Fluorometry, ?????

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Dong-Sun Lee / cat-lab / SWU
2010-Fall Version
Chapter 27
Fluorescence spectrometry
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What is luminescence ? Luminescence is the
emission of photons from electronically excited
state. Luminescence is divided into two types,
depending upon the nature of the ground and the
excited states. In a singlet excited state, the
electron in the higher energy orbital has the
opposite spin orientation as the second electron
in the lower orbital. These two electrons are
said to be paired. Return to the ground state
from an excited singlet state does not require an
electron to change its spin orientation. In a
triplet state these electrons are unpaired, that
is, their spins have the same orientation. A
change in spin orientation is needed for a
triplet state to return to the singlet ground
state.
So
diamagnetic S1
paramagnetic T1
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Types of luminescence (classification according
to the means by which energy is supplied to
excite the luminescent molecule) 1)
Photoluminescence Molecules are excited by
interaction with photons of radiation. ?
Fluorescence Prompt fluorescence S1?
S0 h? The release of electromagnetic
energy is immediate or from the singlet state.
Delayed fluorescence S1? T1? S1? S0
h? This results from two intersystem
crossings, first from the singlet to the triplet,
then from the triplet to the singlet.
? Phospholuminescence T1? S0 h? A
delayed release of electromagnetic energy from
the triplet state. 2) Chemiluminescence The
excitation energy is obtained from the chemical
energy of
reaction. 3) Bioluminescence
Chemiluminescence from a biological system
firefly, sea pansy, jellyfish, bacteria,
protozoa, crustacea. 4) Triboluminescence A
release of energy when certain crystals, such as
sugar, are broken. 5) Cathodoluminescence A
release of energy produced by exposure to cathode
rays 6) Thermoluminescence When a material
existing in high vibrational energy levels emits
energy at a temperature below red heat, after
being exposed to small amounts of thermal energy.
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Fluorescence process A So h? ? S1 or S2
Radiation process Molecular fluorescence
spectrometry is based on the emission of light by
molecules that have become electronically excited
subsequent to the absorption of
visible(400700nm), UV(200400nm), or NIR (700
1100nm) radiation. Excitation process to the
excited state from the ground state is very fast,
on the order of 1015 s. VR vibrational
relaxation, non-radiational process,
1011 s 1010 s. IC internal conversion, S2?
S1 S1? S0 non-radiative process, 1012
s. ST intersystem crossing, S1? T1 F
fluorescence, S1? S0 h? 1010106 s. P
phosphorescence, T1? S0 h? 104 s 104 s.
Jablonski diagram.
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Example showing that phosphorescence comes at
lower energy than fluorescence from the same
molecule. The phosphorescence signal is 10 times
weaker than the fluorescence signal and is only
observed when the sample is cooled.
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(b)
?L
Photoluminescence methods. Absorption of
incident radiation from an external source (a)
causes excitation of the analyte to state 1 or
state 2 (b). Excited species can dissipate the
excess energy by emission of a photon
luminescence (L) or by radiationless processes
(dashed lines) in (b). Emission is isotropic (a),
and the frequencies emitted correspond to the
energy differences between levels (c).
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Emitted radiation ?E
2
E21 h?21 hc/?21
1
E2 h?2 hc/?2
E1 h?1 hc/?1
0
(b)
Sample
?E
Thermal, electrical, or chemical energy
?
?2 ?1 ?21
(a)
(c)
Emission and chemiluminescence(bioluminescence)
methods. In (a) the addition of thermal,
electrical or chemical energy causes
nonradiational excitation of the analyte and
emission of radiation in all directions
(isotropic emission). The energy changes that
occur during excitation (dashed lines) or
emission (soled lines) are shown in (b). The
energies of states 1 and 2 are usually relative
to the ground level and often abbreviated E1 and
E2, respectively. A typical spectrum is shown in
(c).
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Types of fluorescence and emission
processes Stokes fluorescence This is the
reemission of less energetic photons, which have
a longer wavelength than the absorbed photons.
One common cause of Stokes shift is the rapid
decay to the lowest vibrational level of S1.
Furthermore, fluorophores generally decay to
excited vibrational levels of So, resulting in
further loss of vibrational energy. In addition
to these effects, fluorophores can display
further Stokes shifts due to solvent effects and
excited state reactions. In gas phase, atoms and
molecules do not always show Stokes shifts.
Anti-Stokes fluorescence If thermal energy is
added to an excited state or a compound has many
highly populated vibrational energy levels,
emission at shorter wavelengths than those of
absorption occurs. This is often observed in
dilute gases at high temperature. Resonance
fluorescence This is the reemission of photons
possessing the same energy as the absorbed
photons. This type of fluorescence is never
observed in solution because of solvent
interactions, but it does occur in gases and
crystals. It is also the basis of atomic
fluorescence. Rayleigh scattering The emitted
light has the same wavelength as the exciting
light since the absorbed and emitted photons are
of the same energy. Raman scattering This is
a form of inelastic scattering which involve a
change in the frequency of the incident
radiation. Raman scattering involves the gain or
loss of vibrational quantum of energy by
molecules.
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Fluorescence efficiency quantum yield of
fluorescence The ratio of the fluorescence
radiant power to the absorbed radiant power where
the radiant powers are expressed in photons per
second. ? (luminescene radiant power) / (
absorbed radiant power) (number of photons
emitted) / (number of photons absorbed) 1 ? ? ?
0 The higher the value of ?, the greater the
fluorescence of a compound. A non-fluorescent
molecule is one whose quantum efficiency is zero
or so close to zero that thee fluorescence is not
measurable. All energy absorbed by such a
molecule is rapidly lost by collisional
deactivation.
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Fluorescence lifetime Another important property
of fluorescing molecules is the lifetime (?) of
the lowest excited singlet state. The lifetime of
excited state is defined by the average time the
molecule spends in the excited state prior to
return to the ground state. Generally,
fluorescence lifetimes are near 10 nsec. The
quantum yield of fluorescence and ? are related
by ? kf / (kf kd) kf ? where kf is
the rates of fluorescence, kd is the
radiationless rate of deactivation. Fluorescence
lifetime measurement is a valuable technique in
the analysis of multicomponent samples containing
analytes with overlapping fluorescence
bands. Joseph R. Lakowicz , Principles of
Fluorescence Spectroscopy, Plenum Press, New
York, 1983, pp 9-10. Stephen G. Schulman , (Alan
Townshend Edt.), Encyclopedia of analytical
science, Vol. 3, Academic Press, London, pp.
1358-1365.
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Fluorescence related to concentration The
fluorescence radiant power F is proportional to
the absorbed radiant power. F
?(Po P) where ? fluorescence efficiency,
Po incident power, P transmitted power The
relationship between the absorbed radiant power
and concentration can be obtained from Beers
law. P/ Po 10A 10?bC
P Po 10?bC F ? Po
(110?bC) When expanded in a power series, this
equation yields F ? Po (ln?bC)1/ 1! (
ln?bC)2 / 2! ( ln?bC)3 / 3! (ln?bC)4 / 4!
(ln?bC)n /n! If ?bC is 0.05 or less,
only the first term in the series is significant
and equation can be written as F
? Po (ln?bC) kbC where k is a constant equal to
? Poln?. Thus, when the concentrations are very
dilute and not over 2 of the incident radiation
is absorbed, there is linear relationship between
fluorescent power and concentration. When ?bC is
greater than about 1.5, 10?bC is much less than
1 and fluorescence depends directly on the
incident radiation power. F
? Po
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? Po
Fluorescence
Concentration of fluorescing species
Theoretical behavior of fluorescence as a
function of concentration.
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Structural factors affecting fluorescence 1.
Fluorescence is expected in molecules that are
aromatic or multiple conjugated double bonds with
a high degree of resonance stability. 2.
Fluorescence is also expected in polycyclic
aromatic systems. 3. Substituents such as NH3,
OH, F, OCH3, NHCH3, and N(CH3)2 groups,
often enhance fluorescence. 4. On the other hand,
these groups decrease or quench fluorescence
completely Cl, Br, I, NHCOCH3, NO2,
COOH. 5. Molecular rigidity enhances
fluorescence. Substances fluoresce more brightly
in a glassy state or viscous solution. Formation
of chelates with metal ions also promotes
fluorescence. However, the introduction of
paramagnetic metal ions gives rise to
phosphorescence but not fluorescence in metal
complexes. 6. Changes in the system pH, if it
affects the charge status of chromophore, may
influence fluorescence.
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Typical aromatic molecules that do not fluoresce.
Typical aromatic molecules that fluoresce.
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Effect of molecular rigidity on quantum yield.
The fluorene molecule is held rigid by the
central ring, two benzene rings in biphenyl can
rotate to one onother.
Effect of rigidity on quantum yield in complexes.
Free 8-hydroxyquinoline molecules in solution are
easily deactivated through collision with solvent
molecules and do not fluoresce. The rigidity of
the Zn 8-hydroxyquinoline complex enhances
fluorescence.
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Substitution effects on the fluorescence of
benzene. Substituent
Changes in wavelength Changes in
intensity
of fluorescence
of fluorescence Alkyl
None
None OH, CH3, OC2H5
Decrease
Increase COOH
Decrease
Large decrease NH2, NHR, NR2
Decrease
Increase NO2, NO
-
Total quenching CN
None
Increase SH
Decrease
Decrease F, Cl, Br, I
Decrease (F? I)
Increase ( F ? I ) SO3H
None
None Larry G. Hargis ,
Analytical Chemistry-principles and techniques,
Prentice-Hall, 1988, p 435.
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Fluorescence of linear aromatics in a mixture of
ethanol, isopropanol and ether. Compound
? ?ex (nm)
? em (nm) Benzene
0.11 205
278 Naphthalene 0.29
286 321 Anthracene
0.46 365
400 Naphthacene
0.60 390
480
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Fluorescence and environment 1. Temperature A
rise in temperature almost always is accompanied
by a decrease in fluorescence because the
greater frequency of collisions between molecules
increases the probability for deactivation by
internal conversion and vibrational relaxation.
2. pH Changes in pH influence the degree
of ionization, which, in turn, may affect the
extent of conjugation or the aromaticity of the
compound. 3. Dissolved oxygen Dissolved
oxygen often decreases fluorescence dramatically
and is an interference in many fluorometric
methods. Molecular oxygen is paramagnetic (has
triplet ground state), which promotes intersystem
crossing from singlet to triplet states in other
molecules. The longer lifetimes of the triplet
states increase the opportunity for radiationless
deactivation to occur. Other paramagnetic
substances, including most transition metals,
exhibit this same effect. 4. Solvents
Solvents affect fluorescence through their
ability to stabilize ground and excited states
differently, thereby changing the probability and
the energy of both absorption and emission.
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Common problems of fluorescence measurements 1)
Reference materials is as fluorescent as the
sample Contaminating substances Raman
scattering, Rayleigh scattering 2) Fluorescence
reading is not stable Fogging of the cuvet
when the contents are much colder than the
ambient temperature. Drops of liquid on the
external faces of the cuvet. Light passing
through the meniscus of the sample. Bubbles
forming in the solution as it warms.
Quenchers molecular oxygen 3) Sensitivity is
inadequate D.A. Harris, C.L. Bashford ,
Spectrophotometry spectroflurimetry- a
practical approach, IRL Press, Oxford, UK, 1987,
p.18-20.
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Problems with photoluminescence 1)
Self-quenching Self-quenching results when
luminescing molecule collide and lose their
excitation energy by radiationless transfer.
Serious offenders are impurities, dissolved
oxygen, and heavy atoms or paramagnetic species
(aromatic substances are prime offenders). 2)
Absorption of radiant energy Absorption either
of the exciting or of the luminescent radiation
reduces the luminescent signal. Remedies involve
(a) dilution the sample, (b) viewing the
luminescence near the front surface of the cell,
and (c) using the method of standard additions
for evaluating samples. 3) Self-absorption
Attenuation of the exciting radiation a sit
passes through the cell can be caused by too
concentrated an analyte. The remedy is to dilute
the sample and note whether the luminescence
increases or decreases. If the luminescence
increases upon sample dilution, one is working on
the high-concentration side of the luminescence
maximum. This region should be avoided. 4)
Excimer formation Formation of a complex
between the excited-state molecule and another
molecule in th ground state, called an excimer,
causes a problem when it dissociates with the
emission of luminescent radiation at longer
wavelengths than the normal luminescence.
Dilution helps lesson this effect. John A. Dean,
Analytical Chemistry Handbook, McGraw-Hill, 1995,
New York, p.5.55
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Excitation spectrum and emission spectrum The
excitation spectrum is a measure of the ability
of the impinging radiation to raise a molecule to
various excited states at different wavelengths.
An excitation spectrum is recording of
fluorescence versus the wavelength of the
exciting or incident radiation and it is obtained
by setting the emission monochromator to a
wavelength where fluorescence occurs and scanning
the excitation monochromator. An excitation
spectrum looks very much like an absorption
spectrum, because the greater the absorbance at
the excitation wavelength, the more molecules are
promoted to the excited state and the more
emission will be observed. The emission
(fluorescence) spectrum is a measure of the
relative intensity of radiation given off at
various wavelength as the molecule returns from
the excited states to the ground state. The
emission spectrum is recording of fluorescence
versus the wavelength of the fluorescence
radiation, and it is obtained by setting the
excitation monochromator to a wavelength that the
sample absorbs and scanning the emission
monochromator. Since some of the absorbed energy
is usually lost as heat, the emission spectrum
occurs at longer wavelengths (lower energy) than
does the corresponding excitation spectrum. If an
emission spectrum occurs at shorter wavelengths
than the excitation spectrum, the presence of a
second fluorescing species is confirmed. The
absorption and emission spectra will have an
approximate mirror image relationship if the
spacings between vibrational levels are roughly
equal and if the transition probabilities are
similar.
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Energy level diagram showing why structure is
seen in the absorption and emission spectra, and
why the spectra seem roughly mirror images of
each other.
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Excitation and emission spectra of anthracene,
illustrating the mirror-image relationship
between absorption (A) and fluorescence (F),
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Absorption and fluorescence emission spectra of
perylene and quinine. Joseph R. Lakowicz ,
Principles of Fluorescence Spectroscopy, Plenum
Press, New York, 1983, p 3.
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Absorption (black line) and emission (colored
line) spectra of N-methlcarbazole in cyclohexane
solution, illustrating the approximate mirror
image relationship between absorption and
emission.
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Diagram showing why the transition do not exactly
overlap.
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Instrumentation for fluorescence spectroscopy
General layout of fluorescence spectrophotometer
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Schematic diagram of a typical spectrofluorometer.
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1) Light sources a. Gas discharge lamps
Xenon arc lamp High pressure
mercury vapor lamp b. Incandescent lamps
Tungsten wire filament lamp c. Laser tunable
dye laser d. X-ray source for X-ray
fluorescence 2) Wavelength selection devices a.
Filters Absorption filters ---tinted
glass or gelatin containing dyes sandwiched
between glass Interference filters
---thin transparent layer of CF2 or MgF2
sandwiched two parallel,
partially refelecting metal
films b. Monochromators Gratings
Prism
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Cross-sectional view of an interference filter
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Transmittance characteristics of sharp-cut and
bandpass filters.
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Proper choice of primary and secondary filters to
avoid interference from another substance a)
excitation spectra (both substances fluoresce
over same wavelength region, b) fluorescence
spectra (both substances absorb in same
wavelength region).
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3) Sample compartment Fluorescence cells
---- right angle design or small angle(37o)
viewing system Quarz or fused silica
----200 nm 800 nm Glass or plastic
---- 300 nm 4) Detectors
Photomultiplier Photoconductive target
vidicon Return beam vidicon Intensified
target vidicon
Stephen G. Schulman , (Alan Townshend Edt.),
Encyclopedia of analytical science, Vol. 3,
Academic Press, London, pp. 1358-1365.
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Schematic of a fibre optic based multichannel
fluorometer. IDA512 element intensified linear
photodiode array detector, Llens, OF1 and OF2
the excitation and emission fibres. Stephen G.
Schulman , (Alan Townshend Edt.), Encyclopedia of
analytical science, Vol. 3, Academic Press,
London, p 1396.
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Generation of fingerprint excitation-emission
matrix. a) EEM of pure component, compound A, b)
EEM of pure component, compound B, c)fingerprint
EEM of a mixture of compound A and B, d)
isometric projection of fingerprint in c).
Shelly et al., Clinical Chemistry 26, 1127-1132,
1980.
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Applications 1) Direct measurement --- metal
cations as fluorescent chelates 2) Indirect
measurement where the fluorescence of the
substance being determined is measured prior
to and after quenching 3) Indirect measurement
where the fluorescence of the determined
substance is enhanced by the addition of a
reacting material. 4) Tracer techniques ---
bioengineered anlysis.
FISH(fluorescence in situ
hybridization) 5) SFS( spectral fluorescent
signatures)
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4-Bromomethyl-7-methoxycoumarin specific for
carboxylic acid
Fluorescamine, specific for primary and
secondary amines
OPA, specific for N-methylcarbamate and primary
amines
9-fluorenylmethoxycarbonyl(FMOC) primary amine
(ex. Gluphosinate)
Derivatization reactions for fluorescence
detection.
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Examples of naturally fluorescent organic
compounds Compound Wavelength or
Range of ?em(nm) Compound
Wavelength or Range of ?em(nm) Aromatic
hydrocarbons
Drugs

Naphthalene
300-365
Asprine
335 Anthracene
370-460
Codeine
350 Pyrene
370-400
Diethylstibestor
435 1,2-Benzopyrene
400-450
Estrogens
546 Heterocyclic compound

Lysergic acid diethylamide(LSD)
365 Quinoline
385-490
Phenobarbital
440 Quinine sulfate
410-500
Procaine
345 7-Hydroxycoumarine
450
Steroid 3-Hydroxyindol
380-460
Aldosterone
400-450 Dyes

Cholesterol
600 Fluorescein
510-590
Cortisone
580 Rhodamine B
550-700
Prednisolone
570 Methylene Blue
650-700
Testosterone
580 Naphthol
516
Vitamines Coenzymes, nucleic acids,
pyrimidines
Ribofravin(B2)
565 Adenine
380
Cyanocobalamin(B12)
305 Adenosine triphosphate(ATP)
390
Tocopherol(E)
340 Nicotinamide adenine
dinucleotide(NADH)
460 Purine
370 Thymine
380
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Linear calibration curve for fluorescence of
anthracene measured at the wavelength of maximum
fluorescence.
Calibration curve for the spectrofluorometric
determination of tryptophane in soluble proteins
from the lens of a mammalian eye.
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