UV-vis and Fluorescence Spectroscopy

Literature (Selection)
General Texts: M. Klessinfer, J. Michl, Excited States and Photochemistry of Organic Molecules, VCH Publishers, 1995. J.M. Hollas, Modern Spectroscopy, Wiley&Sons, 1996. D.C. Harris, M.D. Bertolucci, Symmetry and Spectroscopy: An Introduction to Vibrational and Electronic Spectroscopy, Dover Publications, 1990. D.C. H.-H. Perkampus, H.C. Grinter, T.L. Threfall, UV-vis Spectroscopy and Its Applications, Springer, 1992. B.J. Clark, T. Frost, M.A. Russell, UV Spectroscopy - Techniques, instrumentation and data handling, Chapman&Hall, 1993. J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Publishers, 1999. M.G. Gore, Spectrophotometry and Spectrofluorimetry: A Practical Approach, Oxford Univ. Press, 2000. L. Brand Fluorescence Spectroscopy, Academic Press, 1997.

Specialized Topics: J.D. Coyle, Introduction to Organic Photochemistry, Wiley&Sons, 1988. N.J. Turro, Modern Molecular Photochemistry, University Science Books, 1991. V. Balzani, F. Scandola, Supramolecular Photochemistry, Ellis Horwood, 1991. J.R. Lakowicz, Topics in Fluorescence Spectroscopy (Vol 1-6), Plenum Publishers, 1997. Data Collections: H.H. Perkampus, UV-vis Atlas of Organic Compounds (Part 1 and 2 ), VCH Publishers, 1992.

UV-vis Range of the Electromagnetic Spectrum

[nm] 10 200 400
420 470 530 580 620 700

750

green

blue

orange

purple

yellow

x-rays

far UV

near UV

red

infrared

visible region [cm1] 106 5104 2.5104 1.3104

c=
(cm ) =
1

E = h =

h c

= hc

1 eV = 8066 cm

= 23 kcalmol 96.5 kJ mol

1

Electronic Transitions
If light with suitable energy hits a molecule in the ground state 1, the energy can be absorbed and the molecule is raised to an electronically excited state 2. The molecule can return to its ground state via spontaneous emission of a photon (light).
2 Absorption Emission 1 2

E = E(1 ) E(2 )
= h
1

The probability for such a transition is expressed by the oscillator strength (= fraction of negative unit charge, which is involved in the transition) and the transition moment M12.

Selection Rule
Not every possible transition is seen in a spectrum > the statements of required characteristics are called selection rules:
Allowed transitions = ground and excited state for a possible transition possess the required characteristics Forbidden transitions = ground and excited state do not meet the characteristics

LaPortes Rule: In centrosymmetric environments transitions can only occur between states of opposite parity
u > g or g > u (d > p, s > p are allowed, but not d > d, s > d etc.)

Spin Selection Rule: Transitions may occur only between energy states with the same spin multiplicity => most possible transitions are actually forbidden! Nevertheless, in certain cases also forbidden transitions can be observed, but their intensity is much weaker.

Jablonski Diagrams

energy A F Ph

Absorption Fluorescence Phosphorescence Radiative process Non-radiative process

IC ISC

Internal Conversion Intersystem Crossing

Vibrational and Rotational Energy Levels

In contrast to single, isolated atoms electronic transitions in molecules appear as broad absorption bands due to the presence of vibrational and rotational energy levels:

Sharp-line absorption typical for isolated atoms in the gas-phase Absorption band with vibrational structure typical for small or rigid molecules Structureless broad absorption typical for large molecules in solution

Etotal = Eel + Evib + Erot

Potential energy curves and vibrational levels of a molecule (rotational sublevels are not shown)

Vibrational and Rotational Energy Levels

The structure of absorption bands depends strongly on the acquisition conditions:

Vibrational structure of the n,* absorption band Electronic, vibrational and rotational energy level diagram of 1,2,4,5-tetrazine: I: Vapor phase spectrum at room temperature II: Spectrum at 77K in isopentane/methycyclohexane matrix III: Spectrum in cyclohexane at room temperature IV: Spectrum in water at room temperature

Franck-Codon Principle
Absorption of light occurs in the range of 1015 s, which is much shorter than the time needed for vibrational changes According to the Franck-Codon principle, the absorption corresponds to a vertical transition of the ground state to the excited state energy hypersurface > all bond lengths, angles, conformations and solvation cages are conserved during the transition

Unsymmetric band resulting from approximately equal equilibrium internuclear distances in the ground and excited state > intense 0 > 0 transition

Symmetric band internuclear distances in the excited state larger than in the ground state > intense 0 > 2 transition

Classification of Electronic Transitions

In organic molecules electronic transitions can be classified by indicating the molecular orbitals involved:
Absorption range for various electronic transitions:

The corresponding transitions can be abbreviated as: > * > * n > * n > *

Labeling of Electronic Transitions

Beside the Kasha nomenclature based on simplified molecular orbitals, there are various other commonly used descriptions for electronic transitions:

Spectrum Acquisition

light source

monochromator

sample

PMT

Lambert-Beer (1760/1852):

I A = log 0 = c l I

A: absorbance

: molar extinction coefficient

l:
path length of sample cuvette

Spectrum Acquisition
Most instruments record the absorbance A as a function of the wavelength Wavenumbers are proportional to the energy of the absorption, and usually the better choice for quantitative interpretations:

UV spectra of azulene 2 in cyclohexane A) log () = f () B) log () = f ( ) C = f () D) = f ( )

Absorption of Isolated Chromophoric Groups (Lowest energy transitions)

Interactions Between Chromophores

A molecule which contains several isolated chromophores will give a UV-vis spectrum which essentially consists of the additive absorptions of the individual groups This is not the case for conjugated chromophores: With increasing conjugation the absorption energy and intensity decreases steadily ( > p*)

H H C O H H O C C O

max = 303 nm = 18

max = 450 nm =5

Olefines and Polyenes

The > * transition in ethylene occurs at 165 nm (max = 16000). Substitution with an atom containing non-bonding electrons (=auxochromic groups, OH, OR, NH2, NHR, SH, SR, Hal) results in a bathochromic shift > the non-binding electrons interact with the -orbitals of the double bond > the energy difference between the highest unoccupied molecular orbital (=HOMO) and the lowest unoccupied molecular orbital (=LUMO) decreases

LUMO energy HOMO

Conjugated Olefins
Conjugation of two or more double bonds results in decreasing energy difference between the HOMO and LUMO:

Table: longest wavelength absorptions in conjugated all-trans polyenes Note: the molar extinction coefficient is increases with increasing size of the polyene

Cis-/trans Isomers
Cis/trans isomers show different absorption spectra due to change of the symmetry properties of the molecule and therefore also of the ground and excited state wavefunctions:

The first overtone of -carotin is found at 340 nm. In the all-trans configuration this transition is symmetry forbidden, whereas in the depicted cis-configuration the transition is allowed. > this leads to the so called cis-peak of the carotines

Empirical Rules
Woodward (1942) as well as Fieser and Scott derived a set of empirical rules for the estimation of the long wavelength maxima in diens:

acyclic, transoid 217 nm

cisoid (homoannular) 253 nm

transoid (heteroannular) 214 nm

Increments:
For each additional conjugated double bond For each exocyclic position of a double bond For each alkyl group For each of the following auxochromic groups: O-alkyl O-acyl S-alkyl N(alkyl)2 Cl Br +30 nm + 5 nm + 5 nm + 6 nm + 0 nm +30 nm +60 nm + 5 nm + 5 nm

Benzene and Aromatic Compounds

A) Energy diagram of the benzene -orbitals B) Energy term diagram: I: max 256 nm, 1A1g > 1B2u (-band) II: max 203 nm, 1A1g > 1B1u (p-band) III: max 184 nm, 1A1g > 1E1u (-band) Absorption spectrum of benzene

Substitution Effects
Long wavelength absorptions of some para-disubstituted benzenes:

The strong bathochromic shift for 4-nitrophenol is due to a charge transfer absorption:

O N O OH

O N O OH

Charge Transfer
UV/vis spectra of o-, m-, and p-nitrophenol: A A) in 10 mM HCl B) in 5 mM NaOH Note: the low energy charge transfer band is not only observed for the ortho and para isomer, but also for the meta isomer

Condensed Aromatic Systems

As compared to benzene the HOMO and LUMO orbitals in condensed aromatic compounds are not degenerate > there are four different electronic transitions possible:

A) Orbital diagram

B) Term diagram

C) Electronic transitions (under consideration of configuration interaction)

Condensed Aromatic Systems

With increasing size the -, p-, and -band of benzene are shifting to longer wavelengths The bathochromic shift leads to absorption in the visible range:
Benzene Tetracene Pentacene Hexacene colorless oange-yellow blue-purple dark green

Carbonyl Compounds
Excitation can occur to the antibonding * or * orbitals With saturated aldehydes and ketones the allowed n > * and > * transitions are observed in the vacuum-UV region > only the forbidden n(p) > * transitions can be observed (275- 300 nm, e = 15-30)

Influence of Auxochromes
Auxochromic substituents (OH, OR, NH2, NHR, SH, SR, Hal) at the carbonyl group increase the energy of the * orbital (-donor), and decrease the n orbital energy (-acceptor) > the n,* transitions are shifted to higher energy (shorter wavelength) Conjugation with a double bond has essentially no effect on the n-orbital energy, but increases the HOMO energy > the ,* transition of enones with increasing chain length is shifted into the visible range

energy