Optical Electronic Spectroscopy 2

Lecture Date: January 28th, 2008

Molecular UV-Visible Spectroscopy

Molecular UV-Visible spectroscopy 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 electronic absorption of UV-Vis radiation.

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 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

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 UV-Vis sources – the 2H lamp:

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

Hamamatsu L2D2 lamps

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 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).

XSubstituent (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

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

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:

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

Intersystem crossings reduce fluorescence (competing process is phosphorescence).

biphenylfluorescence QE = 0.2

fluorenefluorescence QE = 1.0

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:

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 are excited by the laser

– Example: rhodopsin

D

d2

2

2/1 4

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

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 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, 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).

groundstate

excitedstate

virtualstate

Molecular Phosphorescence

Phosphorescence – often used as a complementary technique to fluorescence.

– If a molecule won’t 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, 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

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):

Figure from: http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/lumin1.htm

nitric oxide+ O

O+

-O

ozone nitrogen dioxideO2+NO NO2*

NO2* NO2

hv

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

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 ProblemsOptical Electronic Spectroscopy

Chapter 13:

Problem 13-6Problem 13-13

Further Reading

Review Skoog et al. Chapters 13-15Review Cazes Chapters 5-6

UV-Visible SpectroscopyD. 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.