chapter 27 fluorescence spectrometry dong-sun lee / cat-lab / swu 2010-fall version
TRANSCRIPT
Chapter 27
Fluorescence spectrometry
Dong-Sun Lee / cat-lab / SWU 2010-Fall Version
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.
diamagnetic S1 paramagnetic T1So
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.
Jablonski diagram.
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.
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.
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).
Sample
Luminescence L
Incidentradiation 0
Transmitted radiation
(a)
2
1
0
E21 = h21 = hc/21
E2 = h2 = hc/2
E1 = h1 = hc/1
(b)
L
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).
Sample
Emitted radiation E
Thermal, electrical,or chemical energy
(a)
1
0
E21 = h21 = hc/21
E2 = h2 = hc/2
E1 = h1 = hc/1
2
(b)
E
2 1 21
(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 [(lnbC)1/ 1! – (– lnbC)2 / 2! – (– lnbC)3 / 3! – (lnbC)4 / 4! } – … – (–lnbC)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 (lnbC) = 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
Fluo
resc
ence
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 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.
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
Power
supply
Source Excitation monochromator
Emission monochromator
Detector
Sample cell
Slit
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)
Derivatization reactions for fluorescence detection.
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)
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
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.
