IPC Friedrich-Schiller-Universität Jena 1 Molecular orbital Electronic configuration Electronic states UV/Vis-absorption spectrum 4. Molecular many electron systems: electronic & nuclear movement * : antibinding

IPC Friedrich-Schiller-Universität Jena 2 2 J = 0 J = 1 J = 2 J = 3 J = 4 Rotational levels v = 0 v = 1 v = 0 v = 1 v = 3 v = 4 Vibrational levels Excitation [ s] Internal conversion [ s] Fluorescence [10 -9 s] Intersystem crossing Phosphorescence [10 -3 s] S0S0 S1S1 S2S2 S3S3 S4S4 T1T1 TnTn IR- & NIR- spectroscopy UV-VIS-spectroscopy Microwave- spectroscopy 4. Molecular many electron systems: electronic & nuclear movement Jablonski-Scheme

IPC Friedrich-Schiller-Universität Jena 3  Interpret electronic absorption spectra based on |  | 2 of the vibrational levels  electronic transitions (~ s) are much faster than the vibrational period (~ s) of a given molecule thus nuclear coordinates do not change during transition 5.1 Franck-Condon principle 5. UV-Vis-Absorption

IPC Friedrich-Schiller-Universität Jena 4  Transition dipole moment for a transition between the states |i  and |f  :  For excitation follows:  Electronic transition dipole moment is developed in a rapidly converging Taylor expansion about nuclear displacements from the equilibrium position  Condon approximation neglects higher order terms i.e. electronic transition dipole moment is assumed to be constant i.e. nuclear coordinates correspond to equilibrium geometry  Condon approximation:  Transition dipole moment: B.O.-approximation 5.1 Franck-Condon principle 5. UV-Vis-Absorption

IPC Friedrich-Schiller-Universität Jena 5  = 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: 5.2 Franck-Condon principle 5. UV-Vis-Absorption

IPC Friedrich-Schiller-Universität Jena 6 |i  |f  |i  |f  5.1 Franck-Condon principle 5. UV-Vis-Absorption

IPC Friedrich-Schiller-Universität Jena Molecular electronic transitions 5. UV-Vis-Absorption  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*  * n  *n  * (C=C, C=O) (C=O, C=N, C=S) (–Hal, -S-, -Se- etc.)

IPC Friedrich-Schiller-Universität Jena 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 Fe 2+ )  Cytochromes of respiratory chain  Chlorophyll (green molecules in leaves, central atom Mg) 5. UV-Vis-Absorption  octahedron structure motive  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) Heme-group

IPC Friedrich-Schiller-Universität Jena 9  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 [(Fe 2+ ) ↔ (Fe 3+ )]  -Peaks = sensitive for redox change (analysis of mitochondria) 5.2 Transition metal complexes 5. UV-Vis-Absorption

IPC Friedrich-Schiller-Universität Jena Transition metal complexes  Hemoglobin (iron is always found as Fe 2+ ) Arterial oxygen-loaded blood= light red Blood in veines free of oxygen=deep red Desoxy Hemoglobin (Fe 2+ / 92 pm / high spin) End-on coordination of O 2 (Fe 2+ / 75 pm / low spin) 0,4 A ° B 5. UV-Vis-Absorption

IPC Friedrich-Schiller-Universität Jena 11 Fundamental terms:  Polarimetry, optical rotation, circular birefringence: turning of the plane of linearly polarized light  Optically active molecules exhibit different refractive indices for right n R and left n L polarized light  n R ≠ n L  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 (  R ≠  L ). 5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism 5. UV-Vis-Absorption

IPC Friedrich-Schiller-Universität Jena 12 Polarimetry  What happens if light interacts with chiral molecules?  Enantiomeric molecules interact differently with circular polarized light.  Polarizability  depends on direction of rotation of incoming circular polarized light  Optically active substances exhibit different refractive indices for right n R and left n L polarized light  n R ≠ n L 5. UV-Vis-Absorption 5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism

IPC Friedrich-Schiller-Universität Jena 13 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 5. UV-Vis-Absorption 5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism Sample cell Incoming light Transmitted light phase shift EyEy ExEx

IPC Friedrich-Schiller-Universität Jena 14 Polarimetry: For  follows: Na-D line =589 nm 2-Butanol  =11.2° (Messwert) T=20°C l=1dm   Difference is rather small! Due to the different refractive indices a phase difference  =  L –  R 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  /2 arises It follows: l 5. UV-Vis-Absorption 5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism

IPC Friedrich-Schiller-Universität Jena 15 Polarimetry  The measured angle-of-rotation results in:  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:  in angular degree, length in decimeter(!) and c in g ml UV-Vis-Absorption 5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism

IPC Friedrich-Schiller-Universität Jena 16 Optical rotatory dispersion(ORD)  ORD measures molar rotation [  ] 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  Reason: refractive indices for left and right polarized light change differently with wavelength (rotatory dispersion is proportional to refractive index difference). ORD-spectra of 17ß- and 17  - hydroxy-5  -androstan 5. UV-Vis-Absorption 5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism

IPC Friedrich-Schiller-Universität Jena 17 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 negativ Cotton effect 5. UV-Vis-Absorption 5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism

IPC Friedrich-Schiller-Universität Jena 18 Optical rotatory dispersion(ORD)  Quantitative theoretical correlations between 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). ORD-Spektren von 5  -Spirostan und 5  - Spirostan-3-on 5. UV-Vis-Absorption 5.3 Polarimety & Optical rotatory dispersion & Circular dichroism

IPC Friedrich-Schiller-Universität Jena 19 Circular Dichroism (CD)  Enantiomeric molecules exhibit besides different refractive indices for left and right circular polarized light also different absorption coefficients:  It follows:  left and right circular components ORD : different retardation CD also different absorption  Transmitted light is elliptically polarized. Circular Dichroism 5. UV-Vis-Absorption 5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism EyEy ExEx

IPC Friedrich-Schiller-Universität Jena 20 Circular Dichroism (CD) [10 -1 × deg × cm 2 × g -1 ] [10 × deg × cm 2 × mol -1 ]    The ratio between short and the long elliptical axis is defined as tangent of an angle , the so called ellipticity (tan  = b/a):  a = E R + E L  b = E R - E L  The specific ellipticity is defined as: where  0bs is the experimentally determined ellipticity.  The molar ellipticity is defined as: 5. UV-Vis-Absorption 5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism

IPC Friedrich-Schiller-Universität Jena 21 Circular Dichroism (CD)  Signal heights are displayed either as absorption difference  or as ellipticity [  ].  Molar ellipticity and circular dichroism can be interconverted:  Correlation between ORD and CD:  ORD is based on the different refractive indices of left and right circular polarized light (n R ≠ n L )  CD results from the different absorption behavior for left and right circular polarized light (  R ≠  L )  Connection of both phenomena via Kronig-Kramer relationship:  This relation allows the calculation of an ORD value for a particular wavelength from the corresponding CD spectrum  grad cm 2 dmol -1  5. UV-Vis-Absorption 5.3 Polarimety & Optical rotatory dispersion & Circular dichroism

IPC Friedrich-Schiller-Universität Jena 22 Circular Dichroismus (CD)  Simple model:  For an electronic transition to be CD active the following must be true: µ e is the electronic transition dipole moment (= linear displacement of electrons upon transition into an excited state) µ m 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 transitionMagnetic transitionOptical activity 5. UV-Vis-Absorption 5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism

IPC Friedrich-Schiller-Universität Jena 23 Circular Dichroism (CD)  Application field:  -sheet random coil  -helix 5. UV-Vis-Absorption 5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism

IPC Friedrich-Schiller-Universität Jena 24 Circular Dichroism (CD)  Application field: Typical reference CD spectra: Poly-L-Lysine in different conformations:  -Helix,  -sheet and random coil. Temperature dependent CD spectra of insuline: For increasing temperature the molecule changes form  -helix into the denaturated random coil form with ß-sheet contributions. 5. UV-Vis-Absorption 5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism

IPC Friedrich-Schiller-Universität Jena 25  Vibrational transitions in the IR and NIR  VCD monitors difference in absorption between left and right circular polarized light Vibrational-Circular-Dichroism (VCD) v=0 v=1 5. UV-Vis-Absorption 5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism

IPC Friedrich-Schiller-Universität Jena 26  Determination of the absolute configuration Vibrational-Circular-Dichroism (VCD) (  )-Mirtazapine Advantages VCD vs. CD  Electronic chromophore is not necessary  VCD exhibits more characterisitic bands 5. UV-Vis-Absorption 5.3 Polarimetry & Optical rotatory dispersion & Circular dichroism

IPC Friedrich-Schiller-Universität Jena Fluorescence Spectroscopy

IPC Friedrich-Schiller-Universität Jena 28 J = 0 J = 1 J = 2 J = 3 J = 4 Rotational levels v = 0 v = 1 v = 0 v = 1 v = 3 v = 4 Vibrational levels Excitation [ s] Internal conversion [ s] Fluorescence [10 -9 s] Intersystem crossing Phosphorescence [10 -3 s] S0S0 S1S1 S2S2 S3S3 S4S4 T1T1 TnTn IR- & NIR- spectroscopy UV-VIS-spectroscopy Microwave- spectroscopy 6. Basic concepts in fluorescence spectroscopy

IPC Friedrich-Schiller-Universität Jena 29  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 = Stokes-Shift due to vibrational energy relaxation within electronic excited state 6.1 Stokes-Shift 6. Basic concepts in fluorescence spectroscopy