Analytical Chemistry

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Analytical Chemistry
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Chapter 19Fundamentals of Spectrophotometry
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Properties of Light
Light can be described in terms of particles and
waves.
This is called wave-particle duality.A principle
of quantum mechanics which implies that light
(and, indeed, all other subatomic particles)
sometimes act like a wave, and sometimes act like
a particle, depending on the experiment you are
performing.
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Wave Model
E amplitude of electric field (J) n frequency
(Hz) l wavelength (m) c speed of light (2.998
x 108 m/s in vacuum)
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Particle Model
Light is viewed as particles or packets of energy
called photons.
Energy of a photon
Also written as
New term
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Light Absorption
When light is absorbed by a molecule, the energy
level of the molecule is increased.
Excited States
hn
E
Ground State (lowest energy state)
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Light Emission
When light is emitted by a molecule, the energy
level of the molecule is decreased.
E
hn
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Electromagnetic Spectrum
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Absorbance Measurement
Po
P
Light Source
Light Detector
Sample
l Selector
b
Transmittance
Absorbance
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Absorption Spectrum
Po
P
Light Source
Light Detector
Sample
l Selector
b
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Beer-Lambert Law
Po
P
Light Source
Light Detector
Sample
l Selector
b
Absorbance is directly proportional to
concentration!
Beers Law
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Absorbance vs. Concentration
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Calibration Curve
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Analytical Absorbance Measurement
1) Measure absorbance of naturally occurring
chromophore (e.g. proteins measure A at 280 nm)
2) Add complexing agent to form a colored
complex (e.g. Fe UV-Vis lab). Colorimetric
Analysis
Absorbs strongly at 510 nm
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When Beers Law Fails
1) Light must be monochromatic.
2) Solutions must be relatively dilute (lt 0.01
M).
3) Absorbing molecule must not be involved in a
concentration-dependent equilibrium
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Spectrophotometers
PMT
Single-Beam Instruments
PDA
Double-Beam Instrument
L light source(s), M monochromator(s), C
chopper, B beam splitter, R reference sample,
S test sample and D detector.
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What Happens When Light is Absorbed?
Jablonski Diagram
S1
T1
So
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Singlet vs. Triplet
S1
T1
So
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What Happens When Light is Absorbed?
Jablonski Diagram
S1
T1
So
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Absorption and Emission Spectra
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Fluorescence vs. Phosphorescence
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Fluorescence Spectroscopy
Fluorescence emission is directly proportional to
concentration and incident power.
L light source, Ex.M excitation
monochromator(s), Ex.P excitation polarizer, S
sample, Em.P emission polarizer, Em.M
emission monochromator(s) and D detector.