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
b) Single-beam
e) Double-beam

4. 19.1 The Spectrophotometer -2

5. 19.1 The Spectrophotometer -3

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

7. 19.1 The Spectrophotometer -5

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

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

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

11. 19.1 The Spectrophotometer -9
• Monochromator
nλ = a – b (n = ±1 first-order….)
Grating equation : nλ = d (sinθ – sinφ )
Filters: select a desired wavelength

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

13. 19.1 The Spectrophotometer -10
1) 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
3) Detector
Detector response depends on the λ of the
incident photons.

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

16. 19.1 The Spectrophotometer -13
Photodiode array spectrophotometer :
records the entire spectrum at once.
vs. Dispersive spectrophotometer: one λ 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

17. 19.1 The Spectrophotometer -14
diode array spectrophotometer

18. 19.2 Analysis of a mixture -1
• Absorbance of a mixture :
A = exb[X] + eyb[Y] + …

19. 19.2 Analysis of a mixture -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?
A 465 = ε 465 [ HIn ]
HIn
[ ]
A 465 = ε 465 In −
In −
when [ HIn ] = [ In ] ⇒ ε
− 465
HIn = ε 465 = ε 465
In −
∴ For a mixture :
HIn In −
[ ]
A 465 = ε 465 b [ HIn ] + ε 465 b In −
= ε 465 b ( [ In ] + [ HIn ] )
− −

21. 19.3 Spectrophotometric Titrations -1
apotransferrin + 2Fe3+  (Fe3+)2transferrin
colorless red (465 nm)

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

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

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

25. 19.4
• Geometry of
formaldehyde

26. 19.4 What happens when a molecule
absorbs light ?
• Types of absorbing electrons
Consider formaldehyde: three types of
molecular orbitals
• =σ
H
C O ×=π
H
=n

27. 19.4 What happens absorbs light ?
MO of CH3CO

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

29. 19.4 What happens when a molecule
absorbs light ? -5
1) Singlet / Triplet excited states
ground excited excited
singlet state singlet (S1) triplet (T1)
E: T1 < S1

30. 19.4 What happens when a molecule
absorbs light ? -6
1) Electronic transition of formaldehyde
n → π* (T1), absorption of light at λ = 397 nm
green-yellow
n → π* (S1), absorption of light at λ = 355 nm
colorless (more probable)

31. 19.4 What happens when absorbs
light ?
• Vibrational &
Rotational
states of
CH3CO
(IR and microwave
radiation)

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

33. 19.4 a molecule absorbs light
7) Luminescence procedures : emission
spectrum of M* provides information for
qualitative or quantitative analysis.
 Photoluminescence :
• Fluorescence : S1 → S0,
no change in electron spin. (< 10-5 s)
• Phosphorescence : T1 → S0,
with a change in electron spin. (10-4~102 s)
b. Chemiluminescence :
Chemical reaction (not initiated by light) release
energy in the form of light. ex : firefly.

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

35. 19.4 a molecule absorbs light
1) Absorption &
Emission Spectra

36. 19.5 Luminescence in
analytical chemistry
1) Instrument
• .hνout (photon)
• heat
hνin • breaking a
chemical bond

37. 19.5 Luminescence
• I = kPoC

incident radiation
sensitivity  by P0  or C 
6) more sensitive than Absorption

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

39. 19.5 Luminescence
5) Immunoassarys
 employ anitbody
to detect analyte.
Ex: ELISA

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

41. 19.5 Luminescence
• Environmental
Analysis

42. 19-14

43. 19-15

44. 19-18

45. 19-21

46. 19-22