Topic 4 Optical Electronic Spectroscopy 2

7/29/2019 Topic 4 Optical Electronic Spectroscopy 2

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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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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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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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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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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.

Topic 4 Optical Electronic Spectroscopy 2

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