Transcript 2. Spectrofluorimetry Dr. Hisham E Abdellatef

2. Spectrofluorimetry
Dr. Hisham E Abdellatef
[email protected]
Instruments for Measuring
Absorption of Light….
Fluorescence and Phosphorescence
Excitation Beam
Emitted Beam
Detector
Resonance Fluorescence
• Resonance Fluorescence
– Usually atomic
– Emitted light has same E as excitation light
– Simpler, atomic systems with fewer energy states (vs
molecules) undergo resonance fluorescence
• Not as widely used in analytical chemistry as non-resonance
fluorescence
– Hg analysis is one example
Excitation Beam
Emission (identical E)
Non-resonance Fluorescence
• Typical of molecular fluorescence
• Large number of excited states
– rotational
– vibrational
– etc..
• Molecules relax by ‘stepping’ from one state to another
• Resulting emitted light “shifts” to lower energies
– longer wavelengths = lower energy
Excitation Beam
Emission (lower E, longer  )
Chapter 15 Molecular Luminescence
Important topics in this chapter:
Energy diagram and basic concepts
Fluorescence quantum yield
Fluorescence instrumentation
Homowork in Chapter 15: 1, 2, 3, 4, 6, 7
Luminescence:
1. Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications
Singlet: all electron spins are paired; no energy level splitting
occurs when the molecule is exposed to a magnetic field;
Triplet: the electron spins are unpaired and are parallel;
excited triplet state is less energetic than the corresponding
singlet state.
Diamagnetic: no net magnetic field due to spin paring. The
electrons are repelled by permanent magnetic fields.
Paramagnetic: magnetic moment and attracted to a
magnetic field (due to unpaired electrons).
Ground
Single state
Excited
Single state
Excited
triplet state
Partial energy diagram for a photoluminescent system
Deactivation processes for an excited state:
Vibrational relaxation: fluorescence always involves a
transition from the lowest vibrational states of an excited
electronic state; electron can return to any one of the vibrational
levels of the ground state; 10 -12 s;
Internal conversion: intramolecular processes by which a
molecule passes to a lower-energy electronic state without
emission of radiation.
External conversion: interaction and energy transfer
between the excited molecule and the solvent or other
molecules.
Intersystem crossing: the spin of an excited electron is
reversed and a change in multiplicity of the molecule results.
Phosphorescence: an excited triplet state to give radiative
emission. emission: a photon is emitted.
Comparison of Triplet and Singlet
magnetic effect
electron transition for
emission
radiation induced
excitation
luminescence
life time
Singlet
diamagnetic
more probable
more probable
Triplet
paramagnetic
less probable
(unlikely)
less probable
Fluorescence
short, < 10-5 s to
10-9 s
Phosphorescence
long, 10-5 s to several
seconds or longer
Fluorescence and Phosphorescence
Comparison of Fluorescence and
Phosphorescence
Fluorescence
life time
short, < 10-5s
electron spin
no
excited states
singlet
quantum yield
high
temperature most temperature
Phosphorescence
long, several seconds
yes
triplet
low
low temperature more likely
Resonance fluorescence: absorbed radiation is
re-emitted without a change in frequency.
Stokes shift: molecular fluorescence bands are
shifted to wavelengths that are longer than the
resonance line.
Luminescence:
1. Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications
Variables that affect Fluorescence and phosphorescence
Quantum yield:
the ratio of the number of molecules that
luminescence to the total number of excited molecules.
f = kf/ (kf + ki + kec + kic + kpd + kd)
kf: Fluorescence constant
ki: Intersystem crossing constant
kec: External conversion constant
kic: Internal conversion constant
kpd: Predissociation constant
kd: Dissociation constant
* transitions: high quantum efficiency
F=
kf
kf + knr
Quantum yield = kf / (kf + ki + kec + kic + kpd + kd)
Quantum yield can be close to unity if the radiationless
decay rate is much smaller the the radiative decay.
High quantum yield molecules: rhodamine, fluorescein etc
Effect of structural rigidity: Molecules with rigid structures
have high fluorescence yield.
Nonrigid molecule can undergo low-frequency vibrations. kic
Effect of Concentration on Fluorescence Intensity
Power of fluorescence emission F
F = K’ (I0 –I)
I0 and I are the intensities of excitation lights before and
after absorbed by the analytes. K’ is the constant
related to the quantum yield
I/I0 = 10-ebc
F = K’ I0 (1–10-ebc)
F = 2.3 K’ I0 ebc, (when ebc<0.05)
luminescence in quantitative analysis:
inherent sensitivity (usually three orders of magnitude
better than absorption methods;
Better selectivity than absorption spectroscopy;
The precision and accuracy of photoluminescence
method is usually poorer than spectrophotometer by a
factor of two to five.
Less widely applicable than absorption spectroscopy;
Luminescence Lifetime:
average time the molecule spends in the excited
state prior to return to the ground state
t=
1
Kf + Knr
determines the time available for the fluorophore to interact
with or diffuse in its environment, and hence the information
available from its emission.
Lifetime measurements:
ps or fs lasers used for lifetime measurements;
fluorescence lifetime refers to the mean lifetime of the
excited state, i.e., the probability of finding a given molecule
that has been excited still in the excited state after time t is
exp(-t/t0):
I = I0 e(-t/t0)
precise measurement of the observed lifetime is
important since it can be used to calculate the natural lifetime
t0 (life time in the absence of nonradiative processes, also
called intrinsic lifetime).
For a single exponential decay, 63% of the
molecules have decayed prior to t=t0.
Luminescence:
1. Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications
Mirror images of absorption and fluorescence spectra:
vibrational levels in the ground and excited states have
similar energy gaps, thus absorption and fluorescence spectra
have mirror images (Fig. 15-1).
Figure l.3. Absorption
and fluorescence
emission spectra of
perylene and quinine.
Emission spectra cannot
be correctly presented on
both the wavelength and
wavenumber scales. The
wavenumber
presentation is correct in
this instance.
Wavelengths are shown
for convenience. See
Chapter 3. Revised from
Ref. 5.
Internal conversion: excitation by 1 and 2 produces the same
fluorescence 3.
Qunnine: two absorption bands: 250 nm and 350 nm;
fluorescence at 450 nm.
Figure 15-2
Fluorescence excitation and emission
spectra for a solution of quinine.
Figure 15-5
Fluorescence spectra for
1 ppm anthracene in
alcohol:
(a) excitation spectrum;
(b) emission spectrum.
Figure 15-3 Spectra for phenanthrene: E, excitation; F,
fluorescence; P, phosphorescence. (From W. R. Seitz, in
Treatise on Analytical Chemistry, 2nd ed., P. J. Elving, E. J.
Meehan, and I. M. Kolthoff, Eds., Part I, Vol. 7, p. 169.
New York: Wiley, 1981. Reprinted by permission of John
Wiley & Sons, Inc.)
Luminescence:
1. Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications
Components of a fluorometer:
sources;
wavelength selection: two wavelength selection
devices;
detectors;
sample cell.
Figure 15-4 Components of a fluorometer of a spectrofluorometer.
Figure 15-6
A typical fluouometer.
Farrand Optical Co., Inc.)
(courtesy of
Figure 15-7 A spectrofluorometer. (Courtesy of SLM
Instruments, Inc., Urbana, IL.)
Figure 15-8 (a) Schematic of an optical system for
obtaining a total luminescence spectrum with a twodimensional charge-coupled device. (b) Excitation
and emission spectra of hypothetical compound. (c)
Total luminescence spectrum of compound in b.
Figure 15-9
Schematic of a device for alternately
exciting and observing phosphorescence.
Luminescence:
1. Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications
Fluorescence Sensing
sensing is based on changes in fluorescence signal
either in intensity or in spectrum.
Fluorophore based sensors:
Enzyme based sensors:
Ion sensors
DNA/RNA sensors
neurotransmitter sensors
environmental sensors
Ion Sensors
phosphorimetric methods:
better selectivity;
poorer precision; lower temperature;
heavy atom results in strong phosphorescence
room temperature methods:
deposit analytes on surface: rigid matrix minimize
deactivation of the triplet state by external and internal
conversions;
Using micelles: micelles increase the proximity
between heavy metal ion and the phosphur, thus enhance
phosphorescence.
Chemiluminescence
chemiluminescence is produced when a chemical
reaction yields an electronically excited species, which emits
light as it returns to its ground states.
A + B C* + D
C*  C + hv
NO + O3  NO2* +O2
NO2*  NO2 + hv
Measurements of chemiluminescence is simple:
only detector, no excitation necessary
Figure 15-11 Chemiluminescence emission intensity
as a function of time after mixing reagents.
Preview:
Laser Chapter 7
Homework:
Chapter 7: 6
Instruments for Measuring
Absorption of Light….
Fluorescence and Phosphorescence
Right angle
Excitation Beam
Emitted Beam
Detector
Filter = flurometer
Prism and grating
Fluorescence and Phosphorescence
Excited single state S1 or
S2
Excited
triplet state
phosphorescence
Ground state
Fluorescence
Factors influencing intensity of fluorescence
1.
2.
3.
4.
5.
Concentration of fluorescing species F
Presence of other solutes
pH
Temperature
Photocomposition of sample due to
sunlight
6. viscosity
Disadvantages of fluoremetry
1.
2.
3.
4.
5.
Dilute solution are less stable
Adsorption on the surface of container
Oxidation of fluorescence sample
Photodecomposition
Quenching (even traces of non fluorescent
can quench a fluorescent one in S1 state)
6. It does not exhibit very high precision or
accuracy (2 – 10%)
Difference between fluorometry and
spectrophotometry
difference
Fluorometry
spectrophotometry
nature
Measuring emission
Measuring absorption
sensitivity
Nanogram scale (102 -104
times sensitive)
Microgram
instrumentation
Single beam
Single or double beam
Use 2 filter monochromatic
Only one
Selectivity
more
Less selective
Lambda
maximum
Absorption and emission
Absorption only
equations
F=2.3 QIε. B.C
A = ε. B.C
Calibration
Quinine in dilute H2SO4
Potassium chromate in H2O