Physical Methods In Bioinorganic
Chemistry
1. X-ray spectroscopy: EXAFS, XANES
2. Resonance spectroscopy - Resonance
Raman - RR
3. Electron paramagnetic resonance – EPR
Pulsed EPR: ESEEM, ENDOR
4. Magnetic Spectroscopy -
Magnetic Circular Dichroism
5. Mössbauer Spectroscopy
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1. X-ray spectroscopy: EXAFS, XANES
• Gives M-L distances to high precision
• Gives identities and numbers of M and L
• Gives some information on geometry
• No info on angles, conformations
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X-ray Absorption Spectroscopy in Biology (BioXAS)
It can be applied regardless of the physical state of a sample and is also used for the
characterization of other non-crystalline systems such as metal ions in solution or on catalyst
supports.
At the so-called absorption edge of an element of choice, the XAS shows fine structures, both at
the absorption edge itself as well as above the edge, which are related to the chemical structure.
From the Near Edge Structure (XANES) at and near the absorption edge, information on the valence
state and coordination geometry of the element involved can be derived.
Extended X-ray Absorption Fine Structure (EXAFS) beyond the edge is interpreted to give
information on the type, distance, and number of the surrounding ligands.
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Schematic representation of an X-ray absorption spectrometer
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XAS, EXAFS, XANES
When an atom is bombarded by X-rays
- an electron from a core level is excited to the unoccupied states of the system
- changing the X-ray excitation energy changes the unoccupied state the electron can reach
- EXAFS: extended X-ray absorption Fine Structures
- XANES: X-ray Absorption Near Edge Structure 5
When a photoelectron is ejected
EXAFS “Ripples” from
interference of neighbors
Energy needed to eject
core electron
Considering the wave nature of the ejected
photoelectron and regarding the atoms as point scatterer
Amplitude of a simple picture can be seen in which the backscattered
waves interfere with the forward wave to produce
backscattering is
either peaks or troughs.
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proportional to the
Mn-O ~ 1.4 Å Mn-O ~ 2.2 Å
Various
Intramolecular
Distances
in the Mn4 cluster
Mn-Mn ~ 3.0 Å of Photosystem II,
the O2 evolving
center in
Photosynthesis,
Mn-Ca ~ 3.0 Å
as seen by EXAFS.
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2. Resonance Spectroscopy
Electron paramagnetic resonance – EPR
Gives info on metal ion identity and donor atoms
Some information on bonding character
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Electron Paramagnetic Resonance (EPR)
or
Electron Spin Resonance (ESR)
Ms = +1/2
E = g b B
where the g-value
gives characteristic info.
No magnetic field E is microwave region
B=0 Ms = -1/2,
antiparallel to B more stable
Magnetic field
B≠0
Resonance
Measurement
EPR spectrometer
o X-band: 9-10 GHz
o Q-band (high field): 35 GHz
• vary B field (~3500 Gauss)
to bring resonance;
• E is absorbed by the sample
when the frequency of the
radiation is appropriate to the
energy difference between two
states of the electrons
The working equation permits a large combination of frequency
and magnetic field values, but the great majority of EPR
measurements are made with microwaves in the 9–10 GHz region,
with fields corresponding to about 3500 G.
For a field of 3350 G, spin resonance occurs near 9388.2 MHz for
an electron compared to only about 14.3 MHz for 1H nuclei.
In real systems, electrons are normally not solitary, but are
associated with one or more atoms.
An unpaired electron can gain or lose angular momentum, which
can change the value of its g-factor. This is especially
significant for chemical systems with transition metal ions.
Interactions of an unpaired electron with its environment
influence the shape of an EPR spectral line. Line shapes can
yield many useful information. 11
Derivative Signal
The magnetic moment of a nucleus with a non-zero nuclear spin will
affect the angular moment of any unpaired electron associated with
that atom.
This leads to the phenomenon of hyperfine coupling splitting of
the EPR resonance signal into doublets, triplets and so forth.
The g-factor and hyperfine coupling in an atom or molecule may not
be the same for all orientations of an unpaired electron in an
external magnetic field.
This anisotropy depends upon the electronic structure of the atom
or molecule (e.g., free radical) in question, and so can provide
information about the atomic or molecular orbital containing the
unpaired electron.
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Nuclear Hyperfine Coupling
AH .
CH3 radical
e- localized on C
Hyperfine coupled to 3H (quartet)
AH
.
CH2(OCH3) radical
AH e- localized on C
Larger hyperfine (AH) coupled to
2H (triplet) and smaller hyperfine
(AH) to 3H (quartet)
Isotropic vs. Anisotropic Spectra
Depends on sample type:
- liquid solution room temp
giso
- frozen solution
- powder
- single crystal (oriented)
Depends on symmetry around metal ion g||
g
gyy
gxx gzz
cubic,
octahedral, rhombic,
Oh tetragonal, lower symmetry
D4h
The EPR spin Hamiltonian operator with x,y,z tensors:
Cu-substituted Alcohol Dehydrogenase
Replacement of the catalytic ZnII in
horse liver alcohol dehydrogenase
with CuII produces a mononuclear CuII
chromophore with a ligand set
consisting of two cysteine sulfur, one
histidine nitrogen plus one other atom.
Figures (A), (B), (C) and (E) - rhombic
distortion with g1 > g2 > g3 and the copper
hyperfine splitting of g1 is relatively small
(which we take as evidence for a high copper-
thiolate covalence and extensive ground-state
copper–sulfur orbital mixing), and Figure (D),
the complex with pyrazole - axial species with
g1 > g2 = g3 and where the copper hyperfine
splitting of the g1 line is clearly much
greater.
g3 g2 g1
For the complex with pyrazole the g (g2, g3)
line is split into eight equally spaced
hyperfine lines, which is most easily
VIBRATIONAL SPECTROSCOPIES
Bonds between atoms have quantized vibrational energy levels that can be
probed by methods that employ electromagnetic radiation of the correct
frequency to promote transitions between the different levels.
Vibrational spectroscopies explore the molecular structure by analyzing the
energy of transition between these vibrational levels.
Three different phenomena are associated with vibrational levels and can be
used to probe molecular structure:
1. IR absorption of radiation,
2. Raman scattering of radiation and
3. Fluorescence emission of radiation.
IR absorption can provide structural information on biological
macromolecules.
Resonance Raman
The RR spectroscopy is a particular application of the general Raman
spectroscopy where the incident laser radiation has a frequency that
matches the energy of an electronic transition in the sample.
RR is one of the most versatile methods for the study of metal ion containing
biological molecules.
It requires a very small volume (in the tens of microlitre range) of pure
sample with as high concentration as possible.
The measurement can be made using liquid or frozen solutions of the target
molecule, using solids or even crystals.
Raman Spectroscopy
A scattering technique
Reveals vibrational levels
Complementary selection rules to Infrared Spectroscopy
IR: dipole moment
Raman: polarizability moment
Good for aqueous biological samples; no strong O-H absorption
Laser source
Resonance Raman (RR) = Raman + electronic spectroscopy
If the wavelength of the exciting laser coincides with an electronic absorption of a molecule, the
intensity of Raman-active vibrations associated with the absorbing chromophore are enhanced by a
factor of 100 to 10,000.
RR of [U=O]2+ ion showing
symmetric mode at 835 cm-1 is
dependent on excitation energy
RR gives detailed orbital
and energy information
about two Mo=O model
systems
Contributions to magnetism in biologically relevant ions
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The SQUID is a sensor which can detect smallest changes in the magnetic flux of a magnetic
field and is therefore a highly precise and powerful tool which can measure even
extremely weak signals of any physical quantity related to the magnetic flux (e.g., magnetic
field, current, voltage, and magnetic susceptibility)
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The SQUID is built up of a superconducting ring which is
intercepted by Josephson junctions which are lightly separate
the superconducting ring with normal conducting or even
electric insulating material
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What is measured in the SQUID?
Two types
1.the magnetic susceptibility, χ, which requires a temperature
sweep under a certain applied field (usually smaller than 0.2 T),
2.the magnetization, M, where the magnetic field is swept while
keeping the temperature stable at very low temperatures
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TETRAPYROLLES
Fully conjugated cyclic structure, tetradentate, macrocyclic
ligands
Stable, rigid, planar or nearly planar ring system
Deprotonated forms bind metal ions tightly and size selectively
Extensive conjugation leads to very intense colors (pigments of
life) and potentially to redox activity
PORPHYRIN
S
Paper-Based Heavy Metal Sensors from the Concise
Synthesis of an Anionic Porphyrin: A Practical
Application of Organic Synthesis to
Environmental Chemistry
J. Chem. Educ. 2017, 94, 1137−1142
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BINDING OF O2 BY
MYOGLOBIN
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