UV visible spectroscopy

Basic spectroscopy, introduction, principle, instrumentation of UV visible spectroscopy

1. UV-VISIBLE SPECTROSCOPY Mr. V. M. Patil Associate Professor & PG Teacher Department of Pharmaceutical Chemistry Ashokrao Mane College of Pharmacy, Peth Vadgaon

2. Spectrum and Spectroscopy Spectrum: (a ) Different colors observed when the white light was dispersed through the prism (b ) The changing of light intensity as a function of frequency Spectra- range of electromagnetic energy separated by wavelength Spectroscopy : Study of spectrum, to identify substances OR the study of the light from an object. Spectrometer- an instrument which spreads out light making a spectra.

3. Photometer: An instrument which measures the ratio (or some function of two) of radiant power of two electromagnetic beams . Spectrophotometer: An instrument measures the ratio (or some function of two) of radiant power of two electromagnetic beams over a large wavelength region . Colorimeter: Any instrument which is used to measure absorption in the visible region.

4. An alternate view of the wave shown above Electromagnetic waves can be imagined as a self-propagating transverse oscillating wave of electric and magnetic fields. Electromagnetic Radiation

5. Wavelength λ : It is the distance between two successive maxima on an electromagnetic wave . 1 Ångstrom = 10 -10  meter. Visible light is sometimes also measured in nanometers 1 nanometer = 10 -9  meter = 10 Ångstroms , so in nanometers, the visible band is from 400 to 800 nanometers . Frequency f / v : units: Hz or Fresnel The no. of wavelength units passing through a given point in unit time is called as frequency. Frequency is measured in units of  hertz  (Hz): 1 hertz = 1 wave crest/second . Wave Number ṽ : unit: cm -1 (Kaiser / K) No. of waves per centimeter in vaccum . ṽ = 1/ λ

6. Velocity V : unit: cm/s , m/s It is the product of wavelength and frequency in the medium. wavelength X frequency = Velocity λ X v = V λ X v = c Relation between frequency, velocity & wavenumber:

7. A triangular prism dispersing a beam of white light. The longer wavelengths (red) and the shorter wavelengths (blue) get separated.

8.   Spectroscopic Techniques and Chemistry they Probe   UV- vis UV-vis region bonding electrons   Atomic Absorption UV-vis region atomic transitions (val. e-) FT-IR IR/Microwave vibrations, rotations Raman IR/UV vibrations   FT-NMR Radio waves nuclear spin states X-Ray Spectroscopy X-rays inner electrons, elemental X-ray Crystallography X-rays 3-D structure  

9.   Spectroscopic Techniques and Common Uses   UV-vis UV-vis region Quantitative analysis/Beer’s Law   Atomic Absorption UV-vis region Quantitative analysis Beer’s Law FT-IR IR/Microwave Functional Group Analysis Raman IR/UV Functional Group Analysis/quant   FT-NMR Radio waves Structure determination X-Ray Spectroscopy X-rays Elemental Analysis X-ray Crystallography X-rays 3-D structure Anaylysis  

10. Spectroscopy Emission Absorption Absorption : A transition from a lower level to a higher level with transfer of energy from the radiation field to an absorber, atom, molecule, or solid. Emission : A transition from a higher level to a lower level with transfer of energy from the emitter to the radiation field. If no radiation is emitted, the transition from higher to lower energy levels is called nonradiative decay. M + h v  M* (absorption 10 -8 sec) M*  M + heat (relaxation process) M*  A+B+C (photochemical decomposition) M*  M + h v ( emission) 13

11. Absorbed/Emitted Colors Pretsch / Buhlmann / Affolter / Badertscher , Structure Determination of Organic Compounds

12. Continuous Spectra Radiation is distributed over all frequencies, not just a few specific frequency ranges.

13. Emission Spectra Pattern of bright spectral lines produced by an element.

14. Absorption Spectra Pattern of dark spectral lines where light within a number of narrow frequency ranges has been removed.

15. The situation in which each of the three types of spectra is observed was summarized in a set of rules by Gustav Kirchoff in the 1860’s. These rules are known as “ Kirchoff’s laws ”.

16. Kirchoff’s Laws 1st law : A luminous solid or liquid, or a sufficiently dense gas, emits light of all wavelengths and produces a continuous spectrum of radiation. 2nd law : A low-density hot gas emits light whose spectrum consists of a series of bright emission lines which are characteristic of the chemical composition of the gas. 3rd law : A cool thin gas absorbs certain wavelengths from a continuous spectrum, leaving dark absorption lines in their place superimposed on the continuous spectrum.

17. Photons Electrons may exist only in orbitals having certain specified energies. Atoms can absorb only specific amounts of energy as their electrons are boosted to excited states; atoms emit only specific amounts of energy when their electrons fall back down to lower energy states. The light absorbed or emitted must be in “packets” of electromagnetic radiation containing a specific amount of energy. These packets are called “ PHOTONS .” The energy of a photon is related to the frequency of the electromagnetic energy absorbed or emitted. E

18. Frequency and Energy The energy of a photon is related to the frequency of light emitted or absorbed by E = hf where h = Planck’s constant Recall that wave speed relates frequency and wavelength: v = f  and for light, c = f  so, E  f or E  1/

19. “Every physicist thinks, he knows what a photon is. But I spent my life to find out what a photon is and I still don’t know.” Albert Einstein

20. Terminology The “normal” condition of an atom is called the ground state . minimum energy configuration of the atom If an orbiting electron is given enough energy to escape the atom, the atom is said to be ionized . Between ground state and ionization, the electron can only exist in certain well-defined excited states . Each excited state has a specific energy ( quantized ). Electrons moving from one energy level to another, absorb or emit an amount of energy equal to the difference between the energy levels. The energy is absorbed or emitted in the form of a photon . The energy of the photon is proportional to frequency.

21. Excitation and Emission If an atom is given energy, electron may jump to a more distant orbit. Atoms do not stay in this energized state long. Electron will fall down to a lower orbit, emitting a photon.

22. Quantitative Spectroscopy Lambert's law stated that absorbance of a material sample is directly proportional to its thickness (path length). Much later,  August Beer  in 1852 stated that absorbance is proportional to the concentrations of the attenuating species in the material sample. The modern derivation of the Beer–Lambert law combines the two laws and correlates the absorbance to both the concentrations of the attenuating species as well as the thickness of the material sample.

23. Beer’s Law A = -logT = log(P /P) = ebc T = P solution /P solvent = P/P Works for monochromatic light Compound x has a unique e at different wavelengths cuvette source slit detector

24. Characteristics of Beer’s Law Plots One wavelength Good plots have a range of absorbances from 0.010 to 1.000 Absorbances over 1.000 are not that valid and should be avoided 2 orders of magnitude

25. Lamberts Law: - dI / dt = kI I t = I o e - kt Beer’s Law: I t = I o e - k’c A = ebc e = molar absorptivity (unique for a given compound at l 1 ) b = path length c = concentration I o / I t = Transmittance (T)/ Opacity I t / I o = Absorbance (A)/ Optical density (D)/ Extinction coefficient (E)

26. Effect of Conjugation 29 Molecular structure or environment [Conjugation (substitution) / Auxochrome or change of solvent]can influence λ max and ε . Shift to longer λ  bathochromic /red e.g. Ethylene (170 nm) 1,3 –butadiene (217 nm) Shift to shorter λ  hypsochromic/ blue e .g. aniline (285 nm) and anilinium ion (254 nm) Increase in ε  hyperchromic effect e.g. pyridine (2750) 2-methyl pyridine (3560) Decrease in ε  hypochromic effect e.g. biphenyl (6540) 3- methyl biphenyl (5970) .

27. Terms describing UV absorptions 1.   Bathochromic shift : shift to longer λ, also called red shift. 2.   Hysochromic shift : shift to shorter λ, also called blue shift. 3.   Hyperchromism : increase in ε of a band. 4 .   Hypochromism : decrease in ε of a band.

28. Chromophore and Auxochrome Transitions in UV –Visible spectroscopy are localized in specific bonds or functional groups within a molecule. Chromophore Any group of atoms that absorbs light whether or not a color is thereby produced. These groups are responsible for electronic transitions. These groups will have a characteristic l max and e . e.g. -C-C-, -C=C-, -C=O-, -NO2 etc. Auxochrome These groups does not absorb radiation but increases wavelength towards longer wavelength and higher intensity. These will increase conjugation and there by increases both l max and e. e.g. -OH, -Br, -NH2. 31

29. ABSORBING SPECIES The absorption of ultraviolet or visible radiation by a molecular species M can be considered to be a two-step process, excitation M + h  M* The lifetime of the excited species is brief (10 -8 to 10 -9 s). Relaxation involves conversion of the excitation energy to heat. M* M + h eat The absorption of ultraviolet or visible radiation generally results from excitation of bonding electrons.

30. Electronic Transitions There are three types of electronic transitions. The three include transitions involving: (1)  ,  , and n electrons (2) d and f electrons (3) charge transfer electrons.

31. Types of Absorbing Electrons The electrons that contribute to absorption by a molecule are: (1) those that participate directly in bond formation between atoms; (2) nonbonding or unshared outer electrons that are largely localized about such atoms as oxygen, the halogens, sulfur, and nitrogen. The molecular orbitals associated with single bonds are designated as sigma (  ) orbitals, and the corresponding electrons are  electrons.

32. Molecular orbital is the non-localized fields between atoms that are occupied by bonding electrons. (when two atom orbitals combine, either a low-energy bonding molecular orbital or a high energy anti-bonding molecular orbital results.) Sigma ( ) orbital The molecular orbital associated with single bonds in organic compounds Pi () orbital The molecular orbital associated with parallel overlap of atomic P orbital. n electrons No bonding electrons associated with hetero atoms like O, N, S, Halogens etc. Orbitals in Molecule 35

33. Sigma and Pi orbitals

34. Absorbing species Electronic transitions p, s, and n electrons d and f electrons Charge transfer reactions p, s, and n (non-bonding) electrons

35. Energy The energies for the various types of molecular orbitals differ significantly. The energy level of a nonbonding electron lies between the energy levels of the bonding and the antibonding  and  orbitals. Electronic transitions among certain of the energy levels can be brought about by the absorption of radiation. Four types of transitions are possible:   *, n  *, n  *, and   *.

36. Energy Levels From the molecular orbital diagram, there are several possible electronic transitions that can occur, each of a different relative energy. s* p s p* n Atomic orbital Molecular orbitals Energy s s p n n s * p * p * s * p * Alkanes e ~ 100-1000 (150 nm) Carbonyls e ~ 10-100 (170 nm) Unsaturated e ~ 1000-10000 (180 nm) O, N, S, halogens e ~ 100-3000 (190 nm) Nitro e ~ 1000-10000 (> 220 nm)

37. UV-Visible λ> 180 nm Vacuum UV or Far UV (λ< 180 nm ) 40

38. Electronic transitions

39. Transitions s->s* UV photon required, high energy Methane at 125 nm Ethane at 135 nm

40. s ® s* Transitions An electron in a bonding s orbital is excited to the corresponding antibonding orbital. The energy required is large. For example, methane (which has only C-H bonds, and can only undergo s ® s* transitions) shows an absorbance maximum at 125 nm. Absorption maxima due to s ® s* transitions are not seen in typical UV-VIS spectra (200 - 700 nm)

41. n-> s* Saturated compounds with unshared e - Absorption between 150 nm to 250 nm e between 100 and 3000 L cm -1 mol -1 Shifts to shorter wavelengths with polar solvents Minimum accessibility Halogens, N, O, S

42. n ® s* Transitions Saturated compounds containing atoms with lone pairs (non-bonding electrons) are capable of n ® s* transitions. These transitions usually need less energy than s ® s * transitions. They can be initiated by light whose wavelength is in the range 150 - 250 nm. The number of organic functional groups with n ® s* peaks in the UV region is small . The molar absorptivities are low to intermediate in magnitude and range between 100 and 3000 L cm-1 mol -1.

43. n-> p* and p- >p* Organic compounds, wavelengths 200 to 700 nm Requires unsaturated groups n-> p* low e (10 to 100) Shorter wavelengths p->p* higher e (1000 to 10000)

44. n ® p* and p ® p* Transitions Most absorption spectroscopy of organic compounds is based on transitions of n or p electrons to the p* excited state. These transitions fall in an experimentally convenient region of the spectrum (200 - 700 nm). These transitions need an unsaturated group in the molecule to provide the p electrons.

45. Instrumentation Fixed wavelength instruments Scanning instruments Diode Array Instruments

46. Fixed Wavelength Instrument LED serve as source Pseudo-monochromatic light source No monochrometer necessary/ wavelength selection occurs by turning on the appropriate LED 4 LEDs to choose from photodyode sample beam of light LEDs

47. Scanning Instrument cuvette Tungsten Filament (vis) slit Photomultiplier tube monochromator Deuterium lamp Filament (UV) slit Scanning Instrument

48. Diode array Instrument cuvette Tungsten Filament (vis) slit Diode array detector 328 individual detectors monochromator Deuterium lamp Filament (UV) slit mirror

49. Advantages/disadvantages Scanning instrument High spectral resolution (63000), l / Dl Long data acquisition time (several minutes) Low throughput Diode array Fast acquisition time (a couple of seconds), compatible with on-line separations High throughput (no slits) Low resolution (2 nm)

50. Equipment Left: Equipment diagram Right: Schematic diagram

51. Instrumentation Light source Deuterium lamps Hydrogen lamps Tungston filament lamp Xe arc lamps Sample containers Cuvettes Plastic Glass Quartz Filters / Monochromators Detectors Readout devices

52. Light Source Deuterium Lamps － a truly continuous spectrum in the ultraviolet region is produced by electrical excitation of deuterium at low pressure. (160nm~375nm) Over time, the intensity of light from a deuterium arc lamp decreases steadily. Such a lamp typically has a half-life (the time required for the intensity to fall to half of its initial value) of approximately 1,000 h.

53. Wavelength range : 160 – 325 nm Intensity spectrum of Deuterium lamp

54. Tungsten Filament Lamps － the most common source of visible and near infrared radiation . Range － 400 – 750 nm Consists of piece of tungsten wire heated in a controlled atmosphere. Drawback- Max. radiant energy emitted in the near IR region, only 15% in the visible region at an operating temp. above 2725 C & at 1725 C only 1%. Above 2850 C increases total energy output & shifts the wavelength of max intensity to shorter wavelength.

55. Tungsten Filament Lamp The tungsten-halogen lamp, yields good intensity over part of the UV spectrum and over the entire visible range. This type of lamp has very low noise and low drift and typically has a useful life of 10,000 h.

56. Intensity spectrum of Tungsten Filament Lamp

57. Xenon Lamp : It yields a good continuum over the entire UV and visible regions. However, because the noise from currently available xenon lamps is significantly worse than that from deuterium or tungsten lamps, xenon lamps are used only for applications such as diffuse reflectance measurements, in which high intensity is the primary concern.

58. Xe Lamp (250 – 1000 nm) Intensity spectrum of Xe Lamp

59. Light Source

60. Sample holder French cuvette = "little vessel“ Visible ; can be plastic or glass UV ; must use quartz

61. There are several different types of cuvettes commonly used; each type has different usable  wavelengths  at which its transparency exceeds 80%: Optical  Glass , has an optical wavelength range of 340-2,500nm in which it transmits over 80% along with a matching tolerance of 1% at 350 nm . Plastic , with a wavelength from 380 to 780  nm  (visible spectrum). Fused quartz , with a wavelength below 380  nm  ( ultraviolet spectrum ). UV  quartz has an operational wavelength range of 190-2,500  nm , and a matching tolerance of 1% at 220  nm .

62. Filters : Absorption filters: solid sheet of glass coloured by appropriate pigment / dyed gelatin Interference filters: semitransperant metal film deposited on glass plate then coated with thin layer of dielectric material MgF 2

63. Monochromators : Prism: Normal glass for visual & Quartz for UV Grating: Replica gratings; glass plate coated with alumina; - 15000-30000 lines per square inch are made for UV & visible regions. - made by drawing lines on a glass with a diamond stylus

64. Monochromators These are now superseded by: Diffraction gratings: - made by drawing lines on a glass with a diamond stylus ca. 20 grooves mm -1 for far IR ca. 6000 mm -1 for UV/ vis - can use plastic replicas in less expensive instruments Think of diffraction on a CD http://www.mrfiber.com/images/cddiffract.jpg http://www.ii.com/images/prism.jpg http://www.veeco.com/library/nanotheater_detail.php?type=application&id=331&app_id=34 10 m mx10 m m

65. Monochromators : cont’d Polychromatic radiation enters Second concave mirror focuses each wavelength at different point of focal plane Orientation of the reflection grating directs only one narrow band of wavelengths to exit slit The light is collimated the first concave mirror Reflection grating diffracts different wavelengths at different angles What is the purpose of concave mirrors?

66. Monochromator Czerny-Turner design

67. Grating

68. Monochromators: reflection grating Each wavelength is diffracted off the grating at a different angle Angle of deviation of diffracted beam is wavelength dependent  diffraction grating separates the incident beam into its constituent wavelengths components Groove dimensions and spacings are on the order of the wavelength in question In order for the emerging light to be of any use, the emerging light beams must be in phase with each other Resolution of grating: l Dl =nN Angular resolution: As: d sin( q )+d sin( f )=n l So: n Dl =d cos( f ) Df Therefore: Df/Dl =n/[d cos( f )] What does this mean? n: diffraction order N: number of illuminated groves

69. Components of Optical Instruments Detectors

70. Barrier Layer/Photovoltaic Detector   Principle : This device measures the intensity of photons by means of the voltage developed across the semiconductor layer. Electrons, ejected by photons from the semiconductor, are collected by the silver layer. The potential depends on the number of photons hitting the detector.

71. Barrier Layer/ Photovoltaic Detector

72. Phototube Detector     Principle: This detector is a vacuum tube with a cesium-coated photocathode. Photons of sufficiently high energy hitting the cathode can dislodge electrons, which are collected at the anode. Photon flux is measured by the current flow in the system.

73. Phototube Detector  

74. Photomultiplier Detector : The type is commonly used. The detector consists of a photoemissive cathode coupled with a series of electron-multiplying dynode stages, and usually called a photomultiplier. The primary electrons ejected from the photo-cathode are accelerated by an electric field so as to strike a small area on the first dynode.

75. Principle : The impinging electrons strike with enough energy to eject two to five secondary electrons, which are accelerated to the second dynode to eject still more electrons. A photomultiplier may have 9 to 16 stages, and overall gain of 10 6 ~10 9  electrons per incident photon.

76. Photomultiplier Detector 

77. Schematic Single Beam Spectrophotometer

78. Single Beam Spectrophotometer

79. Schematic Double Beam Spectrophotometer

80. Double Beam Spectrophotometer

81. Time separated double beam

82. HPLC-UV Mobile phase HPLC Pump syringe 6-port valve Sample loop HPLC column UV detector Solvent waste

83. Questions…?