Molecular Luminescence Spectrometry

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Chapter 15

• Molecular Luminescence Spectrometry

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Fluorescence and Phosphorescence
A very sensitive and selective instrumental
technique with some of the lowest LOD's for
molecules that luminesce (not all do!) - down to
single-molecule detection.
diamagnetic diamagnetic paramagnetic
isoelectronic in external magnetic field isoelectronic splits into 3 energy levels
short (ns) lifetimes "allowed" longer (?s or longer) lifetimes "forbidden"
large ? small ?
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Energy Level Diagrams
(Jablonski Diagrams)
ps
ps
Note fluorescence, phosphorescence, and
non-radiative decay can all occur simultaneously
depending on the "quantum yields".
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Quantum Yields
The quantum yield for a given process is the
ratio of the number of molecules that undergo a
particular process to the total number of excited
states.
S1
T1
So
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Vibrational Relaxation
S1 (?)
S1 (?)
collisions
So (?)
So (?)
Kasha's Rule fluorescence in fluid solution
always occurs from the lowest vibrational level
of the 1st excited singlet state.
?
H
e-e
e-v

e-t
C O
.
H
solvent molecules
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Internal Conversion
S3
Favored by overlapping vibrational levels.
S2
S1
So
Intersystem Crossing
Change in spin state from a singlet to a triplet
and vica-versa.
S1
T1
So
Heavy atom effect atoms and small molecules
such as Hg, Br2, I2, and O2 have large
spin-orbit coupling that helps flip spin.
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Luminescence Lifetimes

• unique and useful for identification
• inversely proportional to ?
• Co initial population of the excited state
• k 1st order rate constant (units s-1)

Co
excited state population C ?
???
time ?
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Fluorescence and Structure
Most intense from molecules containing multiple
aromatic rings, ??? transitions, and structural
rigidity.
?f 1.0
no fluorescence
?f 0.2
fluorescence
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Illustration of the Heavy Atom Effect
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Emission and Excitation Spectra
excitation spectra measure luminescence at a
fixed wavelength while scanning the excitation
wavelengths. emission spectra - excite at a fixed
wavelength while scanning the luminescence.
overlap at the 0-0 band.
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Instruments for Measuring Fluorescence and
Phosphorescence
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Xe Arc Lamps
http//en.wikipedia.org/wiki/ImageXenon_short_arc
_1.jpg
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Spectrofluorometers
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Corrected Excitation Spectra
The absorbance compensating cell contains the dye
molecule Rhodamine B which absorbs all
wavelengths equally and emits in a pattern that
mimics the variation in the lamp output. The
corrected excitation spectra are produced by
dividing the raw spectrum with the Rhodamine B
spectrum.
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Phosphorimeters
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Selectivity
The ability to distinguish and identify different
molecules based on differences in luminescence
spectra. Frequently enhanced by cooling the
sample.
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Effect of Concentration on Fluorescence Intensity
Po
Pt
Pf
Converting to base e and expanding e-?bc in a
MacLaurin Series Pf ?fPo(1 2.303e-?bc) Pf
?fPo2.303?bc (2.303?bc)2/2!
(2.303?bc)3/3! ..
Pf ?fPa ?f(Po Pt) ?fPo(1 Pt/Po)
PtPa
from Beers Law
10-?bc
Pf ?fPo 2.303?bc
so Pf ?fPo(1 10-?bc)
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Absorbance vs. Fluorescence Signals
Why fluorescence is inherently more sensitive.
Pf ?fPo 2.303?bc
A -log P/Po
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Comparing UV-Vis to Fluorescence LOD's
anthracene (MW 178)
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Applications

1. Fluorimetric Determination of Inorganic Species
2. Fluorimetric Determination of Organic Species
3. Forensics (latent blood stains - luminol
   chemiluminescence)
4. Fluorescence Imaging Methods

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Fluorimetric Determination of Inorganic Species
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Fluorimetric Determination of Organic Species
Polynuclear Aromatic Hydrocarbons (PAH's) in the
environment.
anthracene
1,2-benzanthracene
perylene
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Applications to Forensics
http//en.wikipedia.org/wiki/Luminol
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Fluorescence Imaging Methods
http//www.microscopyu.com/articles/fluorescence/f
luorescenceintro.html
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