Outline of the EPR Part

• The nature of the EPR experiment• Detection of Signals• Relaxation and Saturation Phenomena• The CW-EPR instrument• Method of Detection• Examples of EPR spectra• Hyperfine coupling effects• Other applications

Electron Paramagnetic Resonance

• Monitors paramagnetic species: transition metals and free-radicals in macromolecules. Examples: mononuclear Fe3+, Cu2+, Ni3+,1+, Co2+, RS•, ROO•, FMN•, RCH2•, PLP•

• Interaction with the magnetic field yields the energy separationof ground and excited states with quantum numbers ms = + ½

• Transitions between the ground and excited states are induced by a microwave radiation (GHz). The energy difference is dependent on:

i) the effect by the other paired electrons in the same atom which cause a spin angular momentum

ii) the presence of a nuclear spin (I) which splits the spin energy levels into 2NI + 1 lines

iii) the nearby presence of other paramagnetic species: coupled systems.

The Zeeman Interaction

)3/1arccos(

2

)1(2

Angle

mh

projection

ssh

Magnitude

S

Basics of the EPR Experiment

SBgH BeZ

128 10 x 274.9

) ( 00232.2

GJ

electronfreeg

B

e

hBgE B

BB g

hB

g

E

Band / GHz λ /cm B / mT

S 3 10.0 107

X 9.75 3.15 348

Q 35 0.86 1250

W 94 0.32 3352

Detection of Signals and Saturation

Tk

BguNNNI

B

Bspins

2~)(

At 9.45 GHz (X-band) for a signal with a linewidth of 0.1 G about 1012 spins (1 pmol) of radicals can be detected. On the other hand for Cu2+ in solution (linewidth ~ 500 G) > 1 nmol is required.For normal free radicals EPR is at least 1000 times more sensitive than NMR.

Effect of Microwave Power and Saturation

Increase in power results in signalsaturation (N- ~ N+). The spin system returns to equilibrium via the spin-lattice relaxation mechanism (rate constant = 1/T1).

Spins with weak coupling to the lattice will relax slowly and are easy to saturate (trace D) with microwave power.

Spin systems with strong lattice coupling are hard to saturate (trace A).The important T1 mechanism is spin-orbit coupling, strong for transition metals and weak for radicals on light atoms (N,O,S,C). Thus T1s are long for free-radicals but short for transition metals.

T1s have an shallow inverse temperature dependence. Hence very low temperatures ( < 20 K) are required for the observation of paramagnetic transition metals.

LowerTemperature

Ideal power range

Basics of the EPR Instrument

Phase Sensitive Detection: EPR Spectra are Recorded in Derivative Mode.

Effect of the Modulation Amplitude on Intensity and Resolution.

The Spin-Orbit Interaction

The coupling of the unpaired electron with other paired electrons withinthe same atom

Treated as spin-orbit:This coupling is anisotropic and gives rise to g1 ≠ g2 ≠ g3

A : isotropic symmetryB and C : axial symmetryD : rhombic symmetry

A : free radicals, some [Fe3S4]1+, Fe3+

B: Ni1+

C: NiFeC species, Co2+

D: all FeS clusters, hemes, highlyresolved radicals

Nuclear Hyperfine Interactions

• Splitting of EPR signals due to spin-nucleus interaction or spin-spin

interaction (less common and better studies by DEER).

• Coupling constant is a tensor expressed in 10-4 cm-1 (3 MHz).

• Since the X-axis of the spectrum is H in Gauss or mT (1mT=10G),

the coupling can also be expressed in Gauss:

zzB SIASBgH

ˆ GaussAg

gMHzA

e

av /)(8025.2)/(

Nucleus Spin % Abundance ~ A /Gauss1H ½ 99.985 500 (iso)

13C ½ 1.11 9055Mn 5/2 100 20759Co 7/2 100 282

63Cu/65Cu 3/2 69.17/30.83 40057Fe ½ 2.12 33

Spin nucleus interaction and quantitation by EPR

Cu2+ as acquired

Single integration

Double integration

S=+1/2

S=+1/2

S=-1/2

-1 0+1

-1 0+1

For Cu2+ (d9) MI = 3/2, 4 lines are observedFor Co2+ (d7) MI = 7/2, 8 linesare observed

Isotopes Nuclear Spins1H, 15N, 13C, 19F,31P, 57Fe, 77Se, 111Cd, 113Cd ½2H, 14N 163Cu, 65Cu, 35,37Cl 3/217O, 95Mo, 97Mo 5/259Co, 77Se (7.5%) 7/2

Small Molecule Case: Nitrosyl Radical

Electron and nuclear spin energy levels for NO(SO3)22-

(Fremy’s salt) in a low magnetic field.

Nitroxyl First-derivative Spectrum

3200 3220 3240 3260 3280

M agnetic F ie ld (gauss)

Spectrum of tempo in fluid solution at X-band at 20oC.

Multi Center Examples: Iron Sulfur Clusters, High

Spin Fe3+2+

What Can We then learn from EPR?

1. Is there a paramagnetic signal?

2. What type of sample conditions produce the signal?

3. How many species are present?

4. dependence of spectra on chemical structure

5. Line shape : Dependence on physical environment – e.g., motion

6. g-value – characteristic for a chemical species

7. Spin-spin coupling – electron-nuclear coupling

8. Spin-spin coupling – electron-electron coupling

9. Relaxation times – effect of oxygen, ROS, other radicals

10. Pulse techniques

11. Biomedical imaging

Introduction to ICP-MS

Inductively Coupled Plasma-Mass Spectrometry (ICP-MS)

30-300 nM ; 1-10 nM ; 0.1-1 nM ; 0.02-0.2 nM ; > 10 pM

Basic Components of an ICP-MS

Diagram of an ICP-MS; Role of CRS

Generation of Matrix and Polyatomic Species

Ar, Ca, Cl and Br can be potential sources of interferences for Se and Fe.Ar, Ca, S, N, O and Cl can interfere for Ni, Co, Cu, Zn and As.

Removal of Polyatomic Species by the ORS.

Elimination of interferences with a Reaction Cell.

Application of ICP-MS to Ionomics of Yeast

(A) vacuolar; (B) mitochondrial; > 2.5 RSD increases in redand > 2.5 RDS decreases in green.

Eide, E.J. et.al. (2005) Gen.Biol. 6: R77.

Salient Points of ICP-MS

1. Useful for most elements which are important for Redox Biology: Se, Fe, MnMo, Cu, Co, Ni, Zn and (maybe) S and P

2. Since it is a form of elemental analysis, it does not distinguish redox states or chemical environments…

3. However, coupled with either GC or HPLC cam be used to identify the specieswhich have different chemical environments (speciation)

4. Applicable to the studies of the effects of gene knockouts, knockdowns, aging,environment, diet on the ion composition within cells (ionomics)

5. Sensitivity is similar to radiolabeling without the complications of disposal, decayhalf lives, cost, etc.