Molecular Luminescence Spectroscopy: Prof. Dr. Ayman H. Kamel

MOLECULAR
LUMINESCENCE
SPECTROSCOPY
Prof. Dr. Ayman H. Kamel

WHAT HAPPENS TO THE ABSORBED EM
ENERGY DETERMINES WHETHER YOU
HAVE…
 Absorbance
 molecule returns to the ground or lower energy state via a non-radiative
transition such as vibration, collision with other molecules, etc. These
give off the energy absorbed rather than the emission of light.
 Fluorescence
 Some energy is lost through various processes (e.g. non-radiative
transitions) and then light is given off.
 Phosphorescence
 The molecule transitions from an excited triplet state to an lower energy
singlet state and gives off light. Non-radiative transitions intervene.
Fluorescence, Phosphorescence, & Chemiluminescence
For UV/Vis need to observe Po
10 – 10 s
-8 -9
A) Introduction and P difference, which limits

M*  M + heat
detection
1.) Theory of Fluorescence and Phosphorescence:
10-5 to 10-8 s fluorescence

10-4 to 10 s phosphorescence
10-14 to 10-15 s
- Excitation of e- by absorbance of h.

- Re-emission of hv as e- goes to ground state.
- Use h2 for qualitative and quantitative analysis
Method Mass detection Concentration Advantages

limit (moles) detection
limit (molar)
UV-Vis 10-13 to 10-16 10-5 to 10-8 Universal
fluorescence 10-15 to 10-17 10-7 to 10-9 Sensitive

2.) Fluorescence – ground state to single state and back.
Phosphorescence - ground state to triplet state and back.
Fluorescence Phosphorescence
10-5 to 10-8 s 10-4 to 10 s
Spins paired Spins unpaired

No net magnetic field net magnetic field
Example of
Phosphorescence
0 sec 1 sec
Jablonski Energy Diagram
Molecules: Numerous vibrational energy levels for each electronic state
S2, S1 = Singlet States Resonance Radiation - reemission at same 

T1 = Triplet State usually reemission at higher  (lower energy)
Forbidden transition: no direct excitation of triplet state because
change in multiplicity –selection rules.
S1 S0

STOKES SHIFT
2 3
Deactivation Processes:
a) vibrational relaxation: solvent collisions
- emission >  excitation (Stokes shift)
- vibrational relaxation is efficient and goes to
lowest vibrational level of electronic state
within 10-12s or less.
- significantly shorter life-time then
electronically excited state
- fluorescence occurs from lowest vibrational
level of electronic excited state, but can go to
higher vibrational state of ground level.
b) internal conversion:
- crossing of e- to lower electronic state.
- S1 to S0 would also happen .
- efficient, therefore many compounds don’t
fluoresce (aliphatic)
- especially probable if vibrational levels of
two electronic states overlap, can lead to
predissociation or dissociation.
- dissociation: direct excitation
(absorption) to vibrational
state with enough energy to break a bond
- predissociation: relaxation to
vibrational state of a lower electronic state
with enough energy to break a bond
c) external conversion:
• deactivation via collision with solvent (collisional quenching)

• decrease collision  increase fluorescence or
phosphorescence
• decrease temperature and/or increase viscosity
• decrease concentration of quenching (Q) agent
d) intersystem crossing:
• spin of electron is reversed
- change in multiplicity in molecule occurs (singlet to triplet)
- enhanced if vibrational levels overlap
- more common if molecule contains heavy atoms (I, Br)
e) Phosphorescence:
Deactivation from an ‘triplet” electronic
state to the ground state producing a
photon
VARIABLES AFFECTING
FLUORESCENCE
Quantum Yield ():
• ratio of the number of molecules that luminesce to the total number of excited
molecules  efficiency
• determined by the relative rate constants (kx)of deactivation processes
 = kf # of Luminescence Photons
L 
# of Absorbance Photons
kf + ki + kec+ kic + kpd + kd
Increase quantum yield by decreasing f: fluorescence I: intersystem crossing

factors that promote other deactivation ec: external conversion ic: internal conversion
processes
pd: predissociation d: dissociation
Types of Transitions:
- seldom occurs from absorbance less
than 250 nm
Notation! 200 nm => 140 kJ/mol breaks many bonds
- fluorescence not seen with   
- typically *   or *  n
Fluorescence Quantum Yield
kF
F 
k F  k ec  k ic  k isc  k pd  k d
kec = external conversion (S1  S0)

kic = internal conversion (S1  S0)
kisc = intersystem crossing (S1  T1)
kpd = predissociation
kd = dissociation
Concentration Dependence
P0 – P : Power of the
F  K ' ( Po  P) excitation beam
absorbed by the
P bc Expanded as system
 10 Maclaurine
Po series
F  K " Po (2.303bc)
F  Kc
If 2.303bc = A < 0.05 then Fluorescence is

proportional to concentration
Effect of Concentration on Fluorescence or
Phosphorescence
At low concentrations: F = 2.3K’bcPo
deviations at higher concentrations can be attributed to absorbance becoming

a significant factor and by self-quenching or self-absorption.
Fluorescence of crude oil

Fluorescence & Structure:
usually aromatic compounds
low energy  * transition
quantum yield increases with number of rings and degree of
condensation.
fluorescence especially favored for rigid structures
fluorescence increase for chelating agent bound to metal.
Examples of fluorescent compounds:
H2
H C
N N
O
N Zn
2
Quinoline indole fluorene 8-hydroxyquinoline
Temperature, Solvent & pH Effects:
- decrease temperature  increase fluorescence (deactivation)
- increase viscosity  increase fluorescence (less collisions)
- fluorescence is pH dependent for compounds with acidic/basic
substituents.
more resonance forms stabilize excited state

H H H H H H
N N N
resonance forms of aniline
Effect of Dissolved O2:

- increase [O2]  decrease fluorescence
- oxidize fluorescing species
- paramagnetic property increase intersystem crossing (spin flipping)
Application of Fluorescence
- detecting inorganic species by adding a fluorophore
Ion Reagent Absorption (nm) Fluorescence (nm) Sensitivity (ppm) Interference

Be, Co, Cr, Cu,
Al3+
Alizarin garnet R 470 500 0.007 F-,NO3-, Ni, PO4-3,
Th, Zr
Be, Co, Cr, Cu,
Al complex of F-,Fe, Ni,PO4-3,
F- Alizarin garnet R 470 500 0.001 Th, Zr
(quenching)
B4O72- Benzoin 370 450 0.04 Be, Sb
2-(0-Hydroxyphenyl)- NH3
Cd2+ 365 Blue 2
benzoxazole
Li+ 8-Hydroxyquinoline 370 580 0.2 Mg
Sn4+ Flavanol 400 470 0.1 F-, PO43-, Zr
B, Be, Sb,
Zn2+ Benzoin - green 10
colored ions
OH
OH HO
N O O OH
C C
HO N N
OH H
O SO3Na
8-Hydroxyquinoline flavanol alizarin garnet R benzoin

COMPONENTS OF FLUORESCENCE
SPECTROPHOTOMETERS
 Light sources
 low pressure Hg lamp
 254, 302, 313 nm lines
 high pressure xenon arc lamp
 lasers
 Wavelength selectors
 filters
 monchromators
 Detectors
 photomultipliers
 CCD cameras
 Cells and sample compartments
 quartz cells
 light tight compartments to minimize stray light
Basic design
• components similar to UV/Vis

• spectrofluorometers: observe
• both excitation & emission spectra.
INSTRUMENTATION
Extra features for phosphorescence
• sample cell in cooled Dewar flask with liquid nitrogen

• delay between excitation and emission
Fluorometers
- simple, rugged, low cost, compact
- source beam split into reference and sample beam
- reference beam attenuated ~ fluorescence intensity
A-1 filter fluorometer

Spectrofluorometer
- both excitation and emission spectra
- two grating monochromators
- quantitative analysis
Perkin-Elmer 204
 Use array detector (CCD) to collect total fluorescence
spectrum
TOTAL FLUORESCENCE
INSTRUMENT
CHEMILUMINESCENCE
- chemical reaction yields an electronically excited species that emits
light as it returns to ground state.
- relatively new, few examples
A + B  C*  C + h
Examples of Chemical Systems giving off light:

Luminol (used to detect blood)
NH2 O NH2
C COO-
NH O2/OH-
+ h + N2 + H2O
NH
C
COO-
O
- phenyl oxalate ester (glow sticks)

sticks
BIOLOGICAL SYSTEMS
Luciferase (Firefly enzyme)
O O
R2
Luciferase Spontaneous
Luciferin + O2 O C C 2
R CO2 + O C* Light
R1
1
R
N
“Glowing” Plants
S Luciferase gene cloned into plants
S
HO
N
O
Luciferin (firefly)
HO
OTHER APPLICATIONS
Determination of nitrogen monoxide
NO + O3 → NO2* + O2
NO2* + → NO2 + h ( = 600 – 2800 nm)
Determination of sulfur
4H2 + 2SO2 → S2* + 4H2O
S2* → S2 + h ( = 384 and 394 nm)

Molecular Luminescence Spectroscopy: Prof. Dr. Ayman H. Kamel

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Molecular Luminescence Spectroscopy: Prof. Dr. Ayman H. Kamel

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MOLECULAR

Prof. Dr. Ayman H. Kamel

A) Introduction and P difference, which limits

10-5 to 10-8 s fluorescence

- Excitation of e- by absorbance of h.

Method Mass detection Concentration Advantages

fluorescence 10-15 to 10-17 10-7 to 10-9 Sensitive

10-5 to 10-8 s 10-4 to 10 s

Spins paired Spins unpaired

S2, S1 = Singlet States Resonance Radiation - reemission at same 

• deactivation via collision with solvent (collisional quenching)

• determined by the relative rate constants (kx)of deactivation processes

kf + ki + kec+ kic + kpd + kd

Increase quantum yield by decreasing f: fluorescence I: intersystem crossing

kec = external conversion (S1  S0)

If 2.303bc = A < 0.05 then Fluorescence is

deviations at higher concentrations can be attributed to absorbance becoming

Fluorescence of crude oil

more resonance forms stabilize excited state

resonance forms of aniline

Effect of Dissolved O2:

Ion Reagent Absorption (nm) Fluorescence (nm) Sensitivity (ppm) Interference

B4O72- Benzoin 370 450 0.04 Be, Sb

8-Hydroxyquinoline flavanol alizarin garnet R benzoin

• components similar to UV/Vis

Extra features for phosphorescence

• sample cell in cooled Dewar flask with liquid nitrogen

A-1 filter fluorometer

Examples of Chemical Systems giving off light:

- phenyl oxalate ester (glow sticks)

Determination of nitrogen monoxide

NO2* + → NO2 + h ( = 600 – 2800 nm)

4H2 + 2SO2 → S2* + 4H2O

S2* → S2 + h ( = 384 and 394 nm)

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