1. Lecture 4, Luminescence, 03-10-2012
PHCM561t WS
Lecture 4 LUMINESCENCE
Quinine
Dr. Rasha Hanafi
© Dr. Rasha Hanafi, GUC
Lecture 4, Luminescence,

2. PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
OBJECTIVES
By the end of this session, the student should be able to:
Define luminescence, fluorescence and phosphorescence.
Explain how luminescence happens
Distinguish absorbance, excitation and emission spectra
Describe the set-up and the components of a fluorometer
Recognize molecular features causing fluorescence
Define parameters that influence molecular fluorescence
State applications of fluorescence spectroscopy
Lecture 4, Luminescence,
© Dr. Rasha Hanafi, GUC
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3. Lecture 4, Luminescence, 03-10-2012
HISTORY
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Lecture 4, Luminescence,

4. WHAT IS MOLECULAR LUMINESCENCE ?
PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
WHAT IS MOLECULAR LUMINESCENCE ?
Chemiluminescence
Phosphorescence
Molecular Fluorescence
BASIC PRINCIPLE:
1st: molecules are excited (outer shell electrons like in absorbance phenomenon)
2nd: excited species give an emission spectrum that provides information for quantitation and qualification
excitation resulting from a chemical reaction
excitation by absorption of photons:
PHOTOLUMINESCENCE
Fluorescence and phosphorescence are alike in that excitation is brought about by absorption of photons. As a consequence, the two phenomena are often referred to by a more general term: photoluminescence.
Fluorescence is short-lived, with luminescence ceasing almost immediately (<10-5 sec) ,while phosphorescence features luminescence from 10-4 to several seconds.
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Lecture 4, Luminescence,
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5. Why Use Molecular Luminescence ?
PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
Why Use Molecular Luminescence ?
ABSORBANCE MEASUREMENT
Changing transmittance from 100% to 99% is hard to measure such a small absorbance because the background is so bright.
WHAT MAKES THESE TECHNIQUES INTERESTING:
sensitivity: ppb range (3 orders of magnitudes higher compared to absorption spectroscopy).
large linear concentration ranges
LIMITING FACTORS:
enormous sensitivity implies purity of analytes (or prior purification)
only few molecules exhibit luminescence, compared to those that absorb UV or visible electromagnetic radiation
T=P/Po=0.99
A= -log T= 0.004
!!!!!!!!!!!!!! Po P
FLUORESCENCE MEASUREMENT
Observing fluorescence of 1% of molecules
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Lecture 4, Luminescence,
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6. SINGLET AND TRIPLET STATES
PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
SINGLET AND TRIPLET STATES
PAULI EXCLUSION PRINCIPLE: “no two electrons in an atom can have the same set of 4 quantum numbers”, in other words: two electrons in the same orbital must have opposite spins (we say: they are "paired": no net magnetic field = the molecule will be "diamagnetic"), molecule with unpaired electrons (e.g. triplet state) possess magnetic moment, are attracted by magnetic field, are called "paramagnetic"
excited singlet state
Excited triplet state is of less energy than excited singlet state.
Singlet to triplet transitions are far less probable than singlet/singlet transitions.
excited triplet state
A molecular electronic state in which all electrons are paired is called a singlet state.
On the other hand the ground state of a free radical, is a doublet state because the odd electron can assume 2 orientations in a magnetic field, which imparts slightly different energies to the system.
When 1 of a pair of a molecule is excited to higher energy level, either a singlet or a triplet state is formed.
Physico-chemical properties of molecules in triplet state can differ significantly from those of singlet state molecules.
Physicochemical properties of molecules in triplet state can differ significantly from those of singlet state molecules.
ground state
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7. ELECTRONIC AND VIBRATIONAL LEVELS
PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
ELECTRONIC AND VIBRATIONAL LEVELS
S0: ground state of a molecule at ambient temperature, all of the molecules in a solution
S1 and S2: excited singlet states
T1: lowest energetic triplet state, usually of less energy than lowest energetic excited singlet state S1.
Same E T1 S1
Each of these states features various vibrational levels – this permits energetic similarity (and even equivalence) of different electronic spin states of a molecule.
Because Singlet / triplet transitions are less probable than singlet / singlet transition (because spin conversion is necessary) , thus the average lifetime of an excited triplet state is 10-4 sec and more, while excited singlet state lifetime is 10-8 to 10-5 s.
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8. PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
DEACTIVATION PROCESS
An excited molecule can return to its ground state by a combination of several mechanistic steps:
Two of these steps, fluorescence and phosphorescence, involve the emission of a photon of radiation.
The other deactivation steps are radiationless processes.
What kind of deactivation does occur?
The favored route to the ground state is always the one that minimizes the lifetime of the excited state.
Thus, if deactivation by fluorescence is rapid with respect to radiationless processes, such emission is observed.
If a radiationless path has a more favorable rate constant, fluorescence is either absent or less intense.
Photoluminescence is limited to a small number of systems incorporating structural and environmental features that cause that the rate of radiationless deactivation be slowed in favor of emission of photons.
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Lecture 4, Luminescence,
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9. PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
ELECTRON TRANSITIONS
Photon absorption rate:
10-14 to s !!
Fluorescence emission rate is directly related to molar absorptivity.   103 to 105: lifetime of excited state to 10-9s; weakly absorbing systems: lifetime 10-6 to 10-5 s.
External conversion involves energy transfer between the excited molecule and the solvent or other solutes (collisional QUENCHING)
Dissociation and pre-dissociation: rupture of chemical bond due to high energy of vibrational state S2 S1 T1
High E, Short λ
Low E, Long λ S0
Energy
vibrational levels
dissociation:
subsequent to internal conversion: electron moves from higher electronic state to an upper vibrational level of a lower electronic state where vibrational energy is high enough to rupture a bond.
In large molecules, probability is appreciable for existence of bonds with strengths less than the electronic excitation energy of chromophors.
pre-dissociation:
absorbed radiation excites electron of a chromophore directly to a sufficiently high vibrational level to cause rupture of a bond. No internal conversion involved.
Paramagnetism of Oxygen:
it is attracted by the magnetic field but does not remain magnetic once it leaves the field. Gaseous oxygen is paramagnetic also but is moving too fast to be affected by the magnets. The reason that it is paramagnetic is because the oxygen molecule has two unpaired electrons. Electrons not only go around the atom in their orbitals, they also spin, which creates a magnetic field. Unpaired electrons spin in the same direction as each other, which increases the magnetic field effect. When the electron in an orbital become paired with another electron in that orbital, the new electron spins in the opposite direction and this cancels the effect of the first electron. Note that according to Valence Shell Electron Pair Repulsion theory (VSEPR), O2 has no unpaired electrons but according to Molecular Orbital (MO) theory it does have unpaired electrons. Since liquid O2 does stick to a magnet, MO theory is better at explaining the behavior.
intersystem crossing (reversal of spin), common in molecules containing heavy atoms or when paramagnetic species are present (O2 in solution)  fluorescence is decreased. λ1 λ2 λ3
absorption
Fluorescence always from lowest vibrational level of an excited electronic state
Internal conversion when 2 levels are sufficiently close energetically.
phosphorescence
vibrational relaxation due to collisions between the molecules of the excited species and those of the solvent
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Lecture 4, Luminescence,
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10. VIBRATIONAL RELAXATION I
PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
VIBRATIONAL RELAXATION I
1st Observation :
Upon excitation different vibrational levels can be achieved, in solution any excess vibrational energy is lost as consequence of collisions between the molecules of the excited species and solvent molecules
Result: Energy transfer to solvent and minuscule warming, lifetime of vibrationally excited species: sec and less.
Consequence: Fluorescence (and Phosphorescence) of an analyte in solution always occurs due to electron transition from a vibrational ground state.
Fluorescence due to any chosen electron transition (from any vibrational ground state) produces less energy than the previous excitation had absorbed.  Fluorescence signals of a molecule (moiety) generally appear at longer wavelengths than the corresponding absorbance signals (STOKES shift).
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Lecture 4, Luminescence,
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11. PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
VIBRATIONAL RELAXATION II
2nd Observation:
Upon luminescence different vibrational levels can be achieved, in solution any excess vibrational energy is lost as consequence of collisions between the molecules of the excited species and solvent molecules
Consequence I: fluorescence (and phosphorescence) of an analyte do not give sharp signals but diffuse bands.
Consequence II: The fluorescence spectrum of an analyte often is more or less similar to its absorbance spectrum.
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Lecture 4, Luminescence,
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12. FRANCK-CONDON-PRINCIPLE
PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
FRANCK-CONDON-PRINCIPLE
Electron transitions are so fast (10-15 s) that each atom has nearly the same position before and after a transition; this is due to the small mass of the electrons (FRANCK-CONDON principle).
Thus, molecular geometry initially is kept, and higher vibrational levels are achieved upon absorption. Upon relaxation to ground state, the same happens: molecular geometry is initially kept, so the vibrational level that is reached first is NOT the ground state.
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Lecture 4, Luminescence,
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13. ABSORBANCE, EXCITATION & EMISSION SPECTRA
PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
ABSORBANCE, EXCITATION & EMISSION SPECTRA
Emission spectra are measured by holding excitation radiation fixed (ex) at one particular wavelength and scanning through the emitted radiation. Graph: emission intensity vs. emission wavelength.
Excitation spectra are measured by varying excitation wavelength and measuring emitted light at one particular wavelength (em): Graph: excitation intensity vs. excitation wavelength. Because the 1st step in generating fluorescence is absorption of radiation to create excited states, an excitation spectrum is essentially identical to an absorbance spectrum taken under the same conditions.
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Lecture 4, Luminescence,
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14. PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
PHOSPHORESCENCE
After intersystem crossing from singlet to triplet state, deactivation can occur by internal or external conversion or by phosphorescence.
Since triplet-to-singlet conversions are comparatively improbable events, the average lifetime of an excited triplet state is 10-4 to 10 sec and more.
Thus, emission from such transition may persist for some time after irradiation has been discontinued.
The other deactivation transitions compete strongly with phosphorescence, so this phenomenon is usually observed at low temperatures, in highly viscous media or at molecules being adsorbed on surfaces. T1 S0
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15. THE SHAPE OF LUMINESCENCE SPECTRA
PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
THE SHAPE OF LUMINESCENCE SPECTRA
I. Phosphorescence and Fluorescence (emission) Spectrum both come at longer wavelengths compared to absorbance spectrum of the same molecule (Stokes shift).
II. Phosphorescence comes at lower energy = at longer wavelengths than fluorescence from the same molecule.
III. Fluorescence (emission) Spectrum of a molecule is more or less similar to its absorbance spectrum.
IV. max of a Fluorescence (emission) Spectrum is slightly shifted to longer wavelengths compared to max of the absorbance spectrum (Frank-Condon principle)
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Lecture 4, Luminescence,
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16. PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
What Do We Make Use Of?
Il (fluorescent intensity)
I0 (radiant intensity)
I (transmitted intensity)
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17. Measurement: Fluorometers
PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
Measurement: Fluorometers
Filter Fluorometers:
rather simple and cheap!
employ absorption or interference filters for limiting ex and em
Spectrofluorometers:
more sophisticated and expensive!
allow generation of both excitation and emission spectra,
employ two grating monochromators
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Lecture 4, Luminescence,
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18. MEASUREMENT: FLUOROMETERS (CONTD.)
PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
MEASUREMENT: FLUOROMETERS (CONTD.)
SPECTROFLUOROMETER
Spectrofluorometer: more sophisticated and expensive, radiation is split after passing first monochromator, reference part passes reference photomultiplier, sample part passes sample and finally sample photomultiplier.
A fluorescence signal produced when “a cell or particle labeled with a fluorochrome” passes through a laser excitation beam. centered while they pass through the laser beam
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Lecture 4, Luminescence,
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19. LIGHT SOURCES OF FLUOROMETERS
PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
LIGHT SOURCES OF FLUOROMETERS
in Spectrofluorimeter:
continuous radiation required
i) 75- to 450W high pressure xenon arc lamp, emitting 300 to 1300 nm
large power supply needed (5 to 20 A at 15 to 30 V)
ii) tunable dye LASERs – comparatively expensive; advantages: suitable for small samples (L or less), if highly monochromatic excitation is required, or for remote sensing
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20. OTHER PARTS OF FLUOROMETERS
PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
OTHER PARTS OF FLUOROMETERS
excitation and emission monochromator:
interference and absorption filters for filter fluorometers
grating monochromators for spectrofluorimeter
sample cell:
cylindrical and rectangular cells of glass or quartz
any fingerprints are even more disturbing than in absorbance spectroscopy
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Lecture 4, Luminescence,
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21. OTHER PARTS OF FLUOROMETERS
PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
OTHER PARTS OF FLUOROMETERS
detector:
the most common transducers are photomultiplier tubes (PMT) run in photon counting mode
the final detector output (fluorescence signal) is the ratio (division!) between the sample beam’s PMT signal intensity and the reference beam’s PMT signal
photon counting mode (applied for low intensity radiation):
analog signal is converted to a train of digital pulses  radiant power is proportional to the number of pulses per unit time.
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Lecture 4, Luminescence,
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22. PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
VARIABLES I
“Quantum yield” or “quantum efficiency”:
ratio of numbers of molecules luminescent to the total number of excited molecules.
Transition types:
normally, ex for fluorescence is >250 nm.
radiation of lower  is too energetic and may cause dissociation or pre-dissociation of the molecules in excited states, thus their deactivation.
As a consequence, either * n or *  transition are responsible for fluorescence, and not *  .
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Lecture 4, Luminescence,
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23. PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
VARIABLES II
For the Intensity I of fluorescent light...
...of the solution of a compound ...at a given concentration and a given wavelength we can write:
 : the molar absorption coefficient at the excitation wavelength. The greater , the more energy is absorbed and can be emitted via fluorescence
I0: intensity of the excitation light the more light is present the higher the fluorescence
Q: the quantum yield of fluorescence, i.e., the fraction of excitation light that is converted into fluorescent light (= the ratio of the emitted and the absorbed quantum.)
K: constant for the equipment used
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Lecture 4, Luminescence,
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24. PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
FLUORESCENCE POWER
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Lecture 4, Luminescence,
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25. PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
FLUORESCENCE SPECTRA
Quinine (1R,3R,4S,8S,9R)
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Lecture 4, Luminescence,
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26. PHCMt561 Instrumental Analysis - WS 12/13 - Lecture 4
SUMMARY
When a molecule absorbs light, it is promoted to an excited state from which it may return to the ground state by radiationless processes or by FLUORESCENCE (singlet singlet) or PHOSPHORESCNECE (triplet  singlet emission).
Any form of luminescence is potentially useful in QUANTITATIVE ANALYSIS, because emission intensity is proportional to sample concentration at low concentration.
An EXCITATION spectrum (a graph of excitation intensity versus excitation wavelength) is similar to an absorption spectrum (a graph of absorbance versus wavelength). An emission spectrum is observed at lower energy than the absorption spectrum due to Stokes shift and Frank Condon Principle.
A molecule that is not fluorescent can be analyzed by attaching a fluorescent group to it (FLUOROPHOR).
Light emitted from a chemical reaction (CHEMILUMINESCENCE) can also be used for quantitative analysis.
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Lecture 4, Luminescence,
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27. Lecture 4, Luminescence, 03-10-2012
REFERENCES
“Quantitative Chemical Analysis” by Harris, 6th edition, Chapter 18, p.422 to 426.
“Principles of Instrumental Analysis”, Skoog, 5th edition, Chapter 15.
Lecture of Dr. Raimund Niess, GUC, 2010.
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Lecture 4, Luminescence,