U V Visible Spectroscopy

Basic idea, Working and Characterization.

1. UV / Visible Spectroscopy Presented By Nandakumar V.G Mphil student Dept.of Opto electronics. K erala University, Kariavattom .

2. C ontents. Introduction Electromagnetic Radiation Basic Principle Electronic Transitions Instrumentation Interpretations Advantages and Disadvantages Applications Conclusion.

3. Introduction The  history of  spectroscopy  began with  Isaac Newton's  optics experiments (1666–1672). Newton applied the word " spectrum " to describe the  rainbow  of  colors  that combine to form white light and that are revealed when the white light is passed through a prism . During the early 1800s, Joseph von  made experimental advances with dispersive  spectrometers  that enabled spectroscopy to become a more precise and quantitative scientific technique. Since then, spectroscopy has played and continues to play a significant role in  chemistry ,  physics  and  astronomy .

4. 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.

5. Four common spectroscopic techniques use to determine structure: UV/Visible Spectroscopy Infrared Spectroscopy (IR) Nuclear Magnetic Resonance Spectroscopy (NMR) Mass Spectrometry (MS or Mass Spec)

6. Electromagnetic Radiation

7. 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.

8. 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.

9. Electromagnetic Radiation The relationship between wavelength & frequency can be written as: c = ν λ As photon is subjected to energy, so E = h ν = h c / λ

10. Electromagnetic Radiation

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

12. Principles of Spectroscopy

13. 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.

14. Principles of Spectroscopy Absorption Spectroscopy: An analytical technique which concerns with the measurement of absorption of electromagnetic radiation. e.g. UV (185 - 400 nm) / Visible (400 - 800 nm) Spectroscopy, IR Spectroscopy (0.76 - 15 μ m)

15. Principles of Spectroscopy 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

16. Interaction of EMR with Matter

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

18. Interaction of EMR with matter 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.

19. Interaction of EMR with matter Rotational Energy Levels: These energy levels are quantized & discrete. The spacing between energy levels are even smaller than vibrational energy levels. ∆E rotational < ∆E vibrational < ∆E electronic

20. E nergy levels

21. Lambert’s Law

22. Lambert’s Law When a monochromatic radiation is passed through a solution, the decrease in the intensity of radiation with thickness of the solution is directly proportional to the intensity of the incident light. Let I be the intensity of incident radiation. x be the thickness of the solution. Then

23. Lambert’s Law So, Integrate equation between limit I = Io at x = 0 and I = I at x=l, We get,

24. Lambert’s Law Where, Absorbance Absorption coefficient Lambert’s Law

25. Beer’s Law

26. Beer’s Law When a monochromatic radiation is passed through a solution, the decrease in the intensity of radiation with thickness of the solution is directly proportional to the intensity of the incident light as well as concentration of the solution. Let I be the intensity of incident radiation. x be the thickness of the solution. C be the concentration of the solution. Then

27. Beer’s Law So, Integrate equation between limit I = Io at x = 0 and I = I at x=l, We get,

28. Beer’s Law Where, Absorbance Molar extinction coefficient Beer’s Law

29. Beer’s Law OR From the equation it is seen that the absorbance which is also called as optical density (OD) of a solution in a container of fixed path length is directly proportional to the concentration of a solution.

30. Principles of UV - Visible Spectroscopy

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

32. In order to obtain a UV-VIS spectrum the sample is ideally irradiated with the electromagnetic radiation varied over a range of wavelength. A monochromatic radiation i.e., a radiation of a single wavelength is employed at a time. This process is called scanning. The amount of the radiation absorbed at each wavelength is measured and plotted against the wavelength to obtain the spectrum. Thus , a typical UV spectrum is a plot of wavelength or frequency versus the intensity of absorption. 6/22/2015 UV-VISIBLE SPECTROSCOPY 36 CHARECTERISTICS OF SPECTRUM

33. The UV spectra of substances are characterised by two major parameters, namely , the position of the maximum of the absorption band called λmax , and the intensity of the bands. The λmax refers to the wavelength of the most absorbed radiation and is a measure of the difference in the electronic energy levels involved in the transition. The intensity on the other hand is indicative of the probability of the transition i.e., whether the transition is allowed or not . It is also is a measure of the concentration of the absorbing species. 6/22/2015 UV-VISIBLE SPECTROSCOPY 37 CHARECTERISTICS OF SPECTRUM

34. The wavelength of the radiation absorbed by an organic molecule is determined by the difference in energy between the ground state and the various excited electronic states of the molecule . In organic molecules that the constituent atoms are bonded through σ and π bonds. In addition, these have nonbonding electrons on the atoms like , N,O,S and halogens etc. There are a number of transitions possible involving the bonding and the nonbonding electrons. 6/22/2015 UV-VISIBLE SPECTROSCOPY 38 ORGANIC ABSORPTION

35. Electronic Transitions

36. The possible electronic transitions can graphically shown as:

37. The possible electronic transitions are

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

39. π electron in a bonding orbital is excited to corresponding anti-bonding orbital π * . Compounds containing multiple bonds like alkenes, alkynes, carbonyl, nitriles, aromatic compounds, etc undergo π → π * transitions. e.g. Alkenes generally absorb in the region 170 to 205 nm. π → π * transition 2

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

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

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

43. Components of spectrophotometer Source Monochromator Sample compartment Detector Recorder INSTRUMENTATION

44. Block Diagram RADIANT SOURCE WAVELENGTH SELECTOR SOLVENT PHOTO- DETECTOR READOUT SAMPLE Fig.-block diagram of instrumentation of UV-spectrophotometer

45. Tungsten filament lamps and Hydrogen-Deuterium lamps are most widely used and suitable light source as they cover the whole UV region. Tungsten filament lamps are rich in red radiations; more specifically they emit the radiations of 375 nm, while the intensity of Hydrogen-Deuterium lamps falls below 375 nm. Problem- Due to evaporation of tungsten life period decreases. It is overcome by using tungsten-halogen lamp. Halogen gas prevents evaporation of tungsten. RADIATION SOURCE

46. - Monochromators generally composed of prisms and slits. The most of the spectrophotometers are  double beam spectrophotometers . The radiation emitted from the primary source is dispersed with the help of rotating prisms. The various wavelengths of the light source which are separated by the prism are then selected by the slits such the rotation of the prism results in a series of continuously increasing wavelength to pass through the slits for recording purpose. The beam selected by the slit is monochromatic and further divided into two beams with the help of another prism. . Monochromator

47. All Monochromators contain the following component parts; • An entrance slit • A collimating lens • A dispersing device (a prism or a grating) • A focusing lens • An exit slit

48. Filters – a)Glass filters- Made from pieces of colored glass which transmit limited wave length range of spectrum. Wide band width 150nm. b)Gelatin filters- Consist of mixture of dyes placed in gelatin & sandwiched between glass plates. Band width 25nm. c)Inter ferometric filters- Band width 15nm Prisms- -Prism bends the monochromatic light. -Amount of deviation depends on wavelength -They produce non linear dispersion.

49. A variety of sample cells available for UV region. The choice of sample cell is based on a) the path length, shape, size b) the transmission characteristics at the desired wavelength c) the relative expense One of the two divided beams is passed through the sample solution and second beam is passé through the reference solution. Both sample and reference solution are contained in the cells. These cells are made of either silica or quartz. Glass can't be used for the cells as it also absorbs light in the UV region. T he thickness of the cell is generally 1 cm. cells may be rectangular in shape or cylindrical with flat ends. Sample and reference cells -

50. Generally two photocells serve the purpose of detector in UV spectroscopy. One of the photocell receives the beam from sample cell and second detector receives the beam from the reference. The intensity of the radiation from the reference cell is stronger than the beam of sample cell. This results in the generation of pulsating or alternating currents in the photocells. Three common types of detectors are used. Barrier layer cell Photo cell detector Photomultiplier cells DETECTORS

51. Barrier layer cells It consist of flat Cu or Fe electrode on which semiconductor such as selenium is deposited. on the selenium a thin layer of silver or gold is sputtered over the surface.

52. 2.

53. 3. P hotomultiplier Photomultipliers have an internal amplification that gives them great sensitivity and a wide spectral range. Light causes emission of electrons from a photocathode which accelerate past a series of dynodes maintained at progressively increasing potentials.

54. Amplifie r Amplifier- The alternating current generated in the photocells is transferred to the amplifier. The amplifier is coupled to a small servometer . Generally current generated in the photocells is of very low intensity, the main purpose of amplifier is to amplify the signals many times so we can get clear and recordable signals .

55. R ecording Device Recording devices- Most of the time amplifier is coupled to a pen recorder which is connected to the computer. Computer stores all the data generated and produces the spectrum of the desired compound.

56. O ver all arrangements

57. Terms used in UV / Visible Spectroscopy

58. 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. NO 2 , N=O, C=O, C=N, C ≡N, C=C, C=S, etc

59. Chromophore To interpretate UV – visible spectrum following points should be noted: Non-conjugated alkenes show an intense absorption below 200 nm & are therefore inaccessible to UV spectrophotometer. Non-conjugated carbonyl group compound give a weak absorption band in the 200 - 300 nm region.

60. Chromophore e.g. Acetone which has λ max = 279 nm and that cyclohexane has λ max = 291 nm. When double bonds are conjugated in a compound λ max is shifted to longer wavelength. e.g. 1,5 - hexadiene has λ max = 178 nm 2,4 - hexadiene has λ max = 227 nm

61. Chromophore Conjugation of C=C and carbonyl group shifts the λ max of both groups to longer wavelength. e.g. Ethylene has λ max = 171 nm Acetone has λ max = 279 nm Crotonaldehyde has λ max = 290 nm

62. 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.

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

64. Absorption & Intensity Shifts

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

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

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

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

69. 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 λ max = 257 nm λ max = 260 nm ε = 2750 ε = 3560 Hyperchromic Effect 3

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

71. Wavelength ( λ ) Absorbance ( A ) Shifts and Effects Hyperchromic shift Hypochromic shift Red shift Blue shift λ max

72. Solution too concentrated Diluted five-fold

73. Solvent: Ethanol Concentration: 15.4 mg L -1 Pathlength : 1 cm UV-visible spectrum of 4-nitroanaline

74. Advantages and disadvantages

75. Applications of UV / Visible Spectroscopy

76. Applications Qualitative & Quantitative Analysis: It is used for characterizing aromatic compounds and conjugated olefins. It can be used to find out molar concentration of the solute under study. Detection of impurities: It is one of the important method to detect impurities in organic solvents. Detection of isomers are possible. Determination of molecular weight using Beer’s law. Detection of unknown compound.

77. References

78. Reference Books Introduction to Spectroscopy Donald A. Pavia Elementary Organic Spectroscopy Y. R. Sharma Physical Chemistry Puri , Sharma & Pathaniya

79. Resources http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/UV-Vis/spectrum.htm http://en.wikipedia.org/wiki/Ultraviolet%E2%80%93visible_spectroscopy http ://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab1.htm

80. Thank You