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Optical Electronic Spectroscopy 2
Lectu re Date: January 28th, 2008
Molecular UV-Visible Spectroscopy
Molecular UV-Visiblespectroscopy can:
Enable structural analysis
Detect molecular chromophore
Analyze light-absorbing properties
(e.g. for photochemistry)
Figures from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv1
Basic UV-Vis spectrophotometers acquire data in the 190-800 nm range and can be designed as flow systems.
Molecular UV-Visible spectroscopy is driven by electronicabsorption of UV-Vis radiation.
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Molecular UV-Vis Spectroscopy: Terminology
UV-Vis Terminology
Chromophore: a UV-Visible absorbing functional group
Bathochromic shift (red shift): to longer wavelengths
Auxochrome: a substituent on a chromophore that
causes a red shift
Hypsochromic shift (blue shift): to shorter wavelengths
Hyperchromic shift: to greater absorbance
Hypochromic shift: to lesser absorbance
Molecular UV-Vis Spectroscopy: Transitions
Classes of Electron transitions
HOMO: highest occupied molecular orbital
LUMO: lowest unoccupied molecular orbital
Types of electron transitions:
(1) , and n electrons (mostly organics)
(2) d and felectrons (inorganics/organometallics)
(3) charge-transfer (CT) electrons
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Molecular UV-Vis Spectroscopy: Theory
Molecular energy levels and absorbance wavelength:
* and
* transitions: high-energy, accessible in vacuum
UV (max
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Molecular UV-Vis Spectroscopy: Absorption
max is the wavelength(s) of maximum absorption (i.e. the
peak position)
The strength of a UV-Visible absorption is given by themolar absorptivity ():
= 8.7 x 1019 P a
where P is the transition probability (0 to 1) governed by selection
rules and orbital overlap,
and a is the chromophore area in cm2
Again, the Beer-Lambert Law:
A =
bc
Molecular UV-Vis Spectroscopy: Quantum Theory
UV-Visible spectra and the states involved in electronic transitionscan be calculated with theories ranging from Huckel to ab initio/DFT.
Example: * transitions responsible for ethylene UV absorptionat ~170 nm calculated with ZINDO semi-empirical excited-states
methods (Gaussian 03W):
HOMOu bonding molecular orbital LUMOg antibonding molecular orbital
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Molecular UV-Visible Spectrophotometers
Continuum UV-Vis sources the2H lamp:
Tungsten lampsused for longer
wavelengths.
The traditionalUV-Vis design
double-beam
grating systems
Figure from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv1
Hamamatsu
L2D2 lamps
Molecular UV-Visible Spectrophotometers
Diode array detectors can acquire all UV-Visiblewavelengths at once.
Advantages: Sensitivity
(multiplex)
Speed
Disadvantages: Resolution
Figure from Skoog, et al., Chapter 13
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Interpretation of Molecular UV-Visible Spectra
UV-Visible spectra can be
interpreted to help determinemolecular structure, but this
is presently confined to the
analysis of electron behavior
in known compounds.
Information from othertechniques (NMR, MS, IR) is
usually far more useful for
structural analysis
However, UV-Vis evidenceshould not be ignored!
Figure from Skoog, et al., Chapter 14
Calculation of Molar Absorption Coefficient
The molar absorption coefficient for each absorbance in aUV spectrum is calculated as follows:
Molar Abs Coeff (AU mol-1 cm-1) = A x mwt / mass x pathlength
Solvent cutoffs for UV-visible work:
Solvent UV Cutoff (nm)
Acetonitrile (UV grade) 190
Acetone 330
Dimethylsulfoxide 268
Chloroform (1% ethanol) 245
Heptane 200
Hexane (UV grade) 195
Methanol 205
2-Propanol 205
Tetrahydrofuran (UV grade) 212
Water 190
Burdick and Jackson High Purity Solvent Guide, 1990
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Interpretation of UV-Visible Spectra
Although UV-Visible spectra are no longer frequently used
for structural analysis, it is helpful to be aware of well-developed interpretive rules.
Examples: Woodward-Fieser rules for max dienes and polyenes
Extended Woodward rules for a,b-unsaturated ketones
Substituted benzenes (max base value = 203.5 nm)
See E. Pretsch, et al., Structure Determination of Organic Compounds, Springer, 2000. (Chapter 8).
X
Substituent (X) Increment (nm)
-CH3 3.0
-Cl 6.0
-OH 7.0
-NH2 26.5-CHO 46.0
-NO2 65.0
Interpretation of UV-Visible Spectra
Other examples: The conjugation of a lone pair on a
enamine shifts the max from 190 nm
(isolated alkene) to 230 nm. The
nitrogen has an auxochromic effect.
See E. Pretsch, et al., Structure Determination of Organic Compounds, Springer, 2000. (Chapter 8).
Figures from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm
Why does increasing conjugation cause bathochromic shifts(to longer wavelengths)?
CH2 HC CH2vs.
~230 nm ~180 nm
H2N H3C
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Interpretation of UV-Visible Spectra
Transition metal
complexes
Lanthanidecomplexes sharp
lines caused by
screening of the f
electrons by other
orbitals
See Shriver et al. Inorganic Chemist ry, 2ndEd. Ch. 14
More Complex Electronic Processes
Fluorescence: absorption ofradiation to an excited state,
followed by emission of radiation to
a lower state of the same
multiplicity
Phosphorescence: absorption ofradiation to an excited state,
followed by emission of radiation to
a lower state of different multiplicity
Singlet state: spins are paired, nonet angular momentum (and no net
magnetic field)
Triplet state: spins are unpaired, netangular momentum (and net
magnetic field)
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Molecular Fluorescence
Non-resonance fluorescence is a phenomenon in which
absorption of light of a given wavelength by a fluorescentmolecule is followed by the emission of light at longer
wavelengths (applies to molecules)
Why use fluorescence? Its not a difference method!
Method Mass detection
limit (moles)
Concentration
detection limit
(M)
Advantage
UV-Vis 10-13 to 10-16 10-5 to 10-8 Universal
fluorescence 10-15 to 10-17 10-7 to 10-9 Sensitive
Molecular Fluorescence: Terminology
Notation: S2, S1 = singlet states, T1 = triplet state
Excitation directly to a triplet state is forbidden by selection rules.
See Skoog Figure 15-1
Jablonski energy diagram:
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Molecular Fluorescence: Terminology
Quantum yield (): the ratio of molecules that luminescence to the
total # of molecules Resonance fluorescence: fluorescence in which the emitted radiation
has the same wavelength as the excitation radiation
Intersystem crossing: a transition in which the spin of the electron isreversed (change in multiplicity in molecule occurs, singlet to triplet).
Enhanced if vibrational levels overlap or if molecule contains heavy
atoms (halogens), or if paramagnetic species (O2) are present.
Dissociation: excitation to vibrational state with sufficient energy tobreak a chemical bond
Pre-dissociation: relaxation to v ibrational state with sufficient energyto break a chemical bond
Stokes shift: a shift (usually seen in fluorescence) to longerwavelengths between excitation and emitted radiation
Predicting the Fluorescence of Molecules
Some things that improve fluorescence: Low energy * transitions
Rigid molecules
Transitions that dont have competition! Example: fluorescence
does not often occur after absorption of UV wavelengths (< 250
nm) because the radiation has too much energy (>100 kcal/mol)
dissociation occurs instead (but see MPE!!!)
Chelation to metals
Intersystem crossings reduce fluorescence (competingprocess is phosphorescence).
biphenylfluorescence QE = 0.2
fluorenefluorescence QE = 1.0
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Predicting the Fluorescence of Molecules
More things that affect fluoroescence:
decrease temperature = increase fluorescence
increase viscosity = increase fluorescence
pH dependence for acid/base compounds (titrations)
Time-resolved fluorescence spectroscopy Study of fluorescence spectra as a function of time (ps to ns)
Fluorescence probes for microscopy: will be covered inthe Surface Analysis and Microscopy lectures (in
conjunction with e.g. confocal scanning microscopy)
Appl ications of Fluorescence
Applications in forensics: trace level analysis of specificsmall molecules
Example: LSD (lysergic acid diethylamide) spectrumobtained with a Fourier-transform instrument and a
microscope, but with no derivitization
M. Fisher, V. Bulatov, I. Schechter, Fast analysis of narcotic drugs by optical chemical imaging, Journal of Luminescence 102103 (2003) 194200
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Appl ications of Fluorescence
Applications in biochemistry:analysis of proteins, enyzmes,
anything that can be tagged
with a fluorophore
In some cases, an externally-introduced label can be
avoided.
In proteins, the stryptophan(Trp), tyrosine (Tyr), and
phenylalanine (Phe) residues
are naturally UV-fluorescent
Example: single -galactosidase
molecules from Escherichia coli
(Ec Gal)
1-photon excitation at 266 nm
Q. Li and S. Seeger, Label-Free Detection of Single Protein Molecules Using Deep UV Fluorescence Lifetime Microscopy. Anal. Chem. 2006, 78, 2732-2737
Another Appl ication of Fluorescence: FRAP
Fluorescence Recovery After Photo-bleaching (FRAP), developed in1974, is a technique for measuring motion and diffusion.
FRAP can be applied at a microscopic level.
FRAP is commonly applied to microscopically heterogeneous systems.
A high power laser first bleaches an area of the sample, after whichthe recovery of fluorescence is monitored with the low power laser.
Recent studies have used a single laser that is attenuated with aPockels cell.
Applications of FRAP have included: Biological systems
Diffusion in polymers
Solvation in adsorbed layers on chromatographic surfaces
Curing of epoxy resins
J. M. Kovaleski and M. J. Wirth, Anal. Chem. 69, 600A (1997).
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Fluorescence Recovery After Photo-bleaching
Spot photobleaching: A spot is bleached, and its subsequent recovery is predicted by:
J. M. Kovaleski and M. J. Wirth, Anal. Chem. 69, 600A (1997).
D. E. Koppel, D. Axelrod, J. Schlessinger, E. Elson, and W. W. Webb, Biophys. J. 16, 1315 (1976).
1 2
2
4/
D
1/2 is the time for the fluorescence to recover 1/2 of its intensity
is the diameter of the spot
D is the diffusion coefficient
depends on the initial amount of fluorophor bleached
Periodic pattern photobleaching Eliminates dependence
Currently the most flexible and accurate FRAP measurement method
Fluorophores: organic fluorescent molecules that areexcited by the laser
Example: rhodopsin
D
d
2
2
2/14
Fluorescence Recovery After Photo-bleaching
J. M. Kovaleski and M. J. Wirth, Anal. Chem. 69, 600A (1997).
B. A. Smith and H. M. McConnell, Proc. Natl. Acad. Sci. USA. 75, 2759 (1978).
A periodic pattern is first photobleached with a high power laser
The recovery of the fluorescence is monitored via a low power laser
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Fluorescence Recovery After Photo-bleaching
J. M. Kovaleski and M. J. Wirth, Anal. Chem. 69, 600A (1997).
B. A. Smith and H. M. McConnell, Proc. Natl. Acad. Sci. USA. 75, 2759 (1978).
Diffusion coefficients can be calculated from periodic pattern
experiments via:
is the time constant of the simple exponential fluorescence recovery
d is the spacing of the lines of the grid
D is the diffusion coefficient
Methods of generating the periodic pattern: Ronchi ruling
Holographic imaging
d
D
2
24
Multiphoton-Excited Fluorescence
Known as MPE (as opposed to theusual 1PE)
Lots of energy required femtosecondpulsed lasers
Multiple low energy photons can beabsorbed, via short-lived virtual states
(lifetime ~ 1 fs). Can get to far-UV
wavelengths without waste
Spatial localization is excellent(because of the high energy needed, it
can be confined to < 1 m3.)
Applications: primarily bioanalytical
J. B. Shear, Multiphoton Excited Fluoroescence in Bioanalytical Chemistry,Anal. Chem., 71, 598A-605A (1999).
ground
state
excited
state
virtual
state
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Molecular Phosphorescence
Phosphorescence often used as a
complementary technique to fluorescence. If a molecule wont fluorescence, sometimes
it will phosphoresce
Phosphorescence is generally longer
wavelength that fluorescence
Some phosphorimeters are pulsed-source,which allows for time-resolution of excited
states (which have lifetimes covering a few
orders of magnitude).
Pulsed sources also help avoid the
interference of Rayleigh scattering or
fluorescence.
Instrumentation similar to fluorescence, butwith cooling dewars and acquisition delays
wavelength
excitation f luorescence phosphorescence
Note that the wavelength
difference between F and P
can be used to measure the
energy difference between
singlet and triplet states
Phosphorescence Studies
Room-temperature Phosphorescence (RTP) Phosphorescence is performed at low temperatures (77K) to avoid
collisional deactivation (molecules hitting each other), which causes
quenching of phosphorescence signal
By absorbing molecules onto a substrate, and evaporating the solvent,the phosphorescence of the molecules can be studied without the need
for low temperatures
By trapping molecules within micelles (and staying in solution), the sameeffect can be achieved
Applications: nucleic acids, amino acids, enzymes, pesticides, petroleum products, and
many more
For more details, see: R. J. Hurtubise, Phosphorimet ry: Theory, Instrumentation , and Applications , Chap. 3, New York, VCH 1990.
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Chemi-luminescence
A chemical reaction that yields an electronically excited
species that emits light as it returns to ground state.
In its simplest form:
A + B C* C + h
The radiant intensity (ICL) depends on the rate of thechemical reaction and the quantum yield:
ICL = CL (dC/dt) = EXEM (dC/dt)
excited states per
molecule reacted
photons per
excited states
Chemi-luminescence and Gas Analysis
Gas analysis see examples in Skoog pg. 375-376. Example: Determination of nitrogen monoxide to 1 ppb
levels (for pollution analysis in atmospheric gases):
Figure from: http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/lumin1.htm
nitric oxide
+ O
O+
-O
ozone nitrogen dioxide
O2+NO NO2*
NO2* NO2
hv
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Chemi-luminescence: Luminol Reactions
Luminol, a molecule that when oxidized can do manythings
Representative uses of luminol: Detecting hydrogen peroxide in seawater1 (indicator of
photoactivity)1
Visualizing bloodstains reaction catalyzed by haemoglobin2
Detecting nitric oxide3
1. D. Price, P. J. Worsfold, and R. F. C. Mantoura,Anal. Chim. Acta, 1994, 298, 121.
2. R . Saferstein, Criminalist ics: An Introduct ion to Forensic Science, Prentice Hall, 1998.
3. J. K Robinson, M. J. Bollinger and J. W. Birks, Anal. Chem. , 1999,71, 5131.
See also http://www.deakin.edu.au/~swlewis/2000_CL_demo.PDF
NH
NH
O
O
NH2
+oxidizing
agent
O
O
NH2
O-
O-
+ hv
Appl ications of Chemi-luminescence
Detection of arsenic in water: Convert As(III) and As(V) to AsH3 via borohydride reduction
pH < 1 converts both As(III) and As(V), pH 4-5 converts only
As(III)
Reacts with O3 (generated from air), CL results at 460 nm
CL detected via photomultiplier tube down to 0.05 g/L for 3 mL
Portable, automated analyzer, 6 min per analysis
See: A. D. Idowu et al.,Anal. Chem., 2006, 78, 7088-7097.
Electrochemiluminescence: species formed at electrodesundergo electron-transfer reactions and produce light
ECL converts electrical energy into radiation
See: M. M. Richter, Chem. Rev. 2004, 104, 3003-3036.
Chemi-luminescence can be applied to fabricatedmicroarrays on a flow chip (biosensor applications) See: Cheek et al.,Anal. Chem., 2001, 73, 5777.
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Homework ProblemsOptical Electronic Spectroscopy
Chapter 13:
Problem 13-6
Problem 13-13
Further Reading
Review Skoog et al. Chapters 13-15
Review Cazes Chapters 5-6
UV-Visible Spectroscopy
D. H. Williams and I. Fleming, Spectroscopic Methods in
Organic Chemistry, McGraw-Hill (1966).
Fluorescence, Phosphorescence, and Chemiluminescence
SpectroscopyK. A. Flectcher et al., Anal. Chem. 2006, 78, 4047-4068.