2008-11-191 Chapter Eleven Ultraviolet-Visible Spectrophotometry Spectrophotometry is one of the...

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Transcript of 2008-11-191 Chapter Eleven Ultraviolet-Visible Spectrophotometry Spectrophotometry is one of the...

  • Slide 1
  • 2008-11-191 Chapter Eleven Ultraviolet-Visible Spectrophotometry Spectrophotometry is one of the most widely used methods of analysis in clinical chemistry, hygiene analysis, medicine analysis. It is used to measure the absorption capabilities of certain compounds with reference to wavelengths of light on the ultraviolet and visible light ranges.
  • Slide 2
  • 2008-11-192 High sensitive, can used microanalysis 10 -3 10 -6 mol/L e.g. There is 10 -8 g Fe in 10ml solution, if titrate it using KMnO 4 standard solution (c=2.010 -4 mol/L), only consumed 0.02ml KMnO 4 standard solution.
  • Slide 3
  • 2008-11-193 11-1 Properties of Light 11-2 Absorption of Light * 11-3 The Spectrophotometer 11-4 Typical Analysis Procedures
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  • 2008-11-194 11-1 Properties of Light Light is electromagnetic wave The dual nature of particles and waves *Wavelength unit: nm *Frequency (Hz) unit of frequency is s -1 Hertz: One oscillations per second is called 1Hz 10 6 s -1 is therefore said to be 10 6 Hz, or one megahertz (MHz). * Wavenumber unit: nm -1, m -1, cm -1
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  • 2008-11-195 The relationship between wavelength and frequency c is the speed of 1ight in vacuum. c=2.99792458 10 8 m s -1
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  • 2008-11-196 E=hv = The light with certain wavelength or frequency have definite energy Photons Planck's constant h=6.62617610 -34 Js One mole of photons is called one einstein.
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  • 2008-11-197 Complement Color: The observed color is said to be the complement of the absorbed color. Any substance that absorbs visible light will appear colored white light yellow green orange red purple blue greenish blue Bluish green
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  • 2008-11-198 Table 11-1 Colors of visible light and complementary hue Wavelength Color Color Absorbed/nm absorbed observed 380-420 Violet Green-yellow 420-440 Violet-blue Yellow 440-470 Blue Orange 470-500 Blue-green Red 500-520 Green Purple 520-550 Yellow-green Violet 550-580 Yellow Violet-blue 580-620 Orange Blue 620-680 Red Blue-green 680-780 Purple Green
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  • 2008-11-199 11-2 Absorption of Light IaIa c b = abc Absorbance Lambert Beer, s law The a bsorbance (A) of a sample is the logarithm of the ratio of I o / I.
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  • 2008-11-1910 = abc LambertBeer, s law A- absorbance a - absorptivity (Lg -1 cm -1 ) b - path length (cm) c - mass concentration (g/L) c mass concentration(g/L) concentration (molL -1 ) a absorptivity (L/gcm ) (Lmol -1 cm -1 ) the molar absorptivity
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  • 2008-11-1911 Transmittance (T) is the ratio of I/I o.
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  • 2008-11-1912 T : range 0 ~ 1 l00T : (the percent transmittance) varies between 0 and 100%. When no light is absorbed T=1=100%, P=P 0 and A=0 I= I 0 and A=0
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  • 2008-11-1913 Lambert Beer, s law, commonly called Beer's law: Absorbance is directly proportional to the concentration of light-absorbing species in the sample and the pathlength of the solution. Lambert Beer, s law is strictly valid for purely monochromatic radiation; that is, for radiation consisting of only one wavelength. Monochromatic light
  • Slide 14
  • 2008-11-1914 Absorption spectrum of iron( ) -o-phenanthroline complex Absorption spectrum depicts what wavelengths of light are absorbed by a sample. One can readily see what wavelengths of light are absorbed (peaks). The maximum absorption at 508nm was characteristic of the complex solution. When the concentration of complex increased, the absorption spectrum increased. max
  • Slide 15
  • 2008-11-1915 11-3 The Spectrophotometer Major Components Light source Wavelength Selector monochromator Sample cell Light detector indicator tungsten lamp a grating phototube cuvette scale
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  • 2008-11-1916 A 1 =bc 1 A 2 =bc 2 11-4 Typical Analytical Procedures Direct comparison method Methods and Applications of Spectrophotometry . Direct comparison method
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  • 2008-11-1917 Standard curve AxAx cxcx c 0 c 1 c 2 c 3 c 4 c 5 A 0 A 1 A 2 A 3 A 4 A 5 . Standard Curve Method The graph should be a straight line.
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  • 2008-11-1918 Analytical Procedures 1) Selection of monochromatic light (Choice of Wavelength) 2) Prepare a reagent blank containing all reagents, but with analyte replaced by distilled water. 3) Prepare a series of standards solution and measure their absorbance 4) Establish a calibration curve 5) the unknowns be prepared using the same procedure as for standards solution 6) Calculate the content of unknown
  • Slide 19
  • 2008-11-1919 Analytical Procedures Choice of max Measure their absorbance Prepare solutions Calculate the content of unknown Establish a calibration curve
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  • 2008-11-1920 Example: A sample in a 1.0 cm cell is determined with a spectrometer to transmit 80% light at a certain wavelength. If the absorptivity of this substance at this wavelength is 2.0, 1) what is the concentration of the substance(g/L). 2) what is the absorbance of the substance
  • Slide 21
  • 2008-11-1921 Solution: The percent transmittance is 80%, and so T=0.80 a=2.0 b=1.0 A=abc = - logT = -log0.80 = 0.0969
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  • 2008-11-1922 Problems: 1.The transmittance of a solution is found to be 35.0%. What is the transmittance if the solution is diluted in half? 2.The transmittance of a solution is found to be 85.0% when measured in a cell whose path length is 1.00cm. What is the transmittance if the path length is increased to 4.00 cm ?
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  • 2008-11-1923 3. A colored substance M has an absorption maximum at 520 nm. A solution containing 2.00 mg M per liter has an absorbance of 0.840 using a 2.00 cm cell. The formula weight of M is 150. (a) Calculate the molar absorptivity of M at 520 nm. (b) How many milligrams of M are contained in 25.00 mL of a solution giving an absorbance of 0.250 at 520 nm when measured with a 1.00 cm cell ? 4. The molar absorptivity of benzoic acid (M=122.1gmol - 1 ) in methanol at 275 nm is about 1950 Lmol -1 cm -1. If it is desired to use an absorbance not exceeding 1.25, what is the maximum allowable concentration in gL -1 that can be used in a 2.00 cm cell.