 111.  The transmitted light strikes the detector and produces electrical signals.  The signals produce by the detector is directly proportional to the intensity of the beam striking its surface.  The output is measured by a micrometer or galvanometer and displayed on the readout device. 122
  112. 112.  The absorbance readings of both the standard and unknown solutions are recorded after adjusting the instrument to 100% transmittance with a blank solution each time whenever the wavelength is changed. 123
  113. 113.  Advantages;  Simple in construction  Easy to operate  Economical 124
  114. 114. 126 Monochromator C H O P P E R Amplifer Entrance slit Radiation Source Exit Slit Reference cell Sample cell Mirror Mirror Detector Detector Readout Device
  115. 115.  Double beam spectrophotometer allows direct measurement ratio of intensities of sample and reference beams respectively.  The design of a double beam spectrophotometer is similar to single beam spectrophotometer except that it contains a beam slitter or chopper. 127
  116. 116.  Chopper or beam slitter is a device consisting of a circular disc. One third of the disc is opaque, one third is transparent and the remaining one third is mirrored.  The chopper splits the monochromatic beam of the light into two beams of equal intensities. 129
  117. 117.  A double beam spectrophotometer can be designed using one or two detectors. 132 Advantages; •It facilitates rapid scanning over wide wavelength region. •Fluctuations due to radiation source are minimized.
  118. 118.  It makes automatic compensations for variations in the wavelength of the incident light.  It does not required adjustment of the transmittance at 0% and 100% at each wavelength.  It gives the ratio of the intensities of the sample and reference beams simultaneously. 133
  119. 119.  Disadvantages;  Construction is complicated.  Instrument is expensive. 134
  120. 120. 1. Detection of Impurities  UV absorption spectroscopy is one of the best methods for determination of impurities in organic molecules. Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw material. By also measuring the absorbance at specific wavelength, the impurities can be detected. APPLICATIONS OF U.V. SPECTROSCOPY:
  121. 121. U.V. SPECTRA OF PARACETAMOL (PCM)
  122. 122. 2. Structure elucidation of organic compounds.  UV spectroscopy is useful in the structure elucidation of organic molecules, the presence or absence of unsaturation, the presence of hetero atoms.  From the location of peaks and combination of peaks, it can be concluded that whether the compound is saturated or unsaturated, hetero atoms are present or not etc.
  123. 123. 3. QUANTITATIVE ANALYSIS  UV absorption spectroscopy can be used for the quantitative determination of compounds that absorb UV radiation. This determination is based on Beer’s law which is as follows. A = log I0 / It = log 1/ T = – log T = abc = εbc Where : ε -is extinction co-efficient, c- is concentration, and b- is the length of the cell that is used in UV spectrophotometer.
  124. 124. BEER’S LAW
  125. 125. 4. QUALITATIVE ANALYSIS  UV absorption spectroscopy can characterize those types of compounds which absorbs UV radiation. Identification is done by comparing the absorption spectrum with the spectra of known compounds.
  126. 126. U.V. SPECTRA'S OF IBUPROFEN
  127. 127. 5. CHEMICAL KINETICS  Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is passed through the reaction cell and the absorbance changes can be observed.
  128. 128. 6. DETECTION OF FUNCTIONAL GROUPS  This technique is used to detect the presence or absence of functional group in the compound  Absence of a band at particular wavelength regarded as an evidence for absence of particular group
  129. 129. BENZENE TOLUNE
  130. 130. 8. EXAMINATION OF POLYNUCLEAR HYDROCARBONS  Benzene and Polynuclear hydrocarbons have characteristic spectra in ultraviolet and visible region. Thus identification of Polynuclear hydrocarbons can be made by comparison with the spectra of known Polynuclear compounds.  Polynuclear hydrocarbons are the Hydrocarbon molecule with two or more closed rings; examples are naphthalene, C10H8, with two benzene rings side by side, or diphenyl, (C6H5)2, with two bond- connected benzene rings. Also known as polycyclic hydrocarbon.
  131. 131. NAPHTHALENE DIPHENYL
  132. 132. 9. MOLECULAR WEIGHT DETERMINATION  Molecular weights of compounds can be measured spectrophotometrically by preparing the suitable derivatives of these compounds.  For example, if we want to determine the molecular weight of amine then it is converted in to amine picrate. Then known concentration of amine picrate is dissolved in a litre of solution and its optical density is measured at λmax 380 nm.
  133. 133.  After this the concentration of the solution in gm moles per litre can be calculated by using the following formula.
  134. 134. 10. AS HPLC DETECTOR  A UV/Vis spectrophotometer may be used as a detector for HPLC.
  135. 135. REFERENCES: 1. Sharma. Y.R. Elementary Organic Spectroscopy. First edition .S.Chand Publisher; 2010. 2. Chatwal G.R. Instrumental methods of chemical analysis. First edition. Himalaya Publisher; 2010. 3. Instrumental analysis by willard and merit

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