Lecture 28,29. Spectrometry

Lecture 28,29. Spectrometry - Spectrophotometer...

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Unformatted text preview: Spectrophotometer Spectrophotometer Spectronic 20 http://abacus.bates.edu/~ganderso/biology/resources/spec20.html Daniel C. Harris, Exploring Chemical Analysis, 2nd ed., W.H. Freeman and Company, New York, 2001. Freeman % Transmittance: Transmittance: Fraction of light that goes through the sample. P: Radiant power of the electromagnetic radiation. T= P P0 Daniel C. Harris, Exploring Chemical Analysis, 2nd ed., W.H. Freeman and Company, New York, 2001. Freeman P0 e.g. P = 0.9 P0 % Transmittance: Transmittance: Fraction of light that goes through the sample. P: Radiant power of the electromagnetic radiation. T= P P0 Daniel C. Harris, Exploring Chemical Analysis, 2nd ed., W.H. Freeman and Company, New York, 2001. Freeman % Transmittance Transmittance Daniel C. Harris, Exploring Chemical Analysis, 2nd ed., W.H. Freeman and Company, New York, 2001. Freeman A: Absorbance A: A = -logT Note: Note: A goes up as T goes down. goes Daniel C. Harris, Exploring Chemical Analysis, 2nd ed., W.H. Freeman and Company, New York, 2001. Freeman A: Absorbance A: A = -logT A = -logT Note: Note: A goes up as T goes down. goes T = 90% => A = 0.046 T = 10% => A = 1.00 T = 1.0% => A = 2.00 T = 0.1% => A = 3.00 Typical spectrometers have the highest accuracy at A ≈ Typical 0.3 to 2.0. 0.3 At A=3.00 there is not enough light transmitted to detect At accurately. accurately. Beer’s Law Beer’s A = abc = ε bc abc a: Absorptivity (g-1 cm-1 L) ε : Molar Absorptivity (M-1 cm-1) b: Path Length (cm) c: Concentration (g/L or M) Gary D. Christian, Analytical Chemistry, 6th ed., John Wiley & Sons, Inc., U.S.A., 2004. Calibration Curve Calibration A plot of A versus λ is plot called a spectrum. called Daniel C. Harris, Exploring Chemical Analysis, 3rd ed., W.H. Freeman and Company, New York, 2005. Freeman Absorption and Color Absorption = white light minus absorbed color = white light minus orange white Daniel C. Harris, Exploring Chemical Analysis, 3rd ed., W.H. Freeman and Company, New York, 2005. Freeman Normalized typical human cone cell Normalized cone responses (S, M, and L types) to monochromatic spectral stimuli monochromatic http://en.wikipedia.org/ QuickTimeª and a decompressor are needed to see this picture. = white light minus absorbed color = white light minus orange white Daniel C. Harris, Exploring Chemical Analysis, 3rd ed., W.H. Daniel Freeman and Company, New York, 2005. Freeman Sample Cells Sample A B C • What types of samples would you use cell C for? What • What types of samples would you use cell B for? What Gary D. Christian, Analytical Chemistry, 6th ed., John Wiley & Sons, Inc., U.S.A., 2004. Sample Cells (Cuvets) Sample Biochemists often use plastic cells. “Quartz” cells made Biochemists of SiO2 are often used in chemistry departments. of • For what types of samples would never use plastic cells? cells? (a) … (b) … Gary D. Christian, Analytical Chemistry, 6th ed., John Wiley & Sons, Inc., U.S.A., 2004. Chapter 19: Molecular Spectroscopy Chapter Instrumentation Instrumentation Spectronic 20 http://abacus.bates.edu/~ganderso/biology/resources/spec20.html Daniel C. Harris, Exploring Chemical Analysis, 2nd ed., W.H. Freeman and Company, New York, 2001. Freeman Molecular Spectroscopy Instrumentation Molecular Daniel C. Harris, Exploring Chemical Analysis, 3rd ed., W.H. Freeman and Company, New York, 2005. Freeman Light Sources Light Deuterium Lamp Tungsten Filament D2 + energy → D2* → D’ + D” + hν Ingle and Crouch, Spectrochemical Analysis Ingle Spectrochemical Light Sources Light Lamp output is temperature dependent: You cannot turn them on and measure right away. Daniel C. Harris, Exploring Chemical Analysis, 2nd ed., W.H. Freeman and Company, New York, 2001. Freeman λ Ranges of Common Sources Ranges Douglas A. Skoog and James J. Leary, Principles of Instrumental Douglas Analysis, Saunders College Publishing, Fort Worth, 1992. Analysis Molecular Spectroscopy Instrumentation Molecular Daniel C. Harris, Exploring Chemical Analysis, 3rd ed., W.H. Freeman and Company, New York, 2005. Freeman Wavelength Selectors Wavelength Prisms QuickTimeª and a decompressor are needed to see this picture. QuickTimeª and a decompressor are needed to see this picture. Gary D. Christian, Analytical Chemistry, 6th ed., John Wiley & Sons, Inc., U.S.A., 2004. Prisms Wavelength Selectors Wavelength Gratings QuickTimeª and a decompressor are needed to see this picture. Gary D. Christian, Analytical Chemistry, 6th ed., John Wiley & Sons, Inc., U.S.A., 2004. http://media-2.web.britannica.com/ Interference Interference Daniel C. Harris, Exploring Chemical Analysis, 3rd ed., W.H. Freeman and Company, New York, 2005. Freeman Diffraction Gratings Diffraction Constructive interference when: nλ = a - b nλ = d(sinθ + sinφ ) Daniel C. Harris, Exploring Chemical Analysis, 3rd ed., W.H. Freeman and Company, New York, 2005. Freeman Diffraction Gratings Diffraction Daniel C. Harris, Exploring Chemical Analysis, 3rd ed., W.H. Freeman and Company, New York, 2005. Freeman Molecular Spectroscopy Instrumentation Molecular Daniel C. Harris, Exploring Chemical Analysis, 3rd ed., W.H. Freeman and Company, New York, 2005. Freeman Light Detector (Transducer) Light Photomultiplier Tube Daniel C. Harris, Exploring Chemical Analysis, 2nd ed., W.H. Freeman and Company, New York, 2001. Freeman Molecular Spectroscopy Instrumentation Molecular Daniel C. Harris, Exploring Chemical Analysis, 3rd ed., W.H. Freeman and Company, New York, 2005. Freeman Slit Function Slit Ingle and Crouch, Spectrochemical Analysis Ingle Spectrochemical Slit Function Slit Ingle and Crouch, Spectrochemical Analysis Ingle Spectrochemical Molecular Spectroscopy Instrumentation Molecular Daniel C. Harris, Exploring Chemical Analysis, 3rd ed., W.H. Freeman and Company, New York, 2005. Freeman Why put the monochromator before the sample cell? sample So why is it OK to put the cell directly after the light source in this setup? the Light Detector (Transducer) Light Photodiode Daniel C. Harris, Exploring Chemical Analysis, 3rd ed., W.H. Freeman and Company, New York, 2005. Freeman Beer’s Law Beer’s A = abc = ε bc abc a: Absorptivity (g-1 cm-1 L) ε : Molar Absorptivity (M-1 cm-1) b: Path Length (cm) c: Concentration (g/L or M) Gary D. Christian, Analytical Chemistry, 6th ed., John Wiley & Sons, Inc., U.S.A., 2004. Analyzing Mixtures Analyzing Gary D. Christian, Analytical Chemistry, 6th ed., John Wiley & Sons, Inc., U.S.A., 2004. Analyzing Mixtures Analyzing A = ε Xb[X] + ε Yb[Y] + ε Zb[Z] + … b[X] b[Y] A1 = ε X1b[X] + ε Y1b[Y] b[X] b[Y] A2 = ε X2b[X] + ε Y2b[Y] b[X] b[Y] ( A1eY2 - A2eY1 ) [X] = b(eX1eY2 - eY1eX2 ) ( A2eX1 - A1eX2 ) [Y] = b(eX1eY2 - eY1eX2 ) Gary D. Christian, Analytical Chemistry, 6th ed., John Wiley & Sons, Inc., U.S.A., 2004. Analyzing Mixtures Analyzing A = ε Xb[X] + ε Yb[Y] + ε Zb[Z] + … b[X] b[Y] A1 = ε X1b[X] + ε Y1b[Y] b[X] b[Y] A2 = ε X2b[X] + ε Y2b[Y] b[X] b[Y] ( A1eY2 - A2eY1 ) [X] = b(eX1eY2 - eY1eX2 ) ( A2eX1 - A1eX2 ) [Y] = b(eX1eY2 - eY1eX2 ) Information from how many wavelengths is needed to determine the Information concentrations of 4 compounds in a mixture of 4 compounds? concentrations Analyzing Mixtures Analyzing ( A1eY2 - A2eY1 ) [X] = b(eX1eY2 - eY1eX2 ) ( A2eX1 - A1eX2 ) [Y] = b(eX1eY2 - eY1eX2 ) Gary D. Christian, Analytical Chemistry, 6th ed., John Wiley & Sons, Inc., U.S.A., 2004. Isosbestic Point Isosbestic Sometimes observed when we change the ratio of [X] and [Y] in a Sometimes mixture where [X] + [Y] = constant. mixture E.g.: * … Isosbestic Point Isosbestic Sometimes observed when we change the ratio of [X] and [Y] in a Sometimes mixture where [X] + [Y] = constant. mixture E.g.: * Titration (if we can ignore the volume change caused by E.g.: addition of titrant). addition Isosbestic Point Isosbestic Why is there an isosbestic point? A = ε Xb[X] + ε Yb[Y] = ε b[X] + ε b[Y] = ε b[X] b[Y] b ([X] + [Y] ) ([X] Isosbestic Point Isosbestic Why is there an isosbestic point? It is not impossible but extremely unlikely that three It compounds would have the same ε at any given wavelength. Deviations from Beer’s Law Deviations A = abc = ε bc abc Typically it works, but sometimes… E.g.: Reasons why a calibration curve for E.g.: naphthyl-2-carboxylic acid may not be linear: naphthyl-2-carboxylic •… •… •… •… COOH According to Beer’s Law… According A = abc = ε bc abc …but you must remember to use it right! UV/Vis spectra depend on the solvent; shifts of a few nm UV/Vis are common and multiples of 10 nm and more are possible. are N O Water 95%; Water 50%; EtOH; Propanol; CH2Cl2; EtOH 5% EtOH 50% Toluene 80% CH2Cl2 20% 20% According to Beer’s Law… According A = abc = ε bc abc …but you must remember to use it right! UV/Vis spectra depend on the solvent; shifts of a few nm UV/Vis are common and multiples of 10 nm and more are possible. are A Ethanol/propanol/butanol methanol acetone chloroform N O nm Molecular Molecular Orbitals Daniel C. Harris, Exploring Chemical Analysis, 3rd ed., W.H. Freeman and Company, New York, 2005. Freeman ...
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