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CHAPTER 15: MOLECULAR
LUMINESCENCE
LUMINESCENCE TECHNIQUES
• Emission of light is used to determine certain properties,e
e.g.structure and concentration, of the emitting species.
• Deactivation processes involved in converting a substance from
excited state to ground state:
• the emission of heat,
• activation of a chemical reaction or
• emission of radiation of the same or a modified wavelength.
• Forms of photoluminescence (luminescence after absorption) are
fluorescence (short lifetime) and phosphorescence (long
lifetime).
• Approximately 10x more sensitive than absorption techniques:ppb
detection limit
• Limited number of systems that photoluminesce.
• Luminescence observed for simple and complex systems and for all
three phases.
Chapter 15 - 2
Theory
Atoms: e.g. dilute Na (g) the
• 3s 3p transition occurs by absorption at l = 5895 and
5890 A. with a lifetime  10-8 sec,
• the electron returns to the ground state isotropically
(isotropically) emitting hn with the wavelength of
emission being the same as the wavelength of
excitation. resonance fluorescence.
• Polyatomic Systems
– Resonance fluorescence observed
– Emission of radiation of longer l (called a Stoke's shift) more
common.. Most fluorescent systems are complex organic
compounds with 1 or more aromatic functional groups so that the
commonly observed transitions are:
 n.
Chapter 15 - 3
EXCITED STATES
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Multiplicity (number of lines observed when the
molecule is placed in a magnetic field) is related
number of unpaired spins in the molecule (S):. M =
2•S + 1.
Most molecules have an even number of electrons
which means that all of their electrons in the ground
state must be paired:
Singlet state (M = 2•0 + 1);. all electrons in ground
state paired.
Doublet state : (M = 2•½ + 1 = 2)a free radical
(substance that has an odd number of electrons);.
electrons can have 2 orientations in the magnetic
field-opposed to the field and aligned.
Triplet state: (M = 2•1 + 1 = 3), excited state in which
excited electron spin is flipped so that the spins are
parallel.
Singlet state
Triplet state
Diamagnetic
Paramagnetic
Probable
Less probable
-8
-13
Lifetime 10 -10 sec
Lifetime up to ≥1 s
Chapter 15 - 4
ENERGY LEVEL DIAGRAM
• Let ground , the first
excited, second excited
etc. electronic states be:
S0, S1, S2 and etc..for all
of the possible singlet
states.
• Triplet states would then
be T1, T2 and so on .
• All electronic state has
several vibrational and
rotational states.
Chapter 15 - 5
Decay Processes
• Internal conversion Movement of electron from one electronic state
to another without emission of a photon, e.g. S2  S1) lasts about
10-12 sec.
• Predissociation internal conversion electron relaxes into a state
where energy of that state is high enough to rupture the bond.
• Vibrational relaxation (10-10-10-11sec)- Energy loss associated
with electron movement to lower vibrational state without photon
emission.
• Intersystem crossing: Conversion from singlet state to a triplet
state. e.g. S1 to T1
• External conversion is a non-radiative process in which energy of
an excited state is given to another molecule (e.g. solvent or other
solute molecules). Related to the collisional frequency of excited
species with other molecules in the solution. Cooling the solution
minimizes this effect.
Chapter 15 - 6
QUANTUM YIELD
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Only a fraction of the photon absorbed result in fluorescence. Fraction
called Quantum yield (efficiency), F:
F
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# emitted
# absorbed
Excite molecule in say the S1 state can undergo a transition back to the
ground state: S1  S0 + hn. The emitted photon is the useful fluorescence
line and takes about 10-6-10-10 sec to occur.
Rate of all processes which involved the absorption or emission of a
photon can be written in terms of a first order rate equation:
Instrumental Analysis,Christian and O’Reilly, p. 251
Chapter 15 - 7
KINETICS OF ADSORPTION
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The intensity of the light absorbed is
.DP = Po - PT = rate of absorption where
Po = photon flux to sample, PT = photon flux out of sample.
At steady state rate of absorption equals rate of fluorescence or:
DP = (kIC + kISC + kf + kQ[Q])[S1]
where kQ = rate constant for quenching process-a second order process
since the [Q] is also important in determining the relative rate of this
process.
Let [S1] = steady state concentration of S1 molecules. The rate of
fluorescence Pf = FDP = F(kIC + kISC + kf + kQ[Q])[S1] and
Pf = kf[S1].
kf
Combine and rearrange: F =
k IC + k ISC + k f + k q [Q]
1
large F means a large kf.
The lifetime of the fluorescing state is given by  f 
kf
The inverse relationship between the rate constant and the lifetime tells us
that a process having a large rate constant has a short lifetime and will have
the largest fluorescence intensity.
Chapter 15 - 8
FLUORESCENCE INTENSITY VS
CONCENTRATION
• Before fluorescence occurs absorption must occur. The
absorption process given by Beer's Law:
- l og
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P
Po
= - log T = A = e  b  C
where e= k/2.303 = molar absorptivity. We will use this
in the development of the fundamental equation for
fluorescence.
Earlier we stated that
Pf = rate of fluorescence = F(kIC + kISC + kf + kQ[Q])[S1] =
FDP = F[Po - PT].
Beers law can be written as:
PT = Po×10-ebC = Po×e-2.303ebC.
Substituting into the fluorescence equation gives:
Pf = F[Po - Po×e-2.303ebC] = FPo[1 - e-2.303ebC].
Chapter 15 - 9
Concentration Dependence 2
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From the mathematics handbook:
x2
x3
xn
+
+  +
2!
3!
n!
We substitute this for the exponential terms to get the following:
ex = 1 + x +
•


(-2.303  e  b  C ) 2 (-2.303  e b  C ) 3
Pf  Po F 1 - 1 + 2.303 e  b  C    
2!
3!






(-2.303  e  b  C )2 (-2.303  e b  C )3
 Po F 2.303 e  b  C +   
2!
3!


For dilute solutions A = ebC is small which will make the squared and higher
powered terms quite small.
e.g. if A = 0.05, then the second term is while the first term is 2.303×0.05 =
0.115.
• The fluorescence equation then reduces to:
• Pf = Po×F×2.303×e×b×C or Pf = K×C or a linear response in the
fluorescence intensity with concentration will be observed as long as the A <
0.05
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Chapter 15 - 10
Fluorescence Intensity vs Conc.
• When performing a fluorometric analysis, Pf is measured
independently of Po so that it is not necessary to determine P0 i.e.
only one measurement of intensity is made.
• Remember that Beer’s law for absorption requires measurement of
both P and P0
• In the fluorescence experiment, one can increase Po and should
expect an increase in Pf. Thus, one can increase the sensitivity to
the analyte by increasing the power of the exciting light.
• In the absorption experiment, one needs the ratio of the input and
output power. Increasing the input power also increases the output
power but does little to the ratio.
• This makes fluorescent techniques inherently more sensitive than
absorption techniques. The detection limits are 10-8M; in
fluorimetry they are 10-12M.
Chapter 15 - 11
F AND TRANSITION TYPE
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In absorption spectrum of organics we can observe the following transitions to excited states: 
;n ;;n 
Fluorescence:  .Same orbitals possible.
., and  n transitions are seldom observed however when the l of the transition is 
240 nm(UV) since the energy of the transition is often high enough to dissociate the molecule.
Less energetic transitions ; n transitions observed.
.F, for the  transition is usually greatest since process has shortest average lifetime and
greatest molar absorptivity.
Other affects on the quantum yield: In our equation describing quantum yield:
kf
.F =
kIC + kISC + kF + k q [Q]
All terms except kf must be minimized to obtain a large fluorescent signal. Chemical structures
that minimize one or more of the these rate constants increase the quantum yield.
Structural rigidity: Decreases the chances of vibrational and rotational de-excitation (which we
have called IC (internal conversion).. Prevents the loss of energy by the internal conversion
process so that the fluorescent yield is higher in rigid molecules.
Ring structures with alternating single and double bonds (conjugation) that are aromatic usually
best fluorescing compounds, although highly conjugated aliphatic compounds may also fluoresce.
Temperature: Raising the temperature of a system increases the collisional frequency between
excited molecules and the solvent which increases the amount of external conversion.
Solvent: Decrease in solvent viscosity also leads to increase in external conversion and a
decrease in fluorescence intensity.
Chapter 15 - 12
PHOTOLUMINESCENT
ANALYSIS
• Since many compounds fluoresce at the same l, fluorescence
cannot be used for qualitative analysis.
• Quantitative analysis of a large number of organic compounds in
particular polycyclic molecules with extensive conjugation possible.
E.g. Vitamin A which has a blue-green fluorescence with lmax » 500
nm in ETOH.
• Often molecules will be polynuclear aromatics such as phenol.
• Inorganic species
– Direct: form a fluorescent complex with organic species and measure
the fluorescent intensity. Fluorometric agents usually polyfunctional
group aromatic compounds.
– Indirect: diminution of fluorescence measured when the ion is added to
a fluorescent solution. Reaction stoichiometry between the ion and the
fluorescent reagent must be known.
Chapter 15 - 13
Fluorescence Problem
5.00 mL of an unknown zinc solution was placed in each of
two separatory funnels and 4.00 mL of 1.10 ppm Zn2+
was added to the second solution. Each was extracted
with three 5 mL aliquots of CCl4 containing an excess of
8-hydroxyquinoline. The extracts were then diluted and
their fluorescence measured with a fluorometer. The
fluorescent intensities were 6.12 for the solution
containing no added zinc and 11.16 for the other
solution. Determine the concentration of the original zinc
solution.
• Strategy:
– Determine concentration of final solution containing the unknown
(Standard Addition Method).
– Determine concentration of the original solution.
Chapter 15 - 14
INSTRUMENTATION
• Source: Hg or Xe arc lamp is used. (continuous radiation in the 250600 nm range is produced).
• Monochromators or filters: needed to select both wavelength of
excitation emission.
• Monochromators are used when dealing with narrow absorption or
emission peaks while filters may be used when peaks are not as
narrow.
• When filters are used, one is limited to wavelength range that
passes through the particular filter used.
• Instruments using filters are called fluorometers while instrument
using monochromators are called spectrofluorimeter.
• Cells and Cell compartments: cylindrical or rectangular (less
scattering rectangular cell); quartz or glass depending upon the
wavelength range needed; Outlet of the sample cell usually 90° from
the inlet.(minimizes source light at detector.
• Detector: Phototube or photomultiplier (small signals)
Chapter 15 - 15
A Fluorometer or
Spectrofluorometer
Chapter 15 - 16
A Sectrofluorometer
Chapter 15 - 17