Spectrometry

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Spectrometry

  1. 1. SPECTROSCOPY
  2. 2. Introduction of Spectrometric AnalysesThe study how the chemical compoundinteracts with different wavelenghts in a givenregion of electromagnetic radiation is calledspectroscopy or spectrochemical analysis.The collection of measurements signals(absorbance) of the compound as a function ofelectromagnetic radiation is called a spectrum.
  3. 3. Energy AbsorptionThe mechanism of absorption energy is different inthe Ultraviolet, Infrared, and Nuclear magneticresonance regions. However, the fundamentalprocess is the absorption of certain amount of energy.The energy required for the transition from a state of lowerenergy to a state of higher energy is directlyrelated to the frequency of electromagnetic radiationthat causes the transition.
  4. 4. Spectral Distribution of Radiant Energy Wave Number (cycles/cm) X-Ray UV Visible IR Microwave 200nm 400nm 800nm Wavelength (nm)
  5. 5. Electromagnetic Radiation V = Wave Number (cm ) -1 λ = Wave Length C = Velocity of Radiation (constant) = 3 x 1010 cm/sec. υ = Frequency of Radiation (cycles/sec) υ 1 V = = C λThe energy of photon:h (Plancks constant) = 6.62 x 10- (Erg×sec) 27 C C E = h υh = υ= C = υλ λ λ
  6. 6. Spectral Properties, Application and Interactions of Electromagnetic Radiation Wave Wavelength Frequency Energy Number V λ υ Type Type Type Radiation spectroscopy Quantum TransitionKcal/mol eV cm-1 cm Hz9.4 x 107 4.9 x 106 3.3 x 1010 3 x 10-11 1021 Gamma Gamma ray ray Nuclear emission X-ray Electronic9.4 x 103 4.9 x 102 3.3 x 106 3 x 10-7 1017 X-ray absorption, (inner shell) emission Ultra9.4 x 101 4.9 x 100 3.3 x 104 3 x 10-5 1015 violet UV absorption Electronic Visible (outer shell)9.4 x 10-1 4.9 x 10-2 3.3 x 102 3 x 10-3 1013 Infrared IR absorption Molecular vibration Molecular rotation9.4 x 10-3 4.9 x 10-4 3.3 x 100 3 x 10-1 1011 Micro- Microwave wave absorption Magnetically Nuclear induced spin Radio magnetic9.4 x 10-7 4.9 x 10-8 3.3 x 10-4 3 x 103 107 states resonance
  7. 7. Spectrum of Radiation
  8. 8. Dispersion of Polymagnetic Light with a PrismPrism - Spray out the spectrum and choose the certain wavelength(λ) that you want by slit. Infrared monochromatic Ray Red Orange Yellow SLIT Polychromatic PRISM Green Ray Blue Violet Ultraviolet Polychromatic Ray Monochromatic Ray
  9. 9. Ultra Violet SpectrometryThe absorption of ultraviolet radiation by molecules isdependent upon the electronic structure of the molecule.So the ultraviolet spectrum is called electronic spectrum.
  10. 10. Electronic ExcitationThe absorption of light energy by organic compoundsin the visible and ultraviolet region involves thepromotion of electrons in σ, π, and n-orbitals from theground state to higher energy states. This is also calledenergy transition. These higher energy states aremolecular orbitals called antibonding.
  11. 11. Antibonding σ* π * Antibonding σ σ π n→ σ → ππ * → * n→* *Energy n Nonbonding Bonding π Bonding σ
  12. 12. Electronic Molecular Energy LevelsThe higher energy transitions (σ →σ*) occur ashorter wavelength and the low energy transitions(π→π*, n →π*) occur at longer wavelength.
  13. 13. Chromophore is a functional group which absorbs acharacteristic ultraviolet or visible region. UV 210 nm Double Bonds 233 nm Conjugated Diene 268 nm Conjugated Triene 315 nm Conjugated Tetraene • • • • σ σ and * orbitals π π and * orbitals
  14. 14. SpectrophotometerAn instrument which can measure the absorbance of asample at any wavelength.Light Lens Slit Monochromator Slits Sample Detector Quantitative Analysis
  15. 15. FluorometerInstrument to measures the intensity of fluorescent light emitted by a sampleexposed to UV light under specific conditions. Emit fluorescent light Antibonding as energy decreases σ π Antibonding n->σ n-> π n Nonbonding Ground state π −>π π Bonding Energy σ −>σ σ Bonding Electrons molecular energy levels UV Light Source Detector Monochromator Monochromator 90°C Sample
  16. 16. Food Compound H 3C S CH2 CH2 CH3
  17. 17. Chromophore is a functional group which absorbs acharacteristic ultraviolet or visible region. UV 210 nm Double Bonds 233 nm Conjugated Diene 268 nm Conjugated Triene 315 nm Conjugated Tetraene • • • • σ σ and * orbitals π π and * orbitals
  18. 18. Beer – Lambert Law Light I0 I Glass cell filled with concentration of solution (C)As the cell thickness increases, the transmitted intensityof light of I decreases.
  19. 19. R- Transmittance I R= I0 - Original light intensity I0 I- Transmitted light intensity  I% Transmittance = 100 x I0 1Absorbance (A) = Log T I0 = Log = 2 - Log%T I ILog is proportional to C (concentration of solution) and is I0 also proportional to L (length of light path through the solution).
  20. 20. A ∝ CL = ECL by definition and it is called the Beer- Lambert Law.A = ECLA = ECLE = Molar Extinction Coefficient ---- ExtinctionCoefficient of a solution containing 1g molecule ofsolute per 1 liter of solution
  21. 21. Absorbance x Liter E = Moles x cmUNITS A = ECL A = No unit (numerical number only) Liter E = Cm x M ole
  22. 22. L = CmC = Moles/Liter Liter MoleA = ECL = ( )x x Cm Cm x Mole Liter
  23. 23. Steps in Developing a Spectrometric Analytical Method1. Run the sample for spectrum2. Obtain a monochromatic 2.0 wavelength for the maximum Absorbance absorption wavelength.3. Calculate the concentration of 0.0 your sample using Beer Lambert 200 250 300 350 400 450 Wavelength (nm) Equation: A = ECL
  24. 24. Spectrometer Reading
  25. 25. ∆ A Slope of Standard Curve = ∆ C x A at 280 nm 1.0 x 0.5 x 1 2 3 4 5 Concentration (mg/ml)There is some A vs. C where graph is linear.NEVER extrapolate beyond point known wherebecomes non-linear.
  26. 26. Spectrometric Analysis Using Standard Curve 1.2 A at 540 nm 0.8 0.4 1 2 3 4 Concentration (g/l) glucoseAvoid very high or low absorbencies when drawing a standardcurve. The best results are obtained with 0.1 < A < 1. Plot theAbsorbance vs. Concentration to get a straight line
  27. 27. Sample CellsUV Spectrophotometer Quartz (crystalline silica)Visible Spectrophotometer Glass
  28. 28. Light SourcesUV Spectrophotometer 1. Hydrogen Gas Lamp 2. Mercury LampVisible Spectrophotometer 1. Tungsten Lamp
  29. 29. Chemical Structure & UV AbsorptionChromophoric Group ---- The groupings of themolecules which contain the electronic system whichis giving rise to absorption in the ultra-violet region.
  30. 30. Chromophoric StructureGroup Structure nmCarbonyl >C=O 280Azo -N = N- 262Nitro -N=O 270Thioketone -C =S 330Nitrite -NO2 230Conjugated Diene -C=C-C=C- 233Conjugated Triene -C=C-C=C-C=C- 268Conjugated Tetraene -C=C-C=C-C=C-C=C- 315Benzene 261
  31. 31. UV Spectrometer Application Protein Amino Acids (aromatic) Pantothenic Acid Glucose Determination Enzyme Activity (Hexokinase)
  32. 32. Flurometric ApplicationThiamin (365 nm, 435 nm)RiboflavinVitamin AVitamin C
  33. 33. Visible Spectrometer Application Niacin Pyridoxine Vitamin B12 Metal Determination (Fe) Fat-quality Determination (TBA) Enzyme Activity (glucose oxidase)
  34. 34. Practice Examples 1. Calculate the Molar Extinction Coefficient E at 351 nm foraquocobalamin in 0.1 M phosphate buffer. pH = 7.0 from thefollowing data which were obtained in 1 Cm cell. Solution C x 10 M 5 Io I A 2.23 100 27 B 1.90 100 32 2. The molar extinction coefficient (E) of compoundriboflavin is 3 x 103 Liter/Cm x Mole. If the absorbance reading(A) at 350 nm is 0.9 using a cell of 1 Cm, what is theconcentration of compound riboflavin in sample?
  35. 35. 3. The concentration of compound Y was 2 x 10 moles/liter and -4 the absorption of the solution at 300 nm using 1 Cm quartz cell was 0.4. What is the molar extinction coefficient of compound Y? 4. Calculate the molar extinction coefficient E at 351 nm foraquocobalamin in 0.1 M phosphate buffer. pH =7.0 from thefollowing data which were obtained in 1 Cm cell. Solution C x 10 M 5 I0 I A 2.0 100 30
  36. 36. Spectroscopy Homework1. A substance absorbs at 600 nm and 4000 nm. What type of energy transition most likely accounts for each of these absorption processes?2. Complete the following table. [X](M) Absorbance Transmittance(%) E(L/mole-cm) L(cm) 30 2000 1.00 0.5 2500 1.002.5 x 10-3 0.2 1.004.0 x 10-5 50 50002.0 x 10-4 150 [X](M) = Concentration in Mole/L
  37. 37. 3. The molar absorptivity of a pigment (molecular weight 300)is 30,000 at 550 nm. What is the absorptivity in L/g-cm. 4. The iron complex of o-phenanthroline (Molecular weight236) has molar absorptivity of 10,000 at 525 nm. If theabsorbance of 0.01 is the lowest detectable signal, whatconcentration in part per million can be detected in a 1-cmcell?

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