1. Chapter 19 Spectrophotometry : Instruments & Application

2. 19.1 The Spectrophotometer –1 Remote sensing of airborne bacteria: Optical fiber coated with antibodies to detect spores of a specific bacterium

3. 19.1 The Spectrophotometer -1 1)Spectrophotometer a) Single-beam b) Double-beam

4. 19.1 The Spectrophotometer -2

5. 19.1 The Spectrophotometer -3

6. 19.1 The Spectrophotometer –4 1) Light source a.Tungsten lamp: Vis. near IR (320 nm~2500 nm) b.Deuterium are lamp: UV (200~400 nm) c.Electric discharge lamp + Hg (g) or Xenon: Vis & UV d.Globar (silicon carbide rod): IR (5000~200 cm -1 ) e.Laser: intense monochromatic sources.

7. 19.1 The Spectrophotometer -5

8. 19.1 The Spectrophotometer -6 2) Monochromator consists: (1) lenses or mirrors: focus the radiation (2) entrance and exit slits: restrict unwanted and control the spectral purity of radiation. (3) dispersing medium: separate the of polychromatic radiation from the source. (a) prism and (b) diffraction grating see Fig 19-2

9. 19.1 The Spectrophotometer -7 a.entrance slit b.collimating mirror or lens c.a prism or grating d.focal plane e.exit slit 2) Monochromator

10. 19.1 The Spectrophotometer -8 Interference of adjacent waves that are (a) 0°, (b) 90 °, and © 180 ° out of phase 2) Monochromator

11. 19.1 The Spectrophotometer -9 nλ = a – b (n =  1 first-order….) Grating equation : nλ = d (sin  – sin  ) Filters: select a desired wavelength 2) Monochromator

12. 19.1 The Spectrophotometer -10 Choosing the bandwidth: exit slit width Resolution Signal 2) Monochromator trade-off

13. 19.1 The Spectrophotometer -10 3)Detector Convert radiant energy (photons) into an electrical signal Ideal detector : high sensitivity, high signal/noise, constant response for λs, and fast response time.

14. 19.1 The Spectrophotometer -11 Detector response depends on the λ of the incident photons. 3) Detector

15. 19.1 The Spectrophotometer -12 Photomultiplier tube: very sensitive detector

16. 19.1 The Spectrophotometer -13 vs. Dispersive spectrophotometer: one  at a time speed (~1s/spetrum) excellent λ repeatability measure λs simultaneously relatively insensitive to errors from stray light relatively poor resolution (1~3 nm) vs 0.1nm Photodiode array spectrophotometer : records the entire spectrum at once.

17. 19.1 The Spectrophotometer -14 diode array spectrophotometer

18. 19.2 Analysis of a mixture -1 1)Absorbance of a mixture : A = e x b[X] + e y b[Y] + …

19. 19.2 Analysis of a mixture -2 2)Isosbestic points : for rxn: X  Y, every spectrum recorded during chemical reaction will cross at the same point. Good evidence for only two principle species in rxn. Ex: HIn  In - + H +

20. 19.2 Analysis of a mixture -3 Why isosbestic point?

21. 19.3 Spectrophotometric Titrations -1 apotransferrin + 2Fe 3+  (Fe 3+ ) 2 transferrin colorless red (465 nm)

22. Ferric nitrilotriacetate [used to avoid Fe(OH)3  ] 19.3 Spectrophotometric Titrations-2

23. Correcting A for the effect of dilution 125 μL ferric nitrilotriancetate 2 mL apotransferrin A = 0.260 A corrected = ? 19.3 Spectrophotometric Titrations-3 ex.at p.408

24. 19.4 What happens when a molecule absorbs light ? 1)Absorbing species : M + hν  M* (lifetime : 10 -8 ~ 10 -9 sec) Relaxation processes : a)M*  M + heat (most common) b)M*  new species (photochemical reaction) c)M*  M + h (fluorescence, phosphorescence)

25. 19.4 Geometry of formaldehyde

26. 2)Types of absorbing electrons Consider formaldehyde: three types of molecular orbitals 19.4 What happens when a molecule absorbs light ?

27. 19.4 What happens absorbs light ? MO of CH 3 CO

28. Four types of electronic transitions σ*σ* π*π* σ π n E < 125 nm 150~250 nm 200~700 nm 19.4 What happens when a molecule absorbs light ?

29. 3)Singlet / Triplet excited states E: T 1 < S 1 ground singlet state excited singlet (S 1 ) excited triplet (T 1 ) 19.4 What happens when a molecule absorbs light ? -5

30. 4)Electronic transition of formaldehyde n   * (T 1 ), absorption of light at λ = 397 nm green-yellow n   * (S 1 ), absorption of light at λ = 355 nm colorless (more probable) 19.4 What happens when a molecule absorbs light ? -6

31. 19.4 What happens when absorbs light ? 5)Vibrational & Rotational states of CH 3 CO (IR and microwave radiation)

32. 6)What happens to absorbed energy 19.4 a molecule absorbs light

33. 7) Luminescence procedures : emission spectrum of M* provides information for qualitative or quantitative analysis. Photoluminescence : a)Fluorescence : S 1  S 0, no change in electron spin. (< 10 -5 s) b)Phosphorescence : T 1  S 0, with a change in electron spin. (10 -4 ~10 2 s) ‚Chemiluminescence : Chemical reaction (not initiated by light) release energy in the form of light. ex : firefly. 19.4 a molecule absorbs light

34. 7)In which your class really shines ? emission spectrum 19.4 a molecule absorbs light

35. 8)Absorption & Emission Spectra

36. 19.5 Luminescence in analytical chemistry 1) Instrument .hν out (photon) ‚heat ƒbreaking a chemical bond hν in

37. 19.5 Luminescence 2) I = kP o C  incident radiation sensitivity  by P 0  or C  3)more sensitive than Absorption

38. 4) Fluorimetric Assay of Selenium in Brazil Nuts –Se is a trace element essential to life: destruct ROOH (free radical) –Derivatized: –Self-absorption: quench 19.5 Luminescence

39. 5) Immunoassarys  employ anitbody to detect analyte. Ex: ELISA

40. 19.5 Luminescence a.pregnancy test. sensitive to < 1 ng of analyte b.Enviromental Analysis. (ppm) or (ppt) pesticides, industrial chemicals, & microbialtoxins.

41. Environmental Analysis 19.5 Luminescence

42. 19-14

43. 19-15

44. 19-18

45. 19-21

46. 19-22