1. Chapter 15 Molecular Luminescence Spectrometry

2. Important topics in this chapter:
Energy diagram and basic concepts
Fluorescence quantum yield
Fluorescence instrumentation

3. Luminescence: Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications

4. 15A THEORY OF FLUORESCENCE AND PHOSPHORESCENCE

5. 15A-1 Excited States Producing
Fluorescence and Phosphorescence

6. Electron Spin Singlet/Triplet Excited States

7. Singlet: all electron spins are paired; no energy level splitting occurs when the molecule is exposed to a magnetic field;
Triplet: the electron spins are unpaired and are parallel; excited triplet state is less energetic than the corresponding singlet state.
Diamagnetic: no net magnetic field due to spin paring. The electrons are repelled by permanent magnetic fields.
Paramagnetic: magnetic moment and attracted to a magnetic field (due to unpaired electrons).
Ground
Single state
Excited
Single state
Excited
triplet state

8. Electronic spin states of molecules. In (a) the ground electronic state
is shown. In the lowest energy, or ground, state, the spins are always paired, and
the state is said to be a singlet state. In (b) and (c), excited electronic states are
shown. If the spins remain paired in the excited state, the molecule is in an excited
singlet state (b). If the spins become unpaired, the molecule is in an excited triplet
state (c).

9. Molecular Fluorescence
Dkkdj
Optical emission from molecules that have been excited to higher energy levels by absorption of electromagnetic radiation.

10. Energy-Level Diagrams for Photoluminescent Molecules

11. Photoluminescence
Band gap determination. The most common radiative transition in semiconductors is between states in the conduction and valence bands, with the energy difference being known as the band gap.
Impurity levels and defect detection. Radiative transitions in semiconductors also involve localized defect levels. The photoluminescence energy associated with these levels can be used to identify specific defects.

12. Photoluminescence
Recombination mechanisms. The return to equilibrium, also known as "recombination," can involve both radiative and nonradiative processes. The amount of photoluminescence and its dependence on the level of photo-excitation and temperature are directly related to the dominant recombination process.
Material quality. In general, nonradiative processes are associated with localized defect levels. Material quality can be measured by quantifying the amount of radiative recombination.

13. FIGURE 15-1 Partial energy-level diagram for a
photoluminescent system.

14. 15A-2 Rates of Absorption and Emission

15. 15A-3 Deactivation Processes

16. Vibration Relaxation
Internal Conversion
External Conversion
Intersystem Crossing
Phosphorescence

17. FIGURE 15-2 Fluorescence excitation and emission spectra
for a solution of quinine.

18. 15A-4 Variables Affecting Fluorescence and Phosphorescence

19. Quantum Yield
Transition Types in Fluorescence
Quantum Efficiency and Transition Type
Fluorescence and Structure

20. Variables that affect Fluorescence and phosphorescence
Quantum yield:
the ratio of the number of molecules that
luminescence to the total number of excited molecules.
f = kf/ (kf + ki + kec + kic + kpd + kd)
kf: Fluorescence constant
ki: Intersystem crossing constant
kec: External conversion constant
kic: Internal conversion constant
kpd: Predissociation constant
kd: Dissociation constant
p ® p* transitions: high quantum efficiency

21. Quantum yield = kf / (kf + ki + kec + kic + kpd + kd)
+ knr
Quantum yield = kf / (kf + ki + kec + kic + kpd + kd)
Quantum yield can be close to unity if the radiationless
decay rate is much smaller the the radiative decay.
High quantum yield molecules: rhodamine, fluorescein etc
Effect of structural rigidity: Molecules with rigid structures
have high fluorescence yield.
Nonrigid molecule can undergo low-frequency vibrations. kic

22. What Affects the fluorescence quantum yield? (1) Excitation l
Short l's break bonds increase kpre-dis and kdis
rarely observed most common
s* ®s p* ® n p* ® p
(2) Lifetime of state
Transition probability measured by e
Large e implies short lifetime
p*®p > p*® n ( s > s)

23. (3) Structure

24. (4) Rigidity

27. TABLE 15-1 Effect of Substitution on the fluorescence of Benzene

28. Effect of Structural Rigidity

30. Temperature and Solvent Effects
Effect of dissolved oxygen

31. Effect of pH on Fluorescence

32. Effect of Concentration on Fluorescence Intensity

33. 15A-5 Emission and Excitation Spectra

34. FIGURE 15-3 Spectra for phenanthrene: E, excitation; F,
fluorescence; P, phosphorescence.

35. 15B INSTRUMENTS FOR MEASURING FLUORESCENCE AND PHOSPHORESCENCE

36. FIGURE 15-4 Components of a fluorometer or spectrofluorometer.
Basic design
components similar to UV/Vis
spectrofluorometers: observe
both excitation & emission spectra.
Extra features for phosphorescence
sample cell in cooled Dewar flask with liquid nitrogen
delay between excitation and emission

37. Fluorometer Schematic

38. 15B-1Components of Fluorometers and Spectrofluorometers

39. FIGURE 15-5
Fluorescence spectra for 1
ppm anthracene in alcohol:
(a) excitation spectrum; (b)
emission spectrum.

40. Components of Fluorometers and Spectrofluorometers
Sources: A more intense source in needed than the tungsten of hydrogen lamp.
Lamps: The most common source for filter fluorometer is a low-pressure mercury vapor lamp equipped with a fused silica window. For spectrofluorometers, a 75 to 450-W high-pressure xenon arc lamp in commonly employed.
Lasers: Most commercial spectrofluorometers utilize lamp sources because they are less expensive and less troublesome to use.

41. Components of Fluorometers and Spectrofluorometers
Filters and Monochromators: Both interface and absorption filters have been used in fluorometers for wavelength selection of both the excitation beam and the resulting fluorescence radiation. Most spectrofluorometers are equipped with at least one and sometimes two grating monochromators.
Transducers: Photomultiplier tubes are the most common transducers in sensitive fluorescence instruments.
Cell and Cell Compartments: Both cylindrical and rectangular cell fabricated of glass or silica are employed for fluorescence measurements.

42. 15B-2 Instrument Designs

43. Fluorometers
Spectrofluorometers

44. FGIURE 15-6 A typical fluorometer.

45. FIGURE 15-7 A spectrofluorometer.

46. Spectrofluormeter Schematic

47. FIGURE 15-8(a) Three-dimensional spectrofluorometer. (a)
Schematic of an optical system for obtaining total luminescence
spectra with a CCD detector.

48. hypothetical compound.
FIGURE 15-8(b) Excitation and emission spectra of a
hypothetical compound.

49. FIGURE 15-8(c) Total luminescence spectrum of compound in (b).

50. Spectrofluorometers Based on Array Detectors
Fiber-Optic Pluorescence Sensors
Phosphorimeters

51. Figure 15-9 Schematic of a device for alternately exciting and observing phosphorescence.

52. 15B-3 Instrument Standardization

53. 15C APPLICATIONS OF PHOTOLUMINESCENCE METHODS

54. 15C-1 Fluorometric Determination
of Inorganic Species

55. Cations Forming Fluorescing Chelates
Fluorometric Reagents

56. Fluorescence Sensing
sensing is based on changes in fluorescence signal
either in intensity or in spectrum.
Fluorophore based sensors:
Enzyme based sensors:
Ion sensors
DNA/RNA sensors
neurotransmitter sensors
environmental sensors

57. Some
fluorometric chelating agents
for metal cations. Alizarin
garnet R can detect Al3+ at
levels as low as μg/mL.
Detection of F– with alizarin
garnet R is based on
Fluorescence quenching of
the Al3+ complex. Flavanol
can detect Sn4+ at the 0.1 –
μg/mL level.

58. and Biochemical Species
15C-2 Methods for Organic
and Biochemical Species

59. TABLE 15-2 Selected Fluorometric Methods for Inorganic Species

60. 15C-3 phosphorimetric methods:

61. better selectivity;
poorer precision; lower temperature;
heavy atom results in strong phosphorescence
room temperature methods:
deposit analytes on surface: rigid matrix minimize deactivation of the triplet state by external and internal conversions;
Using micelles: micelles increase the proximity between heavy metal ion and the phosphur, thus enhance phosphorescence.

62. in Liquid Chromatography
15C-4 Fluorescence Detection
in Liquid Chromatography

63. 15C-5 Lifetime Measurements

64. Figure 15-10

65. 15D Chemiluminescence

66. Measurements of chemiluminescence is simple:
chemiluminescence is produced when a chemical reaction yields an electronically excited species, which emits light as it returns to its ground states.
A + B ® C* + D
C* ® C + hv
NO + O3 ® NO2* +O2
NO2* ® NO2 + hv
Measurements of chemiluminescence is simple:
only detector, no excitation necessary

67. 15D-1 The Chemiluminescence
Phenomenon

68. 15D-2 Measurement of Chemiluminescence

69. FIGURE 15-11 Chemiluminescence emission intensity as a
function of time after mixing reagents.

70. 15D-3 Analytical Applications
of Chemiluminescence

71. Analysis of Gases
Analysis for Inorganic Species
in the Liquid Phase
Analysis for Organic Species

74. Questions and Problems 15-4