Transcript Biophotonics_2011_13 - Friedrich-Schiller

4. Molecular many electron systems: electronic & nuclear movement
Molecular
orbital
Electronic
configuration
Electronic
states
UV/Vis-absorption spectrum
* : antibinding
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4. Molecular many electron systems: electronic & nuclear movement
Jablonski-Scheme
Excitation [10-15 s]
v=1
Rotational levels
J=4
v=0
Microwavespectroscopy
J=3
J=2
J=1
J=0
UV-VIS-spectroscopy
S4
S3
Internal conversion
[10-14 s]
Tn
S2
IR- & NIRspectroscopy
Intersystem crossing
v=4
v=3
Vibrational levels
S1
T1
Fluorescence Phosphorescence
[10-9 s]
[10-3 s]
v=1
v=0
2
S0
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5. UV-Vis-Absorption
5.1 Franck-Condon principle
 Interpret electronic absorption spectra based on
||2 of the vibrational levels
 electronic transitions (~10-16s)
are much faster than the vibrational
period (~10-13s)
of a given molecule
thus nuclear coordinates do not
change during transition
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5. UV-Vis-Absorption
5.2 Franck-Condon principle


= degree of redistribution of electron density during transition
= degree of similarity of nuclear configuration between vibrational
wavefunctions of initial and final states.

 Transition probability is proportional to the square modulus of the overlap integral
between vibrational wavefunctions of the two electronic states
= Franck-Condon-Factor:
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5. UV-Vis-Absorption
5.1 Franck-Condon principle
|f
|f
|i
|i
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5. UV-Vis-Absorption
5.2 Molecular electronic transitions
 Molecular electronic transitions:
valence electrons are excited from one energy level to a higher energy level.
 Electrons residing in the HOMO of a sigma bond can get excited
to the LUMO of that bond:
σ → σ* transition.
 Promotion of an electron from a π-bonding orbital to an antibonding π* orbital:
π → π* transition.
 Auxochromes with free electron pairs denoted as n have their own transitions, as
do aromatic pi bond transitions.
 The following molecular electronic transitions exist:
σ → σ* π → π* n → σ* n → π* aromatic π → aromatic π*
p,p*
(C=C, C=O)
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np*
(C=O, C=N, C=S)
ns*
(–Hal, -S-, -Se- etc.)
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5. UV-Vis-Absorption
5.2 Transition metal complexes
 A biologically very important group of metal complex bonds are the porphyrin
pigments such as:

Hemoglobin (pigment of the blood, central ion Fe2+)
 octahedron structure motive
Heme-group
 The four ligand positions of the base of
the pyramid are occupied by the lone
electron pairs of nitrogen atoms of the
plane porphyrin ring system
 The two corners of the pyramid are
occupied by specific amino acids
(histidine) and/or by an oxygen molecule
(hemoglobin)

Cytochromes of respiratory chain

Chlorophyll (green molecules in leaves, central atom Mg)
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5. UV-Vis-Absorption
5.2 Transition metal complexes
 Cytochrome C:
 Pyramid corners of heme unit are occupied by N-atom of a histidine residue and
S-atom of a mezhionine residue
 Redox change of cytochromes predominatly occurs at the central iron atom
[(Fe2+) ↔ (Fe3+)]
a-Peaks
= sensitive for redox change
(analysis of mitochondria)
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5. UV-Vis-Absorption
5.2 Transition metal complexes
Hemoglobin (iron is always found as Fe2+)
Arterial oxygen-loaded blood
=
light red
Blood in veines free of oxygen
=
deep red
°
0,4 A
End-on coordination of O2
(Fe2+ / 75 pm / low spin)
Desoxy Hemoglobin
(Fe2+ / 92 pm / high spin)
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B
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5. UV-Vis-Absorption
5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism
Fundamental terms:
 Polarimetry, optical rotation, circular birefringence:
turning of the plane of linearly polarized light
 Optically active molecules exhibit
different refractive indices for right nR and left nL polarized light  nR ≠ nL
 Optical rotatory dispersion (ORD):
Wavelength dependency of rotation
 Allows determination of absolute configuration of chiral molecules
 Circular dichroism:
linearly polarized light is transformed into elliptically polarized light upon traveling
through matter
 Different absorption coefficients for left and right circular polarized light
(eR ≠ eL ).
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5. UV-Vis-Absorption
5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism
Polarimetry
 What happens if light interacts with chiral molecules?
 Enantiomeric molecules interact differently with circular polarized light.
 Polarizability a depends on direction of rotation of incoming circular polarized light
 Optically active substances exhibit different refractive indices for right nR and
left nL polarized light  nR ≠ nL
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5. UV-Vis-Absorption
5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism
Polarimetry:
 Linearly polarized light
 Different refractive index for its left and right circular constituents
 Relative phase shift between left and right
 Vector addition yields again linear polarized light with rotated polarization plane
Ey
phase
shift
Ex
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5. UV-Vis-Absorption
5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism
Polarimetry: Due to the different refractive indices a phase difference d = jL –jR
builds up in the active medium which is proportional to the path
length l.
When exiting the medium linear polarized light where the oscillation
plane is rotated by d/2 arises
It follows:
For a follows:
l
a  ( nL  nR )
pl
l
Na-D line
l = 589 nm
2-Butanol
a = 11.2° (measured value)
T = 20°C
l = 1dm

589 10 9 m
(nL  nR )  11.2 deg
 3.66 10 7
180 deg 0.1 m
 Difference is rather small!
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5. UV-Vis-Absorption
5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism
Polarimetry
 The measured angle-of-rotation results in:
a in angular degree, length in
decimeter(!) and c in g ml-1.
 Specific rotation
is a substance specific constant (dependent on temperature
and wavelength) and is a measure for the optical activity of this particular substance.
 Molar rotation is defined as follows:
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5. UV-Vis-Absorption
5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism
Optical rotatory dispersion(ORD)
 ORD measures molar rotation [F] as function of the wavelength!
 If the substance to be investigated has
no electronic absorption within the
investigated spectral region the following
ORD spectra are obtained
ORD-spectra of 17ß- and 17ahydroxy-5a-androstan
 Reason:
refractive indices for left and right
polarized light change differently with
wavelength (rotatory dispersion is
proportional to refractive index
difference).
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5. UV-Vis-Absorption
5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism
Optical rotatory dispersion(ORD)
 Refractive indices for left and right polarized light exhibit anomalous dispersion in the
range of an absorption band
 Cotton effect
Positiv
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negativ Cotton effect
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5. UV-Vis-Absorption
5.3 Polarimety & Optical rotatory dispersion & Circular dichroism
Optical rotatory dispersion(ORD)
ORD-Spektren von 5a-Spirostan und 5a Quantitative theoretical correlations between
Spirostan-3-on
molecular structure and ORD (Cotton effect)
are difficult to derive;
 Empirical investigation are important:
ORD has been successfully applied for
constitution elucidation e.g. to position
carbonyl groups in complex optically active
molecules.
 By comparing ORD curves for structurally
isomeric ketons (reference material needed!)
the keto group can be localized.
ORD curve of molecule (2) is a superposition of a
negative curve i.e. molecular skeleton without a
chromophore (background curve) and a positive
Cotton curve (C=O chromophore).
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5. UV-Vis-Absorption
5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism
Circular Dichroism (CD)
 Enantiomeric molecules exhibit besides different refractive indices for left and
right circular polarized light also different absorption coefficients:
Circular Dichroism
 It follows:
 left and right circular components
ORD : different retardation
CD also different absorption
Ey
Ex
 Transmitted light is elliptically polarized.
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5. UV-Vis-Absorption
5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism
Circular Dichroism (CD)
 The ratio between short and the long elliptical axis is defined as tangent of
an angle , the so called ellipticity (tan  = b/a):
 a = ER + EL
 b = ER - EL
 The specific ellipticity is defined as:

[10-1 × deg × cm2 × g-1]
where 0bs is the experimentally determined
ellipticity.
 The molar ellipticity is defined as:
[10 × deg × cm2 × mol-1]
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5. UV-Vis-Absorption
5.3 Polarimety & Optical rotatory dispersion & Circular dichroism
Circular Dichroism (CD)
 Signal heights are displayed either as absorption difference De or as ellipticity [q].
 Molar ellipticity and circular dichroism can be interconverted:
[q]  [grad cm2 dmol-1]
 Correlation between ORD and CD:
 ORD is based on the different refractive indices of left and right circular
polarized light (nR ≠ nL )
 CD results from the different absorption behavior for left and right circular
polarized light (eR ≠ eL)
 Connection of both phenomena via Kronig-Kramer relationship:
 This relation allows the calculation of an ORD value for a particular wavelength l
from the corresponding CD spectrum
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5. UV-Vis-Absorption
5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism
Circular Dichroismus (CD)
 Simple model:
 For an electronic transition to be CD active the following must be true:
µe is the electronic transition dipole moment
(= linear displacement of electrons upon transition into an excited state)
µm is the magnetic transition moment
(= radial displacement of electrons upon excited state transition)
 Scalar product
is characterized by a helical electron displacement.
 Depending on the chirality of the helix preferably more right or left circular
polarized light will be absorbed, respectively.
Electronic transition
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Magnetic transition
Optical activity
IPC Friedrich-Schiller-Universität Jena
5. UV-Vis-Absorption
5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism
Circular Dichroism (CD)
 Application field:
b-sheet
a-helix
random coil
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5. UV-Vis-Absorption
5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism
Circular Dichroism (CD)
 Application field:
Typical reference CD spectra:
Poly-L-Lysine in different conformations:
a-Helix, b-sheet and random coil.
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Temperature
dependent CD
spectra of insuline:
For increasing
temperature the
molecule changes
form a-helix into the
denaturated random
coil form with ß-sheet
contributions.
IPC Friedrich-Schiller-Universität Jena
5. UV-Vis-Absorption
5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism
Vibrational-Circular-Dichroism (VCD)
 Vibrational transitions in the IR and NIR
v=1
v=0
 VCD monitors difference in absorption between left and right circular
polarized light
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5. UV-Vis-Absorption
5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism
Vibrational-Circular-Dichroism (VCD)
()-Mirtazapine
 Determination of the absolute
configuration
Advantages VCD vs. CD
 Electronic chromophore is
not necessary
 VCD exhibits more
characterisitic bands
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6. Fluorescence Spectroscopy
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6. Basic concepts in fluorescence spectroscopy
Excitation [10-15 s]
v=1
Rotational levels
J=4
v=0
Microwavespectroscopy
J=3
J=2
J=1
J=0
UV-VIS-spectroscopy
S4
S3
Internal conversion
[10-14 s]
Tn
S2
IR- & NIRspectroscopy
Intersystem crossing
v=4
v=3
Vibrational levels
S1
T1
Fluorescence Phosphorescence
[10-9 s]
[10-3 s]
v=1
v=0
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S0
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6. Basic concepts in fluorescence spectroscopy
6.1 Stokes-Shift
= Stokes-Shift due to vibrational energy
relaxation within electronic excited state
 Energy differences between vibrational states which determine vibronic band
intensities are very often the same for ground and electronic excited state
 Emission spectrum = mirror image of absorption spectrum
 Emission bands are shifted bathochromically i.e. to higher wavelengths
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