UV Visible Spectrometry
- 2. T = I0 / It Transmittance T is a ratio of intensity of transmitted light to that of the incident light. A = log I0 / It The more general equation can be written as follows: A = log I0 / It = log 1/ T = – log T = abc = εbc The molar absorption coefficient is a measurement of how strongly a substance absorbs light. The larger its value, the greater the absorption Beer's and Lambert's Law
- 4. INSTRUMENTATION Instruments for measuring the absorption of U.V. or visible radiation are made up of the following components; 1. Sources (UV and visible) 2. filter or monochromator 3. Sample containers or sample cells 4. Detector
- 5. Radiation source It is important that the power of the radiation source does not change abruptly over its wavelength range. The electrical excitation of deuterium or hydrogen at low pressure produces a continuous UV spectrum The mechanism for this involves formation of an excited molecular species, which breaks up to give two atomic species and an ultraviolet photon Both Deuterium and Hydrogen lamps emit radiation in the range 160 - 375 nm Quartz windows must be used in these lamps, and quartz cuvettes must be used, because glass absorbs radiation of wavelengths less than 350 nm.
- 6. Various UV radiation sources a. Deuterium lamp b. Hydrogen lamp c. Tungsten lamp d. Xenon discharge lamp e. Mercury arc lamp Various Visible radiation sources a. Tungsten lamp b. Mercury vapour lamp c. Carbonone lamp
- 7. Filters or monochromators 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 Steps Involved a. Polychromatic radiation enters the monochromator through the entrance slit. b. The beam is collimated, and then strikes the dispersing element at an angle. c. The beam is split into its component wavelengths by the grating or prism. d. By moving the dispersing element or the exit slit, radiation of only a particular wavelength leaves the monochromator through the exit slit.
- 8. Sample containers or sample cells 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 The cell holding the sample should be transparent to the wavelength region to be recorded.
- 9. Quartz or fused silica cuvettes are required for spectroscopy in the UV region. Silicate glasses can be used for the manufacture of cuvettes for use between 350 and 2000 nm. The thickness of the cell is generally 1 cm. cells may be rectangular in shape or cylindrical with flat ends.
- 10. Detectors In order to detect radiation, three types of photosensitive devices are a. photovoltaic cells or barrier- layer cell b. phototubes or photo emissive tubes c. photomultiplier tubes
- 11. Photovoltaic cell Also known as barrier layer or photronic cell. It consists of a metallic base plate like iron or aluminium which acts as one electrode. On its surface, a thin layer of a semiconductor metal like selenium is deposited. Then the surface of selenium is covered by a very thin layer of silver or gold which acts as a second collector tube. When the radiation is incident upon the surface of selenium, electrons are generated at the selenium- silver surface and the electrons are collected by the silver. This accumulation at the silver surface creates an electric voltage difference between the silver surface and the basis of the cell.
- 12. Phototubes Also known as photo emissive cells. A phototube consists of an evacuated glass bulb. There is light sensitive cathode inside it. The inner surface of cathode is coated with light sensitive layer such as potassium oxide and silver oxide. When radiation is incident upon a cathode, photoelectrons are emitted. These are collected by an anode. Then these are returned via external circuit. And by this process current is amplified and recorded.
- 13. The photomultiplier tube It consists of a photoemissive cathode , several dynode and an anode. A photon of radiation entering the tube strikes the cathode, causing the emission of several electrons. These electrons are accelerated towards the first dynode then onwards Each original photon has produced 106 - 107 electrons. The resulting current is amplified and measured. Photomultipliers are very sensitive to UV and visible radiation. They have fast response times. Intense light damages photomultipliers; they are limited to measuring low power radiation.
- 15. Single Beam Spectrometer Benefits of Single Beam Design Less number of optical components results in improved light transmission and reduced noise Affords greater sensitivity due to higher light throughput
- 16. Double Beam Spectrometer Benefits of Double Beam Design Correction of absorbance for solvent blank In single beam design there can be fluctuations in lamp energy between the ratio recording measurements. Correction due to fluctuations in stray light, beam intensity variations and electronic noise are applied in real-time
- 18. Absorption of EMR by Organic molecules Absorption of light result in the promotion of an electron from HOMO to LUMO i.e movement of valence shell electron ,typically from a filled bonding MO to unfilled antibonding MO As conjugation increases HOMO-LUMO gap decreases and the position of absorption shift to longer wavelength In saturated alkyl halide energy required decrease with decrease in electronegativity The conjugated system needs lower energy for excitation because energy gap separation is low
- 19. The Influence of Functional Group in absorption peak Absorption peaks are also influenced by functional groups. The functional groups influence the conjugated systems, causing the absorption peaks to appear at longer wavelengths than the peak wavelength of benzene, although they do not go beyond 400 nm and enter the visible region. The colour of organic compounds, then, is influenced more strongly by the size of the conjugated system.
- 20. Chromophore Functional group not in conjugation with any other group is said to be chromophore Generally groups having unsaturated centers or heteroatoms or both NO2,N=O ,C=O ,C=N ,C=S Auxochrome Don’t have selective absorption above 200nm on its own Along Chromophore shift to a longer wavelength and increase in intensity of absorption peak Acidic: −COOH, −OH, −SO3H Basic: −NH2, −NHR, −NR2
- 21. The Relationship Between Conjugated Double Bond Systems and Absorption Peaks This absorption spectra obtained by dissolving compounds in ethanol and analysing the resulting solutions. The concentrations were adjusted so that the absorption intensities of the components were roughly the same. Peak wavelengths tend to be shifted toward the long wavelength region as the conjugated system gets larger With larger conjugated systems, the absorption peak wavelengths tend to be shifted toward the long wavelength region and the absorption peaks tend to be larger.
- 22. Substance Absorption Peak Molar Absorption Coefficient Ethylene(CH2=CH2) 1.3-butadiene Vitamin A β-carotene 180nm 217nm 328nm 450nm 10000 21000 51000 140000 Benzene Naphthalene Anthracene Naphthacene 255nm 286nm 375nm 477nm 180 360 7100 110000
- 23. Solvent Effect Compound peak could be obscured by the solvent peak So a most suitable solvent is one that does not itself get absorbed in the region under investigation. A solvent should be transparent in a particular region. A dilute solution of sample is always prepared for analysis. Most commonly used solvents are as follows. Solvent λ of absorption Water 191 nm Ether 215 nm Methanol 203 nm Ethanol 204 nm Chloroform 237 nm Carbon tetrachloride 265 nm Benzene 280 nm Tetrahydrofuran 220 nm
- 25. Detection of Impurities 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 Benzene appears as a common impurity in cyclohexane. Its presence can be easily detected by its absorption at 255 nm. Applications of Absorption Spectroscopy (UV, Visible)
- 26. Structure elucidation of organic compounds. To check 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.
- 27. Quantitative analysis Quantitative 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 b is the length of the cell that is used in UV spectrophotometer
- 28. Qualitative analysis Identification is done by comparing the absorption spectrum with the spectra of known compounds UV absorption spectroscopy is generally used for characterizing aromatic compounds and aromatic olefins.
- 29. 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. Quantitative analysis of pharmaceutical substances Many drugs are either in the form of raw material or in the form of formulation They can be assayed by making a suitable solution of the drug in a solvent and measuring the absorbance at specific wavelength. Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at the wavelength 284 nm.
- 30. As HPLC detector The presence of an analyte gives a response which can be assumed to be proportional to the concentration. For more accurate results, the instrument's response to the analyte in the unknown should be compared with the response to a standard; as in the case of calibration curve. Three types - Fixed Wavelength - Variable Wavelength - Diode array
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