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# Spectrophotometry : Instruments & Applications

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### Spectrophotometry : Instruments & Applications

1. 1. Spectrophotometry :Instruments & ApplicationsBy: A. Fadak, R. Mazahernasab1
2. 2. Content• Introduction• Beer-Lambert law• Instruments• Applications2
3. 3. Introduction• The spectrophotometer technique is to measures lightintensity as a function of wavelength.• It does this by:1. diffracting the light beam into a spectrum of wavelengths2. direct it to an object3. receiving the light reflected or returned from the object4. detecting the intensities with a charge-coupled device5. displaying the results as a graph on the detector and thenthe display device .[1],[2]3
4. 4. Introduction4
5. 5. Introduction• Spectrophotometer:a)Single-beamb) Double-beam[4] 5
6. 6. Introdution• compounds absorb light radiation of a specific wavelength.• the amount of light radiation absorbed by a sample ismeasured.• The light absorption is directly related to the concentration ofthe compound in the sample.• As Concentration increases, light Absorption increases,linearly, As Concentration increases, light Transmissiondecreases, exponentially.[3]6
7. 7. Beer-Lambert law• Light Absorbance: (A) = log (I0 / I)= LCƐ• Light Transmission (T) = I/I0 = 10- CLƐ• I0: Light Intensity entering a sample• I: Light Intensity exiting a sample• C: The concentration of analyte in sample• L: The length of the light path in glass sample cuvette• Ɛ: a constant for a particular solution and wavelength7[5]
8. 8. Instruments• Light source: provide a sufficient of lightwhich is suitable for marking a measurement.• The light source typically yields a high output ofpolychromatic light over a wide range of thespectrum.[4]8
9. 9. Instruments• Monochromator : Accepts polychromatic input lightfrom a lamp and outputs monochromatic light.• Monochromator consists of these parts:I. Entrance slitII. Collimating lens or mirrorIII.Dispersion elementIV.Focusing lens or mirrorV. Exit slit [6]9
10. 10. Instruments• Dispersion devices: A special plate withhundreds of parallel grooved lines.• The grooved lines act to separate the white lightinto the visible light spectrum.10The more linesthe smallerthe wavelengthresolution.[5]
11. 11. Instruments• Focusing devices: Combinations of lenses,slits, and mirrors.• relay and focus light through the instrument.[2]11
12. 12. Instruments• Cuvettes: designed to hold samples forspectroscopic experiments. made of Plastic, glassor optical grade quartz• should be as clear as possible, without impuritiesthat might affect a spectroscopic reading.[2]12
13. 13. Instruments• Detectors: Convert radiant energy (photons)into an electrical signal.The photocell and phototube are the simplestphotodetectors, producing current proportionalto the intensity of the light striking Them .[1],[2]13
14. 14. Instruments• Display devices: The data from a detectorare displayed by a readout device, such as ananalog meter, a light beam reflected on a scale,or a digital display , or LCD .• The output can also be transmitted to acomputer or printer. [3]14
15. 15. Applications1. Concentration measurement– Prepare samples– Make series of standard solutions of known concentrations[4]15
16. 16. Applications− Set spectrophotometer to the λ of maximum lightabsorption− Measure the absorption of the unknown, andfrom the standard plot, read the relatedconcentration[4]16
17. 17. Applications2. Detection of Impurities•UV absorption spectroscopy is one of thebest methods for determination of impurities inorganic molecules. [7]17Additional peaks can beobserved due to impuritiesin the sample and it can becompared with that ofstandard raw material.
18. 18. Applications3. Structure elucidation of organic compounds.•From the location of peaks and combination ofpeaks UV spectroscopy elucidate structure oforganic molecules:othe presence or absence of unsaturation,othe presence of hetero atoms.[7]18
19. 19. Applications4. Chemical kinetics•Kinetics of reaction can also be studied usingUV spectroscopy. The UV radiation is passedthrough the reaction cell and the absorbancechanges can be observed.[7]19
20. 20. Applications5. Detection of Functional Groups•Absence of a band at particular wavelengthregarded as an evidence for absence of particulargroup [5]20
21. 21. Applications6. Molecular weight determination•Molecular weights of compounds can bemeasured spectrophotometrically by preparing thesuitable derivatives of these compounds.•For example, if we want to determinethe molecular weight of amine then it is convertedin to amine picrate. [7]21
22. 22. References[1] The principles of use of a spectrophotometer and its application inthe measurement of dental shades[2003][2] Chapter 11 Spectrophotometer[3] Fundamentals of UV-visible spectroscopy, Tony Owen, 1996[4] UV/Visible Spectrophotometer, Mecasys Co., Ltd. , Mar2006[5] Spectrophotometry FUNDAMENTALS (Chapters 17, 19, 20), Dr. G.Van Biesen, Win2011[6] http://www.bio.davidson.edu[7] UNIT: Spectrophotometry, Clinical Chemistry Lab Manual22