1. Molecular Luminescence Spectrometry
Chapter 15
Molecular Luminescence Spectrometry

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

4. Photoluminescence
Light is directed onto a sample, where it is absorbed and imparts excess energy into the material in a process called "photo-excitation." One way this excess energy can be dissipated by the sample is through the emission of light, or luminescence.
The intensity and spectral content of this photoluminescence is a direct measure of various important material properties.

5. 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.
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.

6. PHOTOLUMINESCENCE
Fluorescence – Does not involve change in electron
spin; short lived (less than microsecond). Can be observed at
room temperature in solution.
2. Phosphorescence – Involves change in electron spin.
Long lived (seconds). Can be
observed at low temperature in
frozen or solid matrices.
3. Chemiluminescence – Light emission due to a chemical reaction.

7. Electron Spin
The Pauli exclusion principle states that no two electrons in an atom can have the same set of four quantum numbers. This restriction requires that no more than two must have opposed spin states. Because of spin pairing, most molecules exhibit no net magnetic field and are thus said to be diamagnetic. In contrast, free radical, which contain unpaired electrons, have a magnetic moment are said to be paramagnetic.

8. Singlet/Triplet States
Singlet State: A molecular electrons state in which all electron spins are paired is called a singlet state and no splitting of electronic energy levels occurs when the molecule is exposed to a magnetic field. Net spin S is zero. Spin Multiplicity 2S + 1 = 1.
Doublet State: Free radical (due to odd electron). Net spin S is 1/2. Spin Multiplicity 2S + 1 = 2.
Triplet State: Electron Spins in the ground and excited electronic states are not paired. Net spin S = 1. Spin multiplicity 2S + 1 = 3.

9. JABLONSKI DIAGRAM Solid lines – Radiative process
Vibrational deactivation
Solid lines – Radiative
process
Dashed lines-Nonradiative Process
Intersystem Crossing (Ki)
Absorption
Fl (Kf) Ph
Singlet state
Quenching Kcc
quenching
Internal conversion
Kic

10. Rates of Absorption and Emission...
The rate at which a photon of radiation is absorbed is enormous, the process requiring on the order o f to 10-15s. Fluorescence emission, on the other hand, occurs at a significantly slower rate. Here, the lifetime of the excited state is inversely related to the molar absorptivity of the absorption peak corresponding to the excitation process.

11. Quantum yield or quantum efficiency ():
Quantum yield for a fluorescent process is the ratio of
the number of molecules that fluoresce to the total
number of excited molecules.
For a highly flurorescent molecule such as fluorescein  = 1 and for a nonluminesceing molecule  = 0.
can be defined in terms of the various rate constants
In the Jablonski diagram as
 = kf/(kf + ki + kcc + kic)
Fluorescence (kf); Singlet State quenching (kcc); Intersystem crossing to triplet state from singlet state (ki); Internal conversion (kic)

12. Idealized absorption and emission spectra
The 0-0 transition is common to both absorption and emission. When these transitions overlap we have resonance emission.

13. ABSORPTION AND EMISSIOIN SPECTRA
In the absorption spectra transitions to higher vibrational
energies lead to absorptions at lower wavelengths.
In the emission spectrum transition from the 0 vibrational
level of the excited state to higher vibrational levels of
the ground state lead to emissions at higher wavelengths.
The wavelength maxima for the absorption and emission
spectra under resonance conditions are identical in
accordance with the Franck-Condon principle if the life
time of the excited state is very short..
In many systems the wavelength maxima for the
absorption and emission spectra do not coincide due to
Loss of energy of the excited state by collision with solvent
molecules.

14. ABSORPTION AND EMISSIOIN SPECTRA (CONTD)
Emission Spectrum: Plot of the emission intensity at 90o
to the incident radiation as a function of emission
wavelength for a fixed excitation wavelength.
Excitation Spectrum: Plot of the emission intensity at
a fixed emission wavelength at 90o to the incident
radiation as a function of excitation wavelength.
Fluorescence lifetimes: – 10-6 s.
Phosphorescence lifetimes: – seconds.

15. Sample Excitation and Emission Spectra
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

16. Sample Spectra
Excitation (left), measure luminescence at fixed wavelength
while varying excitation wavelength. Fluorescence (middle)
and phosphorescence (right), excitation is fixed and record
emission as function of wavelength.
Phosphorescence is susceptible to O2 and collisions with solvent molecules. Triplet states are rapidly deactivated under these conditions. For many molecules phosphorescence can only be observed at low temperatures in frozen matrices in the absence of
O2. (RTP application)
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

17. FLUORESCENCE AND STRUCTURE
1. Fluorescence from singlet states of - * have more intensity than those from n- * transitions as the molar absorptivities for - * absorptions are much higher than those for n- * absorptions.
2. Simple heterocycles do not exhibit fluorescence.
The n-*singlet quickly converts to the n- * triplet and no
Fluorescence is observed.

18. FLUORESCENCE AND STRUCTURE (CONTD)
3. Fusion of heterocycles to benzene rings increases the molar absorptivity for n- * absorptions and shortens the life time of the n- * singlet preventing its conversion to triplet. This increases fluorescence quantum efficiency.

19. STRUCTURAL RIGIDITY
Flurorescence is favored in molecules with structural
rigidity. The quantum yields for fluorescence for
fluorene and biphenyl are 1 and 0.2 respectively.
The increased rigidity of fluorene stabilizes the - *
singlet state leading to higher quantum yield.
Chelation also can lead to increased fluorescnece.

20. HEAVY ATOM EFFECT
A very significant influence on the fluroescence quantum
yield of the benzene ring which is due to - * singlet
states is observed with halogen substitution. The
quantum yield decreases with the atomic number of the
halogen. This called the heavy atom effect.
The probability for intersystem crossing increases with
increasing atomic number of the halogen which reduces
fluorescence.
Substitution of carboxylic acid or carbonyl group on
benzene generally inhibits fluorescence due to the
n- * states being lower in energy than the - * states
and these do not fluoresce efficiently.

21. TEMPERATURE AND SOLVENT EFFECTS
Quantum yield of fluorescence of most molecules decreases with increasing temperature due to collisional deactivation of the singlet state.
2. Fluorescence is decreased by solvent containing heavy atoms such as as those containing halogens.
Heavy atoms promote intersystem crossing to the triplet state. This decreases fluorescence quantum yield but increases phosphorescence quantum yield.
4. Solvent Viscosity – lower viscosity, lower quantum yield.
5. Concentration:
Self-quenching due to collisions of excited molecules.
Self-absorbance when fluorescence emission and absorbance
wavelengths overlap.

22. Effect of Concentration on Fluorescence Intensity

23. 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.

24. 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.

25. Fluorometer Schematic

26. Fluorometer Figure

27. Spectrofluormeter Figure

28. ABSORBANCE VS. LUMINESCENCE
ABSORBANCE LUMINESCENCE
Most compounds absorb All compounds that absorb do not emit light. (good selectivity)
Narrow liner dynamic range Large linear dynamic range
Not very sensitive to impurities Very sensitive to impurities, 1~ order of magnitude better than in absorption spectrometry
Absorbance monitored along Luminescence monitored
the axis of the incident at 90o to the incident
radiation radiation
While emission occurs in all directions only the photons emitted at 90o to the incident radiation are monitored to avoid interference from transmitted photons.