EPR and methologies Flashcards
What is EPR?
Electron Paramagnetic Resonance.
It is a spectroscopic technique that detects species that have UNPAIRED electrons.
What is ES(M) R ?
Electron Spin (Magnetic) Resonance
List some
Unpaired e- species:
1) Radicals
2) Transition metals
3) Defects in material
How does EPR work? Differences between
A spin of an electron gives it a magnetic Moment. A magnetic field has two orientations (+1/2 or -1/2)
In DIAmagnetic species all electrons paired and so overall it has NO SPIN
PARAmagnetic species have at least one UNPAIRED electron and so have spin. EPR deals with unpaired.
Where there is no magnetic fields, the e- have SAME ENERGY and so SAME number of UNPAIRED e- in both states (one + and - means no net spin) but where there is an magnetic field (B0), the e- don’t have the same energy
What is spectroscopy
The differene beteen the 2 energy levels (low energy -e- and high energy +e-.
E difference is proportional to the magnetic field; increase field, increases difference
The absolute energy difference between the high and low energy states is what determines the population distribution between the states.
No E difference in the absence of a field.
What is the Boltzmann distribution?
This determines the the ratio/proportion of e- in high and low states; the equation shows particles partitioning between energy states.
The higher the frequency, the greater SENSITIVITY to the technique used.
The population difference in EPR is greater than NMR , so EPR is more sensitive
Describe the difference between the two energy states.
If it orients itself parallel to the field it is in a low energy state(-1/2)
If it orients itself anti-parallel to the field it is in a high energy state (+1/2) It takes (greater) energy to turn a magnet around AGAINST the field,
What exactly is detected by the EPR spectrum?
At equilibrium, there is an small excess of number of spins in the LOW ENERGY state.
The state of resonance is detected: where the electron absorbs or emits the correct amount of electromagnetic radiation (at the correct frequency) in order to move between energy levels.
ie, the movement from low to high.
How can an electron can be brought into a state of resonance?
change in energy depends on magnetic field and so e- moves if:
1) fixed frequency and vary the field- CONTINUOUS WAVE (CW) technique; magnetic field increases E change only at fixed frequency a which point it will show an absorption peak
2) fixed field, vary the frequency- DOMAIN/PULSED technique
describe CW EPR type resonance spectrums
Spectrum : x axis magnetic field, y axis absorbance.
When y=zero this corresponds to absorbance peak maximum.
the gradient of the absorbance peak (derivative of the spectrum)
Electrons are shown to relax FASTER 9than protons
describe pulsed EPR type resonance spectrum
Resonance only
occurs when correct
frequency is obtained
Frequency applied as a pulse of short high energy microwaves.
instruments produce short enough pulses to excite about 100MHz of spectrum; the size of the pulse determines on how much of spectrum to excite.
However this technique is difficult, required frequencies are hard to obtain so CW is used.
In Pulsed ERP
Each e- is represented by a vector in either +z (low energy) or -z (high energy) the NET magnetization determines the direction of the magnetic field (B0)
If field is applied, the net magnetisation will turn 45 degrees, then 90 degrees at which point it will align with B1 direction.
At B1 net magnetisation, PHASE COHERENCE happens.
In pulsed EPR
To revert the B1 state to the equilibrium state through RELAXATION
T) spin-lattice relaxation (longitudinal) where z magnetization decreases.
Tii) spin-spin relaxation (transverse) where xy magnetisation and so increase in Z
Ti always LONGER than Tii
In Pulsed EPR
DEADTIME is the length of time for excess energy to DISSIPATE from the system before detection; the time in between initial pulse and measurement
Pulsed EPR relies on spin echo experiment; spin echo recovery sequence (90x-t-180y-t- echo)
1) apply 90 degree pulse (so z direction to y direction) and wait time (t) in which they go clockwise from y
2) apply pulse in 180 degree on the y axis so e- are flipped and wait or time in which they travel back o start point y.
Tii at this stage, spread out called dephasing
3) echo- when in phase (e- direction same a SIGNAL is seen
How is EPR different from NMR?
1) Uses gigahertz frequencies instead of megahertz
2) More sensitive (Boltzmann distribution)
3) Requires weaker magnets.
4) Has much broader lines due to much faster relaxation rate.
5) Pulsed EPR must be carried out at much lower temperature (50K cf 293K)
6) Pulsed EPR experiments are based on spin echo pulses
7) At the moment can not excite whole spectrum, but NMR can.
List the outline of instruments used in EPR
1) source- microwave bridge
2) sample (in cavity in magnetic field
3) detector (microwave bridge
4) spectrum, outputted to computer
The HIPER project instrumentation aims to produce no deadtime and so maximal sensitivity, and ins pulses that excite whole nitroxide spectrum (similar to NMR) with high powered field and power.
What are some uses of EPR?
1) structural biology - determines their PRIMARY SEQUENCES of nucleic acids and proteins which are polymeric chemicals
This is anylysed through the use of :
i) X-ray Crystallography
ii) NMR (size restriction)
iii) Small angle x-ray scattering (in solution)
iv) Circular dichroism
v) EPR
EPR requires an unpaired electron, most systems do not have this so the molecule of interest must be LABELED
Paramagnetic
Paramagnetism is a form of magnetism whereby certain materials are attracted by an externally applied magnetic field, and form internal, induced magnetic fields in the direction of the applied magnetic field.
Molecule that do not contain paramagnetic species are EPR slient.
Advantage: easier to interpret simple systems by spin labels. but disadvantage as there are fewer accessible systems
How does spin labeling work?
the reactivity is unique o a specific location in molecule (RNA/DNA/Protein) and posses a chemistry that suits its chemistry; the impact of the modification and its ability to be removed is also important.
Nitroxides
they have a STABLE UNPAIRED e- where higher density on the N-O bond.
It can be inactivated and made silent by reducing agents; vitamin C reduces radicals;so where N-O molecules are radical but where N-OH molecules are nonradical
Spin labeling in DNA
DNA oligomers (60 nucleotides) are modified AFTER they are made.
Synthesis incorporates a 5-iodo 2’deoxyuridine which can be controlled for selection
Post synthetic incorporation requires 2’-aminouridine
4 amino TEMPO converted to an isocyanate
Isocyanate reacted with purified DNA; this reaction (amine and isocyante very fast and specific)
Spin label linker still has rigid peptide bond character
Sonogashira reaction is a cross-coupling reaction
used in organic synthesis to form carbon–carbon bonds.
It is very specific and high yeilding. The spin label attached with RIDID LINKER
Spin labeling in proteins
The protein must be MUTATED.
MTSL agent is used for very specific condition to form a DISULPHIDE BOND with the protein.
The linker is flexible
Label itself is SMALL
