Optical Electronic Spectroscopy 2
Lecture Date: January 28th, 2008
Molecular UV-Visible Spectroscopy
 Molecular UV-Visible spectroscopy is driven by electronic
absorption of UV-Vis radiation.
 Molecular UV-Visible
spectroscopy can:
– Enable structural analysis
– Detect molecular chromophore
– Analyze light-absorbing properties
(e.g. for photochemistry)
 Basic UV-Vis spectrophotometers acquire data in the 190800 nm range and can be designed as “flow” systems.
Figures from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv1
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 f electrons (inorganics/organometallics)
(3) charge-transfer (CT) electrons
Molecular UV-Vis Spectroscopy: Theory
 Molecular energy levels and absorbance wavelength:
  * and   * transitions: high-energy, accessible in vacuum
UV (max <150 nm). Not usually observed in molecular UV-Vis.
n  * and   * transitions: non-bonding electrons (lone pairs),
wavelength (max) in the 150-250 nm region.
n  * and   * transitions: most common transitions observed in
organic molecular UV-Vis, observed in compounds with lone pairs
and multiple bonds with max = 200-600 nm.
Figure from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm
Molecular UV-Vis Spectroscopy: Theory
 d/f orbitals – transition metal complexes
– UV-Vis spectra of lanthanides/actinides are particularly sharp, due
to screening of the 4f and 5f orbitals by lower shells.
– Can measure ligand field strength, and transitions between dorbitals made non-equivalent by the formation of a complex
 Charge transfer (CT) – occurs when electron-donor and
electron-acceptor properties are in the same complex –
electron transfer occurs as an “excitation step”
– MLCT (metal-to-ligand charge transfer)
– LMCT (ligand-to-metal charge transfer)
– Ex: tri(bipyridyl)iron(II), which is red – an electron is exicted from
the d-orbital of the metal into a * orbital on the ligand
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 the
molar 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 transitions

can be calculated with theories ranging from Huckel to ab initio/DFT.
Example:   * transitions responsible for ethylene UV absorption
at ~170 nm calculated with ZINDO semi-empirical excited-states
methods (Gaussian 03W):
HOMO u bonding molecular orbital
LUMO g antibonding molecular orbital
Molecular UV-Visible Spectrophotometers
 Continuum UVVis sources – the
2H lamp:
Hamamatsu
L2D2 lamps
 Tungsten lamps
used for longer
wavelengths.
 The traditional
UV-Vis design –
double-beam
grating systems
Figure from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv1
Molecular UV-Visible Spectrophotometers
 Diode array detectors can acquire all UV-Visible
wavelengths at once.
 Advantages:
– Sensitivity
(multiplex)
– Speed
 Disadvantages:
– Resolution
Figure from Skoog, et al., Chapter 13
Interpretation of Molecular UV-Visible Spectra
 UV-Visible spectra can be
interpreted to help determine
molecular structure, but this
is presently confined to the
analysis of electron behavior
in known compounds.
 Information from other
techniques (NMR, MS, IR) is
usually far more useful for
structural analysis
 However, UV-Vis evidence
should not be ignored!
Figure from Skoog, et al., Chapter 14
Calculation of Molar Absorption Coefficient
 The molar absorption coefficient for each absorbance in a
UV 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
Interpretation of UV-Visible Spectra
 Although UV-Visible spectra are no longer frequently used
for structural analysis, it is helpful to be aware of welldeveloped 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)
X
Substituent (X)
Increment (nm)
-CH3
3.0
-Cl
6.0
-OH
7.0
-NH2
26.5
-CHO
46.0
-NO2
65.0
See E. Pretsch, et al., Structure Determination of Organic Compounds, Springer, 2000. (Chapter 8).
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.
H3C
H2N
CH2
vs.
~230 nm
HC
CH2
~180 nm
 Why does increasing conjugation cause bathochromic shifts
(to longer wavelengths)?
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
Interpretation of UV-Visible Spectra
 Transition metal
complexes
 Lanthanide
complexes – sharp
lines caused by
“screening” of the f
electrons by other
orbitals
See Shriver et al. Inorganic Chemistry, 2nd Ed. Ch. 14
More Complex Electronic Processes
 Fluorescence: absorption of



radiation to an excited state,
followed by emission of radiation to
a lower state of the same
multiplicity
Phosphorescence: absorption of
radiation to an excited state,
followed by emission of radiation to
a lower state of different multiplicity
Singlet state: spins are paired, no
net angular momentum (and no net
magnetic field)
Triplet state: spins are unpaired, net
angular momentum (and net
magnetic field)
Molecular Fluorescence
 Non-resonance fluorescence is a phenomenon in which
absorption of light of a given wavelength by a fluorescent
molecule 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
 Jablonski energy diagram:
 Notation: S2, S1 = singlet states, T1 = triplet state
 Excitation directly to a triplet state is forbidden by selection rules.
 See Skoog Figure 15-1
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 is
reversed (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 to

break a chemical bond
Pre-dissociation: relaxation to vibrational state with sufficient energy
to break a chemical bond
 Stokes shift: a shift (usually seen in fluorescence) to longer
wavelengths between excitation and emitted radiation
Predicting the Fluorescence of Molecules
 Some things that improve fluorescence:
– Low energy   * transitions
– Rigid molecules
– Transitions that don’t 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
biphenyl
fluorescence QE = 0.2
fluorene
fluorescence QE = 1.0
 Intersystem crossings reduce fluorescence (competing
process is phosphorescence).
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 in
the Surface Analysis and Microscopy lectures (in
conjunction with e.g. confocal scanning microscopy)
Applications of Fluorescence
 Applications in forensics: trace level analysis of specific

small molecules
Example: LSD (lysergic acid diethylamide) spectrum
obtained 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 102–103 (2003) 194–200
Applications 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 Application of Fluorescence: FRAP
 Fluorescence Recovery After Photo-bleaching (FRAP), developed in
1974, 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 which
the recovery of fluorescence is monitored with the low power laser.
– Recent studies have used a single laser that is attenuated with a
Pockel’s 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).
Fluorescence Recovery After Photo-bleaching
 Spot photobleaching:
– A spot is bleached, and its subsequent recovery is predicted by:
1/ 2
–
–
–
–
 2 
  
 4D
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
d2
 1/ 2  2
4 D
 Fluorophores: organic fluorescent molecules that are
excited by the laser
– Example: rhodopsin
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).
Fluorescence Recovery After Photo-bleaching


A periodic pattern is first photobleached with a high power laser
The recovery of the fluorescence is monitored via a low power laser
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).
Fluorescence Recovery After Photo-bleaching
 Diffusion coefficients can be calculated from periodic pattern
experiments via:

–
–
–
d2
4 2 D
 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
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).
Multiphoton-Excited Fluorescence
 Known as MPE (as opposed to the
usual 1PE)
 Lots of energy required – femtosecond
pulsed lasers
excited
state
 Multiple low energy photons can be
absorbed, via short-lived “virtual states”
(lifetime ~ 1 fs). Can get to far-UV
wavelengths without “waste”
 Spatial localization is excellent
virtual
state
ground
state
(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).
Molecular Phosphorescence
 Phosphorescence – often used as a
complementary technique to fluorescence.
– If a molecule won’t fluorescence, sometimes
it will phosphoresce
excitation
fluorescence
phosphorescence
– 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, but
with cooling dewars and acquisition delays
wavelength
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 same
effect can be achieved
 Applications:
– nucleic acids, amino acids, enzymes, pesticides, petroleum products, and
many more
For more details, see: R. J. Hurtubise, Phosphorimetry: Theory, Instrumentation, and Applications, Chap. 3, New York, VCH 1990.
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 the
chemical reaction and the quantum yield:
ICL = CL (dC/dt) = EX EM (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):
O+
NO
nitric oxide
+
-
O
NO2*
O
+
O2
nitrogen dioxide
ozone
NO2*
NO2
hv
Figure from: http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/lumin1.htm
Chemi-luminescence: Luminol Reactions
 Luminol, a molecule that when oxidized can do many

things…
Representative uses of luminol:
– Detecting hydrogen peroxide in seawater1 (indicator of
photoactivity)1
– Visualizing bloodstains – reaction catalyzed by haemoglobin2
– Detecting nitric oxide3
NH2
NH2
O
NH
O
O-
oxidizing
+
agent
+
O
NH
O
hv
-
O
1. D. Price, P. J. Worsfold, and R. F. C. Mantoura, Anal. Chim. Acta, 1994, 298, 121.
2. R. Saferstein, Criminalistics: An Introduction 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
Applications 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 electrodes
undergo 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 fabricated
microarrays on a flow chip (biosensor applications)
– See: Cheek et al., Anal. Chem., 2001, 73, 5777.
Homework Problems
Optical 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
Spectroscopy
K. A. Flectcher et al., Anal. Chem. 2006, 78, 4047-4068.