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SPECTROSCOPY
Light interacting with matter as an
analytical tool
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X-ray:
core electronexcitation
UV:
valanceelectronic
excitation
IR:
molecularvibrations
Radio waves:
Nuclear spin states(in a magnetic field)
Electronic Excitation by UV/Vis Spectroscopy :
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Spectroscopic Techniques andChemistry they Probe
UV-vis UV-vis region bonding electrons
Atomic Absorption UV-vis region atomic transitions (val. e-)
FT-IR IR/Microwave vibrations, rotations
Raman IR/UV vibrations
FT-NMR Radio waves nuclear spin states
X-Ray Spectroscopy X-rays inner electrons, elemental
X-ray Crystallography X-rays 3-D structure
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Spectroscopic Techniques and Common Uses
UV-vis UV-vis regionQuantitative
analysis/Beers Law
Atomic Absorption UV-vis regionQuantitative analysis
Beers Law
FT-IR IR/Microwave Functional Group Analysis
Raman IR/UVFunctional Group
Analysis/quant
FT-NMR Radio waves Structure determination
X-Ray Spectroscopy X-rays Elemental Analysis
X-ray Crystallography X-rays 3-D structure Anaylysis
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Different Spectroscopies
UV-vis electronic states of valence e/d-orbital transitions for solvated transitionmetals
Fluorescence emission of UV/vis by certainmolecules
FT-IR vibrational transitions of molecules
FT-NMR nuclear spin transitions X-Ray Spectroscopy electronic transitions
of core electrons
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Quantitative Spectroscopy
Beers Law
Al1 = el1bc
e is molar absorptivity (unique for a
given compound at l1)
b is path length
c concentration
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Beers Law
A = -logT = log(P0/P) = ebc
T = Psolution/Psolvent = P/P0
Works for monochromatic light
Compound x has a unique e at different
wavelengths
cuvette
source slit
detector
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Characteristics of
Beers Law Plots One wavelength
Good plots have a range of
absorbances from 0.010 to 1.000
Absorbances over 1.000 are not that
valid and should be avoided
2 orders of magnitude
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Standard Practice
Prepare standards of knownconcentration
Measure absorbance at lmax Plot A vs. concentration
Obtain slope
Use slope (and intercept) to determinethe concentration of the analyte in theunknown
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Typical Beers Law Plot
y = 0.02x
0
0.2
0.4
0.6
0.8
1
1.2
0.0 20.0 40.0 60.0
concentration (uM)
A
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UV-Vis Spectroscopy
UV- organic molecules
Outer electron bonding transitions
conjugation
Visible metal/ligands in solution
d-orbital transitions
Instrumentation
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Characteristics of UV-Vis spectra of
Organic Molecules Absorb mostly in UV unless highly
conjugated
Spectra are broad, usually to broad forqualitative identification purposes
Excellent for quantitative Beers Law-
type analyses The most common detector for anHPLC
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Molecules have quantized energy levels:
ex. electronic energy levels.
energy
hv
energy
}= hv
Q: Where do these quantized energy levels come from?
A: The electronic configurations associated with bonding.
Each electronic energy level(configuration) has
associated with it the many
vibrational energy levels we
examined with IR.
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Broad spectra
Overlapping vibrational and rotational
peaks Solvent effects
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Molecular Orbital Theory
Fig 18-10
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2s 2s
s
s*
s
s*
p*
p
2p 2pn
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C C
s
s*
hv
s
s*
s s*
C C
H
H
HH
HH
lmax
= 135 nm (a high energy transition)
Absorptions havinglmax < 200 nm are difficult to observe because
everything (including quartz glass and air) absorbs in this spectral
region.
Ethane
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C C
s
s*
hv
p
p*
s
s*
p
p*
p p*
Example: ethylene absorbs at longer wavelengths: lmax = 165 nm = 10,000
= hv=hc/l
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s
s*
hv
p
p*
n
s
s*
p
p*
n
C O
p*n
The n to pi* transition is at even lower wavelengths but is notas strong as pi to pi* transitions. It is said to be forbidden.
Example:
Acetone: ns*lmax = 188 nm ; = 1860
np* lmax = 279 nm ; = 15
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C C
C C
C O
C O
H
ss* 135 nm
pp* 165 nm
ns* 183 nm weak
pp* 150 nm
ns* 188 nmnp* 279 nm weak
l
A
180 nm
279 nm
C O
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C C
HOMO
LUMO
Conjugated systems:
Preferred transition is between Highest Occupied Molecular Orbital(HOMO) and Lowest Unoccupied Molecular Orbital (LUMO).
Note: Additional conjugation (double bonds) lowers the HOMO-
LUMO energy gap:
Example:
1,3 butadiene: lmax = 217 nm ; = 21,000
1,3,5-hexatriene lmax = 258 nm ; = 35,000
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O
O
O
Similar structures have similar UV spectra:
lmax = 238, 305 nmlmax = 240, 311 nm lmax = 173, 192 nm
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Lycopene:
lmax = 114 + 5(8) + 11*(48.0-1.7*11) = 476 nm
lmax(Actual) = 474.
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Metal ion transitions
Degenerate
D-orbitals
of naked Co
D-orbitalsof hydrated Co2+
Octahedral Configuration
E
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Co2+
H2O
H2O
H2O
H2O
H2O
H2
O
Octahedral Geometry
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Instrumentation
Fixed wavelength instruments
Scanning instruments
Diode Array Instruments
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Fixed Wavelength Instrument
LED serve as source
Pseudo-monochromatic light source
No monochrometer necessary/ wavelength selection
occurs by turning on the appropriate LED 4 LEDs to choose from
photodyode
sample
beam of light
LEDs
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Scanning Instrument
cuvette
Tungsten
Filament (vis)
slit
Photomultiplier
tube
monochromator
Deuterium lamp
Filament (UV)
slit
Scanning Instrument
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sources
Tungten lamp (350-2500 nm)
Deuterium (200-400 nm)
Xenon Arc lamps (200-1000 nm)
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Monochromator
Braggs law, nl = d(sin i + sin r)
Angular dispersion, dr/dl = n / d(cos r)
Resolution, R = l/l=nN, resolution is
extended by concave mirrors to refocus
the divergent beam at the exit slit
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Sample holder
Visible; can be plastic or glass
UV; you must use quartz
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Single beam vs. double beam
Source flicker
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Diode array Instrument
cuvette
Tungsten
Filament (vis)
slit
Diode array detector
328 individual detectors
monochromator
Deuterium lampFilament (UV)
slit
mirror
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Advantages/disadvantages
Scanning instrument High spectral resolution (63000),l/l
Long data acquisition time (severalminutes)
Low throughput
Diode array
Fast acquisition time (a couple of
seconds), compatible with on-lineseparations
High throughput (no slits)
Low resolution (2 nm)
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HPLC-UV
Mobile
phase
HPLC
Pump
syringe
6-port
valveSample
loop
HPLC
column
UV
detector
Solvent
waste