Ch18 Fundamentals of Spectrophotometry Part I An Introduction to Optical Spectroscopy

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Ch18Fundamentals of SpectrophotometryPart I
An Introduction to Optical Spectroscopy
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CONTENTS

• 1. Overview of Optical Spectroscopy
• 1) Electromagnetic Radiation
• 2) Wave-Particle Duality
• 3) Electromagnetic Spectrum
• 4) Types of Quantum Transition
• 5) Spectroscopies without Energy Exchange
• 6) Spectroscopies Involving Energy Exchange
• 2 Photometer
• 1) Classification
• 2) Conceptual Block Diagram of Spectrometer

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• 3. Basic Components of Spectroscopic
  Instrumentation
• 1) Sources of Energy
• 2) Wavelength Selector
• 3) Sample Holder/Matrix
• 4) Detectors
• 5) Signal Processors (omitted)

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Overview of Optical Spectroscopy
1) Electromagnetic Radiation
(a) Plane-polarized electromagnetc radiation
(b) 2D representation of electric factor
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2) Wave-Particle Duality
? wavelength ? frequency v light
speed C 3x108 m/s (in vacuum) n refractive
index In vacuum n 1 ? wavenumber
(cm1) ?E energy gap h Planks constant,
6.626x1034Js
Light measurement Power (P) The flux of energy
per unit time. Intensity (I) -The flux of energy
per unit time per area.
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? change between different medium, ? remains
constant Speed of light c/n, n (usually n gt
1) is the refractive index of the medium
Fig 24-2, p.713
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3) Electromagnetic Spectrum
?-ray
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Vacuum ultraviolet (VUV) 120185 nm Ultraviolet
(UV) 185400 nm Visible 400700 nm Near
infrared regions (NIR) 700850 nm. Far infrared
(FIR) 251000 ?m.
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4) Types of Quantum Transition
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5) Spectroscopies without Energy Exchange
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6) Spectroscopies Involving Energy Exchange
(1) Classification
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(2) Glossary for Spectroscopies Involving Energy
Exchange
i) Optical spectroscopy (Involving Energy
Exchange) Methods based on the absorption,
emission, luminescence of electromagnetic
radiation that is proportional to the amount of
analyte in the sample. ii) Absorption
spectroscopy Measuring the quantized energy
absorbed by atoms/molecules. iii) Emission
spectroscopy Exciting atom by heat (thermal),
then, the emitted quantized energy from excited
state to ground states is measured.
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• iv) Photoluminescence Exciting atom/molecule by
  light, then, the emitted quantized energy is
  measured.
• Fluorescence The ground state with the same spin
  as excited state.
• Phosphorescence The ground state with the
  opposite spin as excited state.
• v) Chemoluminescence (chemiluminescence) The
  luminescence (emission light) is the result of a
  chemical reaction.

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(3) Energy transition process illustrate
i) Absorption process
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Internal Energy of Molecules
EtotalEtransEelecEvibErotEnucl Eelec
electronic transitions (UV, X-ray) Evib
vibrational transitions (Infrared) Erot
rotational transitions (Microwave) Enucl nucleus
spin (nuclear magnetic resonance) or (MRI
magnetic resonance imaging)
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Electronic transitions

• There are three types of electronic transition
• which can be considered
• Transitions involving p, s, and n electrons
• Transitions involving charge-transfer electrons
• Transitions involving d and f electrons

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Absorbing species containing p, s, and n electrons

• Absorption of ultraviolet and visible radiation
  in organic molecules is restricted to certain
  functional groups (chromophores) that contain
  valence electrons of low excitation energy.

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10.6
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Vacuum UV or Far UV (?lt190 nm )
UV/VIS
? h c/?E
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s s Transitions

• An electron in a bonding s orbital is excited to
  the corresponding antibonding orbital. The energy
  required is large. For example, methane (which
  has only C-H bonds, and can only undergo s s
  transitions) shows an absorbance maximum at 125
  nm.

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n s Transitions

• Saturated compounds containing atoms with lone
  pairs (non-bonding electrons) are capable of n
  s transitions. These transitions usually need
  less energy than s s transitions. They can be
  initiated by light whose wavelength is in the
  range 150 - 250 nm. The number of organic
  functional groups with n s peaks in the UV
  region is small.

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n p and p p Transitions

• Most absorption spectroscopy of organic compounds
  is based on transitions of n or p electrons to
  the p excited state.
• These transitions fall in an experimentally
  convenient region of the spectrum (200 - 700 nm).
  These transitions need an unsaturated group in
  the molecule to provide the p electrons.

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ii) Emission or Chemoluminescence process
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iii) Photoluminescence process
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2. Photometer
Photometer A any instrument for detecting
scattered light intensity, absorption, or
fluorescence.
1) Classification (1) Spectrometer An
instrument used to measure properties of light
that uses filters to isolate the excitation
wavelengths. (2) Spectrophotometer A photometer
for measuring absorbance of UV, Vis, NIR, that
equipped a monochromator to select the wavelength.
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• Fluorometer An instrument for measuring
  fluorescence that uses filters to isolate the
  excitation and emission wavelengths.
• Spectrofluorometer an instrument for measuring
  fluorescence that uses a monochromator to select
  the excitation and emission wavelengths.
• Luminator An instrument for measuring
  chemiluminescence (bioluminescence).

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2) Conceptual Block Diagram of Spectrometer
(1) For absorption measurement
Light Source
Wavelength selector
Detector
Signal processor and readout
Sample
(2) For emission measurement
Wavelength selector
Detector
Signal processor and readout
Sample
Thermal
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(3) For fluorescence measurement
Light Source
Wavelength selector
Sample
Wavelength selector
Signal processor and readout
Detector
(4) For chemiluminescence measurement
Sample Reagent
Signal processor and readout
Wavelength selector
Detector
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3. Basic Components of Spectroscopic
Instrumentation

• i) Sources of Energy

Common Sources of Electromagnetic Radiation for
Spectroscopy
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Deuterium Lamps
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Tungsten Lamp
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??
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2) Wavelength Selector
(1) Filter A wavelength selector that uses
either absorption, or constructive and
destructive interference to control the range of
selected wavelengths. a) Absorption filter b)
Interference filter
(2) Monochromator A wavelength selector that
uses a diffraction grating or prism, and that
allows for a continuous variation of the nominal
wavelength. a) Diffraction grating b) Prism
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Diffraction Grating A diffraction grating is the
tool of choice for separating the colors in
incident light.                                 
                                                  
                                              
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Monochromator grating
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(3) Polychromator A wavelength selector in which
different directions of the dispersed light (by
prism or diffraction grating), simultaneously
detected by a multi-detector (e.g., photodiode
array, PDA)
(4) Interferometer A device that allows all
wavelengths of light to be measured
simultaneously. The signal shows a function of
the moving mirrors position. The result is
called an interferogram, or a time domain
spectrum. The time domain spectrum is converted
by Fourier Transform, to the normal frequency
domain spectrum.
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Ch18Fundamentals of Spectrophotometry Part II
UV-Visible Spectrophotometry
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CONTENTS
1. Basics of Atomic UV/Vis Light
Absorption 2. Basics of Molecular UV/Vis Light
Absorption 1) Energy levels 2) Example of
UV-Vis spectra 3) UV/Vis absorption by organic
compounds 4) UV/Vis absorption by inorganic
compounds 3. Molecular UV/Visible
Spectrophotometer 1) Single beam
instrument 2) Double beam instrument 3) Phodedio
de array (PDA) spectrophotometer 4. Absorbance
and Concentration 1) Transmittance and
Absorbance 2) Beers Law 3) Quantitative
Application of Beers Law 4) Limitations to
Beers Law
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Basics of optical spectroscopy

• Scattering
• Absorption spectroscopy
• Fluorescence detection

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1. Basics of Atomic UV/Vis Light Absorption
1) Description When a atom absorbs specific
quantized UV/Vis radiation, it undergoes a change
in its valence electron configuration
e
h?
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color wheel
complementary colors are diametrically opposite
each other
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Electronic transitions

• There are three types of electronic transition
• which can be considered
• Transitions involving p, s, and n electrons or
  valance electrons
• Transitions involving charge-transfer electrons
• Transitions involving d and f electrons

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I-I Transitions involving valance electrons
Sodium (Na) for example electron configuration
in ground state is Ne3s1. Partial energy level
diagram of unoccupied, higher energy atomic
orbitals
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3) Atomic spectrum (line spectra) of Na consists
of a few, discrete absorption lines corresponding
to transitions between the ground state. Na for
example 3s?3p and 3s?4p etc.
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I-2 Transitions involving p, s, and n electrons
2. Basics of Molecular UV/Vis Light Absorption

Vibration level
Electronic Excited
1) Energy levels Molecular UV absorptions are
generally broad band (band spectra) because
vibrational and rotational levels are
"superimposed" on the electronic levels.

Vibration level
Electronic Excited

Vibration level
Electronic Ground state
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2) Example of UV-Vis spectra
Gaseous phase
Nonpolar solvent
Analyte 1,2,4.5-tetrazine
Polar solvent
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3) UV/Vis absorption by organic compounds
valence electron transition molecular orbitals
energy level of molecular orbitals
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Molecular orbitals of Chromophore
Chromophore The unsaturated organic functional
groups in a molecule responsible for the
absorption of a particular wavelength of light,
i.e., gives color to the molecule.
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Molecular Orbitals (MO).
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Experiments show O2 is paramagnetic
No unpaired e-
Should be diamagnetic
Molecular orbital theory bonds are formed from
interaction of atomic orbitals to form molecular
orbitals.
10.6
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Energy levels of bonding and antibonding
molecular orbitals in hydrogen (H2).
A bonding molecular orbital has lower energy and
greater stability than the atomic orbitals from
which it was formed.
An antibonding molecular orbital has higher
energy and lower stability than the atomic
orbitals from which it was formed.
10.6
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10.6
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Li2
10.6
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Two Possible Interactions Between Two Equivalent
p Orbitals
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10.6
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10.7
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paramagnetic nature of oxygen
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HF
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Formaldehyde for example
Ground state
Excited state
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MO of formaldehyde
n 4 e p 2 e ? 6 e Total 12 e 6 MOs
HOMO?LUMO HOMO Highest Occupied Molecular
Orbital LUMO Lowest Unoccupied Molecular
Orbital In this example n(So)?p p(T1) less
probable p(S1) More probable T1 excited
triplet state S1 excited singlet state
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Vacuum UV or Far UV (?lt190 nm )
UV/VIS
? h c/?E
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s s Transitions

• An electron in a bonding s orbital is excited to
  the corresponding antibonding orbital. The energy
  required is large. For example, methane (which
  has only C-H bonds, and can only undergo s s
  transitions) shows an absorbance maximum at 125
  nm.

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n s Transitions

• Saturated compounds containing atoms with lone
  pairs (non-bonding electrons) are capable of n
  s transitions. These transitions usually need
  less energy than s s transitions. They can be
  initiated by light whose wavelength is in the
  range 150 - 250 nm. The number of organic
  functional groups with n s peaks in the UV
  region is small.

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n p and p p Transitions

• Most absorption spectroscopy of organic compounds
  is based on transitions of n or p electrons to
  the p excited state.
• These transitions fall in an experimentally
  convenient region of the spectrum (200 - 700 nm).
  These transitions need an unsaturated group in
  the molecule to provide the p electrons.

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1,3-butadiene  
CH2CH-CHCH2


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Delocalized molecular orbitals are not confined
between two adjacent bonding atoms, but actually
extend over three or more atoms.
10.8
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Electron density above and below the plane of the
benzene molecule.
10.8
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II Transitions involving d and f electrons
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4) UV/Vis absorption by inorganic compounds
(1) Ligand field theory For transition metal
ions, with the interaction of a complexing ligand
or solvent molecule, the d- or f- orbitals split
into two or more groups that differ in energy.
The absorption is due to valence electrons in the
metal ions d-or f- orbitals.
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III Charge transfer For particular complex,
absorbing a photon with producing an excited
state species described in terms of the transfer
of an electron from the ligand (L) to metal (M),
or metal to the ligand. e.g., h? MnLm ?
Mn1Lm1
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3. Absorbance and Concentration
1) Transmittance and Absorbance
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2) Beers Law
C Analyts concentration b Light path length
Beers Law A abC a absorptivity, unit is of
cm1conc1. Analyte in molar concentration A
?bC ? molar absorptivity, unit is of cm1M1

• Beers law is the linear relationship between a
  samples absorbance and concentration.
• Values for a or ? depend on the wavelength of
  electromagnetic radiation.
• Wavelengths corresponding to maxima absorbance in
  the spectra called ?max.

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Beer's Law
A ebc Where A is absorbance (no units, since A
log10 P0 / P )e is the molar absorbtivity with
units of L mol-1 cm-1b is the path length of the
sample - that is, the path length of the cuvette
in which the sample is contained. We will express
this measurement in centimetres.c is the
concentration of the compound in solution,
expressed in mol L-1
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3) Quantitative Application of Beers Law
Example The following data was obtained from an
optical absorption instrument with a cell path
length 1 cm. (a) Find the molar absorptivity
coefficient. (b) Determine the concentration of
an unknown solution that has an absorbance of
1.52.
Solution

• Y 201.85X ?bC
• (a) ?201.85 cm1M1
• (b) C 0.0075 M

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Limitations of the Beer-Lambert law
1. deviations in absorptivity coefficients at
high concentrations (gt0.01M) due to electrostatic
interactions between molecules in close proximity
2.scattering of light due to particulates in
the sample 3.fluoresecence or phosphorescence
of the sample 4. changes in refractive index at
high analyte concentration 5.shifts in chemical
equilibria as a function of concentration 6.
stray light
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4. UV/Visible Spectrophotometer
1) Single beam instrument
2) Double beam instrument
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3) Phodediode array (PDA) spectrophotometer
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Instrumentation
Schematic of a single beam uv-vis
spectrophotometer
                                               
                             
?? ??? ?? ??? ???
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UV-Visible Spectrometer
Prism or
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Basic Components of Spectroscopic Instrumentation

• i) Sources of Energy

Common Sources of Electromagnetic Radiation for
Spectroscopy
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Deuterium Lamps
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Tungsten Lamp
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??
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2) Wavelength Selector
(1) Filter A wavelength selector that uses
either absorption, or constructive and
destructive interference to control the range of
selected wavelengths. a) Absorption filter b)
Interference filter
(2) Monochromator A wavelength selector that
uses a diffraction grating or prism, and that
allows for a continuous variation of the nominal
wavelength. a) Diffraction grating b) Prism
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MonochromatorCzerny-Turner design
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3) Sample Holder/Matrix
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3) Sample Holder/Matrix
(1) Feasibility of cuvette materials
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(2) Feasibility of preparation solvent
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Applications Protein Quantification
   Diluting the BSA sample 12, the absorbance
maxima again produced a peak around 280nm with
half the peak value of undiluted BSA. This shows
a direct correlation between absorbance value and
protein concentration.
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Quantification of nucleic acids
Quantification of nucleic acids is commonly used
in molecular biology to determine the
concentrations of DNA or RNA present in a
mixture, as subsequent reactions or protocols
using a nucleic acid sample often require
particular amounts for optimum performance
Because DNA and RNA absorb ultraviolet light,
with an absorption peak at 260nm wavelength,
spectrophotometers are commonly used to determine
the concentration of DNA in a solution. Inside a
spectrophotometer, a sample is exposed to
ultraviolet light at 260 nm, and a photo-detector
measures the light that passes through the
sample. The more light absorbed by the sample,
the higher the nucleic acid concentration in the
sample.
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260280 ratio lacks sensitivity for protein
contamination in nucleic acids
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Ch18Fundamentals of Spectrophotometry Part III
Molecular Luminescence Spectroscopy
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Contents
1. Photoluminescence energy diagram 1) Basic
view of luminescence 2) Basic relaxation types
of excited state 3) Lifetime, relaxation, and
multiplicity 4) Energy transitions in
fluorescence and phosphorescence 5) Category of
nonradiative relaxation 2. Photoluminescence
spectra 1) Phosphorescence intensity
equation 2) Fluorescence intensity
equation 3) Instrumentation for Fluorescence
3. Chemiluminescence
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1) Basic view of luminescence
1. Luminescence energy diagram

• Lifetime of an analyte in the excited state (A)
• 108104 s for electronic excited states
• 1015 s for vibrational excited states.

Figure 18-13
2) Basic relaxation types of excited
state (1) Nonradiative relaxation, e.g.,
vibrational deactivation, excess energy is
released to solvent molecules A ? A
heat (2) Involve a decomposition reaction A ?
X Y (3) Released as a photon of electromagnetic
radiation (photoluminescence) A ? A h?
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(4) Basic energy level diagram
Nonradiative relaxation
Photoluminescence
Absorption
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Page 392 18-6 Luminescence Figure 18-15
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3) Lifetime, Relaxation, and Multiplicity
(1) Lifetime The length of time that an
analyte stays in an excited state before
returning to a lower-energy state. (2) Relaxation
Any process by which an analyte in excited
state returns to a lower-energy by radiation
(called radiative relaxation, e.g.,
photoluminescence), or by releasing heat (called
nonradiative relaxation). (3) State of spin
multiplicity a) Singlet state A electron
configuration in which the spins of two electrons
are oriented antiparallel, (total electron spin
number of 0). b) Triplet state A electron
configuration in which the spins of two electron
particles are oriented parallel, (total electron
spin number of 1).
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4) Energy transitions in fluorescence and
phosphorescence
S0
S1
T1
(1) Fluorescence Emission of a photon when the
analyte returns to a lower-energy state with the
same spin as the higher energy state, i.e.,
S1?S0, in which the electron life time in the
excited state is 105108 s. (2) Phosphorescence
Emission of a photon when the analyte returns
to a lower-energy state with the opposite spin as
the higher-energy, i.e., T1?S0, in which the
electron life time in the excited state is
104104 s.
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(3) Energy level diagram Deactivation of an
excited state molecule
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5) Category of nonradiative relaxation

• (1) Vibrational relaxation (vr) A nonradiative
  relaxation when a excited molecule nonradiatively
  loses vibrational energy in a same electronic
  level, lifetime is rapid (1013 to 1011 s).
• (2) Internal conversion (ic) A radiationless
  relaxation in which the analyte moves from a
  higher electronic level to a lower electronic
  level without spin change, e.g., S1 ? S0.
• (3) Intersystem crossing (isc) A nonradiative
  relaxation when a excited molecule moves from a
  higher electronic level to a different
  multiplicity lower electronic level, e.g., S1 ?
  T1.
• (4) External conversion (ec) A form of
  radiationless relaxation in which energy is
  transferred to the solvent or sample matrix.

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2. Photoluminescence spectra
Figure 18-16,-17
E excitation (absorption) spectrum F
Fluorescence spectrum P Phosphorescence spectrum
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1) Phosphorescence intensity equation
(1) Phosphorescence is favorable for molecules
where the lowest energy absorption is a n??
transition. (2) Intensity of phosphorescence (Ip)
is expressed as Ip 2.303kFpP0?bC
kC C analyts concentration b light path
length ? molar absorptivity k efficiency
constant of collecting and detecting the
emission P0 excitation incident power Fp
number of photons emitted/number of photons
absorbed (quantum yield).
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2) Fluorescence intensity equation
(1) Fluorescence is generally observed with
molecules where the lowest energy absorption is a
p ? p transition, and those chromophores are
called fluors or fluorephores. (2) For low
concentrations of the fluorescing species, where
ebC is less than 0.01, the intensity of
fluorescence (If) is expressed as If
2.303kFfP0ebC kC C analyts
concentration b light path length e molar
absorptivity k efficiency constant of
collecting and detecting the emission P0 excitat
ion incident power Ff number of photons
emitted/number of photons absorbed
(quantum yield).
Figure 18-23
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3) Instrumentation for Fluorescence
(1) Fluorometer an instrument for measuring
fluorescence that uses filters to select the
excitation and emission wavelengths.
(2) Spectrofluorometer an instrument for
measuring fluorescence that uses a monochromator
to select the excitation and emission
wavelengths.
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3. Chemiluminescence
1) Chemiluminescence The luminescence which is
produced when a chemical reaction yield and
electronically excited intermediates following
radiative relaxation and return to its ground
state or transfer its energy to another
species. e.g., A B ?C D C excited
intermediate C ? C h?

2) Luminator
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3) Examples of chemiluminescence analysis

• Analyzing H2O2

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b) Analyzing ATP
Luciferin is used as substrates by the enzymes
firefly luciferase, emit light which may be
measured or detected, the level of ATP may be
quantitated as follows Luciferin ATP O2
? (Firefly Luciferase) Oxyluciferm
AMP pyrophosphate CO2 light This assay is
extremely sensitive allowing detection at the pM
to fM level.
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Examples All Exercises A, B, C Problems 14,
6-10, 16 Draw Conceptual Block Diagrams of
Spectrometer !!
End of Chapter 18