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Ultraviolet (uv) and visible spectroscopy ppt

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Instrumentation and principle of Ultraviolet (uv) and visible spectroscopy

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Ultraviolet (uv) and visible spectroscopy ppt

  1. 1. Ultraviolet (UV) and Visible Spectroscopy HemaT MSc Biochemistry Bharathiar University 2018-2020
  2. 2. What is Spectroscopy? ■ Spectroscopy is a necessary tool for structure determination. ■ Organic chemists use spectroscopy as a necessary tool. ■ The first spectroscope was invented in 1859 by the German chemist Robert Wilhelm Bunsen and the German physicist Gustav Robert Kirchhoff. ■ Spectroscopy may be defined as the study of the quantized interaction of electromagnetic radiations with matter.
  3. 3. Difference between spectrometer and spectrophotometer: ■ SPECTROMETER: An optical spectrometer ( spectrograph or spectroscope) is an instrument used to measure properties of light over a specific portion of the electromagnetic spectrum, typically used in spectroscopic analysis to identify materials. ■ SEPCTROPHOTOMETER: A spectrophotometer is an analytical instrument used to quantitatively measure the transmission or reflection of visible light, UV light or infrared light.
  4. 4. What is electromagnetic spectrum? ■ Electromagnetic spectrum covers a very wide range of electromagnetic radiations from cosmic rays to radio waves at the other end. ■ The arrangement of all types of electromagnetic radiations in order of their wavelengths or frequencies is known as complete electromagnetic spectrum.
  5. 5. Ultraviolet (UV) andVisible Spectroscopy ■ Ultraviolet (UV) andVisible Spectroscopy deals with the recording of the absorption of radiations in the ultraviolet and visible regions of the electromagnetic spectrum. ■ The ultraviolet region extends from 10 to 400 nm. ■ Near ultraviolet (quartz) region (200- 400 nm) ■ Far or vacuum ultraviolet region (10- 200nm) ■ The visible region extends from 400 to 800 nm.
  6. 6. Ultraviolet (UV) and Visible Spectroscopy
  7. 7. Principle of UV-Vis spectroscopy ■ UV-Visible spectroscopy follows the Beer- Lambert Law. ■ Beer’s Law The intensity of a beam of monochromatic light decreases exponentially with the increase in concentration of the absorbing substance. ■ Lambert’s Law When a beam of light is allowed to pass through a medium, the rate of decrease of intensity with the thickness of medium is directly proportional to the intensity of the light. A= Ecl
  8. 8. Instrumentation ■ Radiation source: Hydrogen- discharge lamp is the most commonly used source of radiation in the UV region. A deuterium- discharge lamp is used in its place when more intensity is desired. ■ Monochromator: It disperses the radiations obtained from the source into their separate wavelengths. – Prism – Grating made up of quartz ■ Detectors: These have photocells or photo multiplier tubes which generate voltage proportional to the radiation energy that strikes them.
  9. 9. ■ Amplifier : The spectrometer has balancing electronic amplifier which subtracts the absorption of the solvent from that of the solution electronically. ■ Recorder : It automatically records the spectrum as a plot of wavelengths of absorbed radiations against absorbance or molar absorptivity.
  10. 10. Sample handling ■ UV- visible spectra are usually recorded either in very dilute solutions or in the vapour phase. ■ The sample is dissolved in some suitable solvent which does not itself absorb radiation in the region under investigation. ■ Commonly used solvents are cyclohexane, 1, 4- dioxane, water and 95% ethanol. ■ The chosen solvent should be inert to the sample.
  11. 11. Theory of U-VVisible Spectroscopy ■ U-V visible absorption spectra originate from electronic transitions within a molecule. ■ These transitions involving promotion of valence electrons from the ground state to the higher-energy state(excited state) are called electronic excitations and are caused by the absorption of radiation energy in the UV-visible regions of the electromagnetic spectrum. ■ Since various energy levels of molecules are quantized, a particular electronic excitation occurs only by the absorption of specific wavelength of radiation corresponding to the required quantum of energy.
  12. 12. ElectronicTransitions ■ According to molecular orbital theory, the excitation of a molecule by the absorption of radiation in the UV-visible regions involves promotion of its electrons from a bonding, or non bonding (n)orbital to an antibonding orbital. ■ σ - σ * transition The transition or promotion of an electron from a bonding sigma orbital to the associated antibonding sigma orbital is σ - σ * transition. It is a high energy process because σ bonds are generally very strong. ■ n - σ * transition Transition or promotion of an electron from a non-bonding orbital to an antibonding sigma orbital is designated as n - σ * transition. Compounds containing non bonding electrons on a heteroatom are capable of absorption due to n - σ *Transitions.These transitions require lower energy than σ- σ* transitions
  13. 13. ■ π- π* transition The transition or promotion of an electron from a π bonding orbital to a π antibonding orbital is designated π- π* transition.These type of transitions occur in compounds containing one or more covalently unsaturated groups like C=C,C=O,NO2 etc., π- π*Transitions require lower energy than n - σ * transitions. ■ n - π* transition The transition or promotion of an electron from a non-bonding orbital to a π antibonding orbital is designated n - π*.This transition reqires lowest energy.
  14. 14. Relative energies of electronic transitions
  15. 15. Formation of Absorption Bands ■ Since the energy required for each electronic transition is quantized, the UV-visible spectrum is expected to exhibit a single, discrete line corresponding to each electronic transition. ■ Broad absorption bands are usually absorbed.
  16. 16. Designation of Absorption Bands ■ UV-visible absorption bands may be designated by the type of electronic transition from which they originate. ■ K-Bands: These bands originate from π- π* transitions in compounds having π- π* conjugated system. ■ R-Bands:These bands originate from n - π* transitions of a single chromophoric group. ■ B-Bands:These bands originate from π- π* transitions in aromatic or heteroaromatic compounds. ■ E-Bands: Similar to B-Bands , these are characteristic of aromatic and heteroaromatic compounds and originate from π- π* transitions of the ethylenic bonds present in the aromatic ring.
  17. 17. Absorption and Intensity Shifts ■ Bathochromic Shift or Effect. The shift of an absorption maximum to a longer wavelength due to the presence of an auxochrome or solvent effect is called a bathochromic shift or red shift. ■ Hypsochromic Shift or Effect. The shift of an absorption maximum to a shorter wavelength is called hypsochromic or blue shift.This is caused by the removal of conjunction or change in the solvent polarity. ■ Hyperchromic Effect: An effect which leads to an increase in absorption intensity Emax is called hyperchromic effect.The introduction of an auxochrome usually causes hyperchromic shift. ■ Hypochromic Effect: An effect which leads to a decrease in absorption intensity Emax is called hypochromic effect.This is caused by the introduction of a group which distorts the chromophore.
  18. 18. Applications of Ultraviolet andVisible Spectroscopy ■ Detection of a functional group (Chromophore). ■ Detection of Conjugation and Elucidation of its nature. ■ Study of Extent of Conjugation. ■ Distinction between Conjugated and Unconjugated Compounds. ■ Study of Strain. ■ Determination of Configurations of Geometrical isomers. ■ Study ofTautomerism. ■ Confirmation of Suspected Phenols and Aromatic Amines. ■ Study of Structural Features in Different Solvents.
  19. 19. Thank You