电子自旋共振顺磁共振经典讲座

1、An introduction to Electron Spin Resonance (ESR), November 7, 2007An Introduction to Electron Spin Resonance (ESR)Boris Dzikovski, ACERT, Cornell University The application field The basic ESR experiment. Some theoretical background. Nitroxide spin labels. Some examples for extraction of parameters

2、of molecular dynamics from ESR spectra Site directed spin labeling (SDSL) ESR distance measurementsAn introduction to Electron Spin Resonance (ESR), November 7, 2007ESR is a spectroscopic technique that detects chemical species that have unpaired electrons : Transition metal ions and complexes Mn2+,

3、 Cu2+, Gd 3+ etc. Simple inorganic compounds: O2 , NO, NO2 . Short-lived intermediate radicals OH, H, F etc. in kinetics study Defects in crystals Electrons trapped in radiation damaged sites Stable organic radicals Triplet states Biological applications: Paramagnetic cofactors: iron sulfur, copper

4、proteins Free radicals of biological origin and their spin-trapping products Spin-labelingEnergy-level diagram for two spin states as a function of applied field H. This represents the simplest ESR transition (e.g., free electrons).“Allowed” EPR transitions occur when DDMs = 1 (Ms is the magnetic sp

5、in quantum number of the spin state).The equation describing the absorption (or emission) of microwave energy between two spin states is D DE = = hu =u = gb bH where: D DE is the energy difference between the two spin statesh is Plancks constantu u is the microwave frequencyg is the Zeeman splitting

6、 factor (2.0023 for free electron) b b is the Bohr magnetonH is the applied magnetic field.An introduction to Electron Spin Resonance (ESR), November 7, 2007HAn introduction to Electron Spin Resonance (ESR), November 7, 2007RelaxationEvolution of a spin system is describedby Bloch equations:T1- spin

7、-lattice or longitudinal relaxation timeT2- spin-spin or traverse relaxation timeWhen properly integrated, the Bloch equations will yield the X, Y, and Z components of magnetization as a function of time.Stationary solution in the rotating frame gives a lorentzian line 202222)(11)(HHTTHF=)(21exp(2)(

8、202222HHTTHF=Gaussian line= inhomogeneous broadeningESR linewidth:HkTeD=211222111TTT=)/(1076. 17Gsrade=k=1 Lorentz2ln=kGaussMx, My Mz magnetization components in the rotating frame0= eH0 the Larmor FrequencyAn introduction to Electron Spin Resonance (ESR), November 7, 2007ESR and NMR are very differ

9、ent methods!electronprotonratioRest massme =9.1094*10-28 gmp =1.6726*10-24 g5.446*10-4Chargee=-4.80286*10-10ESUe=4.80286*10-10ESU-1Angular momentumh/4h/41Magnetic dipole momentmS=-ge meSge= 2.002322me=eh/4mec =9.274*10-21 erg/GmS=-gN mNSgN= 5.5856mN=eh/4mNc =5.0504*10-24 erg/G1836.12Frequency: Facto

10、r 1000 larger in EPR ! (GHz instead of MHz)Coupling strength: Factor 1 000 000 larger in EPR ! (MHz instead of Hz)Relaxation Times: Factor 1000 000 smaller in EPR ! (ns instead of ms)= much higher techniqual requirements, but unique sensitivity to molecular motionSensitivity : Factor 1 000 000 bette

11、r than in NMR ! (1nM instead of 1mM ) An ideal case, thoughAn introduction to Electron Spin Resonance (ESR), November 7, 2007The Basic ESR Experiment (conventional ESR)SourceCirculatorDetectorelectromagnetModulation coilsResonator (cavity)An introduction to Electron Spin Resonance (ESR), November 7,

12、 2007The Basic ESR Experiment (conventional ESR)ESR is done from 1 to 300+GHz 30mT-10T or 30cm-1mm, up to 2000+ GHzMachines are classified according to their source frequency : Commonly used X-band at 9.5 GHzL (1.5), S (3.0), C (6.0), Ku (17), K (23), Q (36), V (50), W (95), D(140), G(180)Field modu

13、lation is used to encode the spectrum 1st derivative lineshapeUse microwave transmission linesDo spectroscopy with a few microwatts to a few milliwatts of powerSolid state Gunn diode or DRO or tube klystron sourcesTemperatures from 4K (heme and non-heme iron) to 310K+ (in vivo/vitro) Sensitivity : I

14、ncreases as (frequency)2, but limited by sample size, field homogeneity and component construction problems. Practically (at X-band): detect 1011 spins, a detectable concentration of 10-9M. Unlike NMR a large proportion of machines are still cw. That is they do not use pulsed detection methods An in

15、troduction to Electron Spin Resonance (ESR), November 7, 2007Field modulationAn introduction to Electron Spin Resonance (ESR), November 7, 2007A commercial 9GHz systemAn introduction to Electron Spin Resonance (ESR), November 7, 2007Commercial 95GHzSpectrometer (3T)An introduction to Electron Spin R

16、esonance (ESR), November 7, 2007The g-factor:DE = hu = gbHThe field at each spin influenced by local magnetic fields, not just the external field : Heft = H + Hlocal so Heff = (1-s)H = (g/ge)H-This field is induced by H, and so depends on the external field H -g is an effective Zeeman factor, shifte

17、d from the electron g-factor, ge -The shift in g is akin to the chemical shift of NMR -The local induced field comes from the orbital motion of electrons, spin-orbit coupling mixes J, L and S and shifts g, the shift can can be g2. g is thus characteristic of different electronic structures and is al

18、so known as the Land splitting factor:Light atoms, anic and first row transition metals with a single unpaired electron can have g close to 2.0 Heavier atoms, and molecules or atoms with more than one unpaired electron can have g-values very different from 2 An introduction to Electron Spin R

19、esonance (ESR), November 7, 2007g-value Flavin semiquinone, ubiquinone, ascorbate, etc 2.0030 - 2.0050 Nitroxide spin labels and traps 2.0020 - 2.0090 sulphur radicals : S-S, S-H 2.02 - 2.06 MoV (in aldehyde oxidase) 1.94 Cu2+ 2.0 - 2.4 Fe3+ (low spin) 1.4 - 3.1 Fe3+ (high spin) 2.0 - 10 g-values fo

20、r some biologically important paramagnetic compoundsAn introduction to Electron Spin Resonance (ESR), November 7, 2007The unpaired electron, which gives us the EPR spectrum, is very sensitive to local fields in its surroundings. Local fields arising from magnetic nuclei are permanent and independent

21、 of H. Interaction with neighboring nuclear magnetic dipoles gives the nuclear hyperfine interaction and hyperfine splitting ACorresponds to the NMR coupling constant J A splittings are independent of the external field.For several equivalent nuclei n, (2nMIM + 1) transitions are observed for a nucl

22、eus M with a spin I The relative intensities are given by Pascals triangle for I = 11 11 2 11 3 3 11 4 6 4 11 5 10 10 5 11 6 15 20 15 6 11 7 21 35 35 21 7 1A - the hyperfine splittingAn introduction to Electron Spin Resonance (ESR), November 7, 2007I=1/2, 2I+1=2I=1, 2I+1=3Organic radicals in the liq

23、uid phaseCyclooctatetraen anionObservation of the 1:8:28:56:70:56:28:8:1 spectrum shows that eightprotons are equivalentButadien ion in liquid NH3Two sets of equivalent protons: 2 and 4Pyrazine anionNa+ is the counterionK+ is the counterionAn introduction to Electron Spin Resonance (ESR), November 7

24、, 2007The figures are taken from the textbook by Wertz&BoltonAn introduction to Electron Spin Resonance (ESR), November 7, 2007Anisotropy in g and AMany measurements are made in the solid state in EPR spectroscopy. The ability of EPR to obtain useful information from amorphous (glassy) and polycryst

25、alline (powders) as well as from single crystal materials has attracted much biology and biochemistry research Usually : gX, gY, gZ are not all equal, so g is anisotropic. Same for AX, AY, AZ.For EPR the local symmetry at an unpaired electron center is categorised as : Cubic. If x = y = z is cubic (

26、cubal, octahedral, tetrahedral) No anisotropy in g and A. Uniaxial (Axial). If x = y, and z is unique. Linear rotation symmetry (at least 3-fold). Two principal values each for g and A. For an arbitrary orientation:Rhombic. Three unequal components for g and A For an arbitrary orientation:22222cossi

27、nIIggg=222222222cossinsincossinZZYYXXgggg=An introduction to Electron Spin Resonance (ESR), November 7, 2007gx=2.0091, gy=2.0061, gz=2.0023The field shift between the X- and Z- orientations is DH=h/gxb- h/gz b hDg/4b11GAn introduction to Electron Spin Resonance (ESR), November 7, 2007gx=2.0091, gy=2

28、.0061, gz=2.0023I=1/2, Ax= 6.2, Ay = 6.3, Az=33.6An introduction to Electron Spin Resonance (ESR), November 7, 2007gx=2.0091, gy=2.0061, gz=2.0023I=1, Ax= 6.2, Ay = 6.3, Az=33.6An introduction to Electron Spin Resonance (ESR), November 7, 2007Powder and glass spectra S=1/2, I=0, gx=gygz Axially symm

29、etric g-factor2/12222sincos=bbgghghHIIeffris the angle between a given symmetry axis and themagnetic field directionThe given solid angle W W is defined to be the ratio of the surface area A to the total surface area on the sphere: W= W= A/4r2 dW=2r2sind/4r2= sind/2ddHHPsin)(ddHHP/sin)(bcos)()sincos

30、()(222/32222gggghHPIIIIbcos)(1)(223ggHhHPIIrAn introduction to Electron Spin Resonance (ESR), November 7, 2007Axial Lineshape Rhombic Lineshape An introduction to Electron Spin Resonance (ESR), November 7, 2007EPR Middle-EarthQ-band ESR spectrum of molecularoxygen at reduced pressure500015000 G10000

31、The rotational angular momentum, which is quenched in the liquid or solid phases couples strongly to the electronic spin and orbital angular momentaESR signals around usEPR dosimetry:ToothpasteHuman hairAn introduction to Electron Spin Resonance (ESR), November 7, 2007Determination of the accumulate

32、d radiationdose by ESR of tooth enamelAn introduction to Electron Spin Resonance (ESR), Nitroxide spin labelsNONONOCH3CH3CH3H3CCH3H3CHOThe g- and A-tensor frame for anitroxide radicalAn introduction to Electron Spin Resonance (ESR), November 7, 2007An introduction to Electron Spin Resonance (ESR), g

33、x=2.0091, gy=2.0061, gz=2.0023I=1, Ax= 6.2, Ay = 6.3, Az=33.69.4 GHzAn introduction to Electron Spin Resonance (ESR), November 7, 2007An introduction to Electron Spin Resonance (ESR), Angular averaging in the case of S=1/2, I=1, gx=2.0089, gy=2.0061, gz=2.0027, Ax= 5.2, Ay = 5.2, Az=31.0, X-BandAn i

34、ntroduction to Electron Spin Resonance (ESR), November 7, 2007component I=+1is spread over DA- hDg/4b component I=0is spread over hDg/4bcomponent -1is spread over DA+ hDg/4bThe figures are from the monograph by Kuznetsov An introduction to Electron Spin Resonance (ESR), High field EPR spectroscopy i

35、s the g-resolved spectroscopy,the regions corresponding different orientations of the magneticaxis relative to the external magnetic field do not overlapAn introduction to Electron Spin Resonance (ESR), November 7, 2007gx=2.0091, gy=2.0061, gz=2.0023I=1, Ax= 6.2, Ay = 6.3, Az=33.6170 GHzAn introduct

36、ion to Electron Spin Resonance (ESR), November 7, 2007An introduction to Electron Spin Resonance (ESR), November 7, 2007Nitroxyl tumbling correlation timeAs the molecule tumbles, the smaller splitting for mI = 0 is averaged more effectively than the larger splittings, which causes differences in the

37、 linewidths of the three hyperfine lines: An introduction to Electron Spin Resonance (ESR), November 7, 2007Nitroxyl LineshapesAs the tumbling correlation time decreases, the extent of averaging of anisotropic features increases and the spectrum approaches the 3-line signal that is characteristic of

38、 rapid tumbling.In the motional narrowing region, the dependence of the width of an individual hyperfine line on the nuclear spin state (mI) can be expressed as 2 )(ImCmBAmBII=DX-bandRotation correlation times between 10-11 and 10-6 are detectable by ESRAn introduction to Electron Spin Resonance (ES

39、R), November 7, 2007=) 1( I)0( I) 1( I)0( I21BThe parameters B and C are related to peak-to-peak amplitudes, I(mI) by:=2) 1( I)0( I) 1( I)0( I21CThe high-field line has mI = -1. Tumbling correlation times are calculated from B and C using1o2oB18bg32CDb=andWhere1ooB1b15B4og32BDDb=)ggg(31gzyxo=b=oogB)

40、(5 . 0(yxzggg=Db)AA(5 . 0A(32byxz=DBo is the peak-to-peak width of the center line Hyperfine values (A) are in radians/s The calculation assumes isotropic tumblingCalculation of tumbling times in the case of fast isotropic tumblingAn introduction to Electron Spin Resonance (ESR), November 7, 2007NOO

41、HSample Calculation gx = 2.0094, gy = 2.0059, gz = 2.0023Ax = 2 18x106, Ay = 2 22.5x106, Az = 2 103x106 rad/sI(+1) = 13.5, I(0) = 16.4, I(-1) = 3.4 (arbitrary units)DBo = 3.52 Gauss b = 9.274x10-21 erg/G= 9.2449x109 s-1 h=6.626x10-27 erg s = 2.1x10-9 s from B or = 2.3x10-9 s from CThe disagreement i

42、s an indication of the approximate nature of this calculation.4-OH-TEMPO (tempol) in 9:1 glycerol:waterDetermination of microviscosity:kTV=(Stocks-Einstein)Extremely useful in oversaturated/overcooled disperse systems.Example: testing photographic materialsAn introduction to Electron Spin Resonance

43、(ESR), November 7, 2007g- and A- tensors are sensitive to the local polarity TEMPO in Emulsion: toluene/SDS/waterNOOHgx gy gz giso Ax Ay Az AisoToluene 2.00986 2.00626 2.00222 2.00617 6.2 7.0 34.3 15.6Water/glyc 2.00878 2.00604 2.00215 2.00565 6.9 7.9 37.4 17.4An introduction to Electron Spin Resona

44、nce (ESR), November 7, 2007TEMPO in the LQ phase of DLPC Partially A-resolvedg-resolved spectraAn introduction to Electron Spin Resonance (ESR), November 7, 2007Orientational resolution of HF ESR for nitroxide spin labels One of the main virtues of HF ESR over ESR at conventional microwave frequency

45、 is the excellent orientational resolution for nitroxide spin labels. At HF, once motion is discernable in the spectrum, one can discern about which axis the motion occurs.Spin labeled fatty acids in solid cyclodextrinsCOOHNOOCOOHON ONOOCOAn introduction to Electron Spin Resonance (ESR), November 7,

46、 2007Z-rotation vs. slow motionAveraging real tensor componentsgxx, gyy, gzz, Axx, Ayy, AzzAveraging effective tensor components(gxx+ gyy)/2, (gxx+ gyy)/2, gzz (Axx+ Ayy)/2, (Axx+ Ayy)/2 AzzX-rotationAn introduction to Electron Spin Resonance (ESR), November 7, 2007Averaging effective tensor compone

47、ntsgxx, (gyy+ gzz)/2, (gyy+ gzz)/2, Axx, (Ayy+ Azz)/2, (Ayy+ Azz)/2Diffusion tilt angleY-rotationAn introduction to Electron Spin Resonance (ESR), November 7, 2007Averaging effective tensor components(gxx+ gzz)/2, gyy, (gxx+ gzz)/2, (Axx+ Azz)/2, Ayy, (Axx+ Azz)/2An introduction to Electron Spin Res

48、onance (ESR), November 7, 2007OOOOOPOOOONOONOONOzRCCCCH2CH2CH2CH2NCH3CH3CH3_+xmzmSide viewUpper viewym16-PCOOOOOPOOONOCCCHCH2CH2CH2CH2NH3CCH3_+xmymzRzdDPPTCONOzdxmymzmCSLZ-orderingX-orderingY-orderingLipid spin labelsAn introduction to Electron Spin Resonance (ESR), November 7, 2007ESR is one of the

49、 most powerful tools in lipid researchPhase state of lipidsInteraction of lipid with proteins, formation of lipoproteids, boundary lipid etc. Domains in model and biological membranesDiffusion studies in the membrane phasePolarity profiles in membranesMembrane permeation profiles for oxygen and para

50、magnetic ionsESR on aligned membranesSimulation of angular dependent spectra is much freer of ambiguity, compared to vesiclesSpin-labeled gramicidin A in DPPC, 220 CAligned membraneVesiclesApplication of aligned membranes allows extracting information on relative orientation of diffusion and magneti

51、c axes, which can not be obtained from vesiclesAn introduction to Electron Spin Resonance (ESR), November 7, 2007An introduction to Electron Spin Resonance (ESR), November 7, 2007Spin labeled Gramicidin A in DPPC at 170 GHzgx=2.0091, gy=2.0061, gz=2.0023I=1, Ax= 6.2, Ay = 6.3, Az=33.6All orientation

52、s of the membrane normal relative to the magnetic field are averaged in vesicles:NOFor a macroscopically disordered sample the orientation of the nitroxidemoiety manifests itself as a result of anisotropic molecular motion around the principal axis of the molecular frameNOOCOCOHOONOnnXZ9 GHz170 GHzM

53、OMD: microscopic order macroscopic disorder. An important case in biologyAn introduction to Electron Spin Resonance (ESR), November 7, 2007Spin labeling. Peptides and proteinsRNOOHNHOO+DCCONOROON ONitroxides are introduced into proteins as reporter groups to provide information about local environme

54、nt, overall tumbling rate of the protein or/and segmental mobility, accessibility of the labeling site for polar/non-polar molecules, distance measurements to other spin labels, co-factors, membrane surface. Labeling of the hydroxyl groupNOCH2SSO2CH3ProteinSH+SCH2NOSProteinMTSL spin label is cystein

55、e specific. SDSL = site directed spin labeling is introducing cysteines into the protein molecule by point mutations with following MTSL labeling.Cysteine mapping of the protein molecule.An introduction to Electron Spin Resonance (ESR), November 7, 2007T4 lysozyme an example of successful EPR mappingMultif