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SKA6014
ADVANCED ANALYTICAL CHEMISTRY
TOPIC 4Optical Electronic Spectroscopy 2
Azlan Kamari, PhDDepartment of Chemistry
Faculty of Science and Mathematics
Universiti Pendidikan Sultan Idris
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Molecular UV-Visible Spectroscopy
Molecular UV-Visible
spectroscopy can:
Enable structural analysis
Detect molecular chromophore
Analyse 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 electronic
absorption 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 thatcauses a red shift
Hypsochromic shift (blue shift): to shorter wavelengths
Hyperchromic shift: to greater absorbance
Hypochromic shift: to lesser absorbance
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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) dand 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: 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 d-
orbitals 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
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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 the
molar absorptivity ():
= 8.7 x 1019P a
where Pis the transition probability (0 to 1) governed
by selection rules and orbital overlap,
and ais the chromophore area in cm2
Again, the Beer-Lambert Law:
A =bc
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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 forethylene 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 the
2H lamp:
Tungsten lamps
used for longer
wavelengths.
The traditional
UV-Vis design
double-beamgrating systems
Figure from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv1
Hamamatsu
L2D2 lamps
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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
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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
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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
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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 ofwell-developed interpretive rules.
Examples:
Woodward-Fieser rules formax 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
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Interpretation of UV-Visible Spectra
Other examples:
The conjugation of a lone pair on a
enamine shifts the maxfrom 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 metalcomplexes
Lanthanide
complexes sharp
lines caused byscreening of the f
electrons by other
orbitals
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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 netmagnetic field)
Triplet state: spins are unpaired,
net angular 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? It is 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
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Molecular Fluorescence: Terminology
Notation: S2, S1 = singlet states, T1 = triplet state
Excitation directly to a triplet state is forbidden by selection
rules
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 is
reversed (change in multiplicity in molecule occurs, singlet to triplet).
Enhanced if vibrational levels overlap or if molecule containsheavy 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
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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 (competing
process 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 in
the Surface Analysis and Microscopy lectures (in
conjunction with e.g. confocal scanning microscopy)
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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 derivation
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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Applications of Fluorescence
Applications in biochemistry:
analysis of proteins, enyzmes,
anything that can be tagged witha 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
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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
Pockels 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:
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 are
excited by the laser Example: rhodopsin
D
d2
2
2/1
4
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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).
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
dis 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
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Multiphoton-Excited Fluorescence
Known as MPE (as opposed to the
usual 1PE)
Lots of energy required femtosecond
pulsed lasers
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
(because of the high energy needed, itcan 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 theinterference of Rayleigh scattering or
fluorescence.
Instrumentation similar to fluorescence, but
with cooling dewars and acquisition delays
wavelength
excitation fluorescence phosphorescence
Note that the wavelength
difference between F and P
can be used to measure the
energy difference between
singlet and triplet states
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Phosphorescence Studies
Room-temperature Phosphorescence (RTP)
Phosphorescence is performed at low temperatures (77K) to avoidcollisional 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 needfor 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
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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 the
chemical reaction and the quantum yield:
ICL = CL (dC/dt) = EXEM (dC/dt)
excited states per
molecule reacted
photons per
excited states
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Chemi-luminescence and Gas Analysis
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
Ch i l i L i l R i
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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
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
NH
NH
O
O
NH2
+oxidizing
agent
O
O
NH2
O-
O-
+ hv
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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 fabricatedmicroarrays on a flow chip (biosensor applications)
See: Cheek et al.,Anal. Chem., 2001,73,5777.