Transcript Fluorometry, 형광분석법

Dong-Sun Lee / cat-lab / SWU
2010-Fall Version
Chapter 27
Fluorescence spectrometry
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
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.
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(400~700nm), UV(200~400nm), or
NIR (700 ~ 1100nm) radiation. Excitation
process to the excited state from the ground
state is very fast, on the order of 10–15 s.
VR: vibrational relaxation,
non-radiational process, 10–11 s ~10–10 s.
IC : internal conversion, S2 S1 S1 S0
non-radiative process, 10–12 s.
ST : intersystem crossing, S1 T1
F : fluorescence, S1 S0 + h 10–10~10–6 s.
Jablonski diagram.
P : phosphorescence, T1 S0 + h
10–4 s ~104 s.
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.
2
Luminescence
L
E21 = h21 = hc/21
1
E2 = h2 = hc/2
E1 = h1 = hc/1
0
(b)
Incident
radiation
0
Sample
(a)
Transmitted
radiation

L
2
1
21

(c)
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).
Emitted
radiation
E
2
E21 = h21 = hc/21
1
E2 = h2 = hc/2
E1 = h1 = hc/1
0
(b)
Sample
E
(a)
Thermal,
electrical,
or chemical
energy
2
1
21

(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).
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.
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.
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.
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
Beer’s law.
P/ Po = 10–A = 10–bC
P = Po 10–bC
F =  Po (1–10–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
 Po
Concentration of fluorescing species
Theoretical behavior of fluorescence as a function of concentration.
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.
Typical aromatic molecules that do
not fluoresce.
Typical aromatic molecules that fluoresce.
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.
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
NO2, NO
Decrease
-
Increase
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.
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
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.
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.
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
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.
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.
Excitation and emission spectra of anthracene,
illustrating the mirror-image relationship between
absorption (A) and fluorescence (F),
Absorption and fluorescence
emission spectra of perylene
and quinine.
Joseph R. Lakowicz , Principles of
Fluorescence Spectroscopy, Plenum
Press, New York, 1983, p 3.
Absorption (black line) and emission (colored line) spectra of N-methlcarbazole
in cyclohexane solution, illustrating the approximate mirror image relationship
between absorption and emission.
Diagram showing why the transition do not exactly overlap.
Instrumentation for fluorescence spectroscopy
Sample cell
Power
supply
Source Excitation
monochromator
Slit
Emission monochromator
Detector
Data processor
General layout of fluorescence spectrophotometer
Schematic diagram of a typical spectrofluorometer.
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
Cross-sectional view of an interference filter
Transmittance characteristics of sharp-cut and bandpass filters.
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).
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.
Schematic of a fibre optic based multichannel fluorometer.
IDA=512 element intensified linear photodiode array detector, L=lens, 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.
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.
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)
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.
Examples of naturally fluorescent organic compounds
Compound
Wavelength or Range of em(nm)
Aromatic hydrocarbons
Naphthalene
300-365
Anthracene
370-460
Pyrene
370-400
1,2-Benzopyrene
400-450
Heterocyclic compound
Quinoline
385-490
Quinine sulfate
410-500
7-Hydroxycoumarine
450
3-Hydroxyindol
380-460
Dyes
Fluorescein
510-590
Rhodamine B
550-700
Methylene Blue
650-700
Naphthol
516
Coenzymes, nucleic acids, pyrimidines
Adenine
380
Adenosine triphosphate(ATP)
390
Nicotinamide adenine
dinucleotide(NADH)
460
Purine
370
Thymine
380
Compound
Wavelength or Range of em(nm)
Drugs
Asprine
Codeine
Diethylstibestor
Estrogens
Lysergic acid diethylamide(LSD)
Phenobarbital
Procaine
Steroid
Aldosterone
Cholesterol
Cortisone
Prednisolone
Testosterone
Vitamines
Ribofravin(B 2)
Cyanocobalamin(B 12)
Tocopherol(E)
335
350
435
546
365
440
345
400-450
600
580
570
580
565
305
340
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.
Q n A
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