This document discusses spectrophotometry and luminescence techniques. It describes the basic components of a spectrophotometer including the light source, monochromator, and detector. The monochromator uses prisms or diffraction gratings to separate wavelengths and can be used to measure absorption across the spectrum. Luminescence techniques like fluorescence and phosphorescence can provide information about electronic transitions and are used in analytical applications like immunoassays and environmental analysis due to their high sensitivity.

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