Vij D.R. Handbook of Applied Solid State Spectroscopy

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with the magnetic field perpendicular to the complex plane occur at low field.

By setting the magnetic field in the low field region of the EPR spectrum

(indicated by “a” in Figure 4.9a), the perpendicular orientation is selected.

The ENDOR spectrum taken at this field value (Figure 4.9b, line a) has a

crystal-like character. It consists of two

N hyperfine lines, each split by

quadrupolar coupling. Thus the nitrogen Ã and Q coupling tensor compo-

nents with respect to the symmetry axis of g and the Cu hyperfine tensors

were determined.

Figure 4.9 Cu(pic)

in Zn(pic)

4 H

O powder. (A) EPR spectrum. The magnetic field settings

chosen for the ENDOR spectrum are indicated by arrows. (B)

N ENDOR spectra referring to

the field settings indicated in the EPR spectrum. For the magnetic field set to a the ENDOR

spectrum has a crystal like character. c and d as well as e and f differ only by a small shift in

the field. From [7].

However, all the components of the hyperfine tensor cannot be determined

form the crystal-like spectra. Additionally, when the g and the hyperfine

tensors are not coaxial, the hyperfine component thus measured, in general,

does not correspond to a principal value.

Several theoretical approaches for analyzing ENDOR spectra taken at

arbitrary field values and corresponding to multiple orientations, have been

reported [8, 9]. Consequently, ENDOR data can be used to characterize quite

general cases of hyperfine coupling. The elements of the hyperfine tensor Ã

4. ENDOR Spectroscopy

176

and its relative orientation to the g tensor frame can be deduced from the

variation of the hyperfine coupling value at different field settings across the

EPR spectrum. These techniques are called angle or orientation selection.

In the angle selection method, ENDOR spectra are recorded at each point

in the EPR powder pattern including the turning points. From crystal-like

spectra approximate principal values are obtained. Simulation of selected

spectra is then carried out by varying the respective parameters. For a series

of metalloenzymes, ENDOR lines obtained with the angle selection method of

many nuclei including

14,15

Fe,

63,65

Cu, and

95,97

have given valuable information about the sites of the metal center and a

bound ligand or substrate [10].

The angle selection method has been applied to many different systems:

transition metal complexes [11] in which the g tensor anisotropy dominates;

nitroxide radical [12] with strongly anisotropic nitrogen hyperfine couplings;

and organic biradicals [13] in which the magnetic anisotropy results from the

electron-electron coupling (D) tensor.

Besides the requirement of considerable anisotropy of the magnetic

interaction in the system, another important condition must be fulfilled for

one to observe crystal-like spectra and apply the angle selection method:

Saturation of particular EPR transitions should be possible. This condition is

satisfied when the process causing transfer of saturation from one orientation

to another is slow compared to the spin-lattice relaxation rate. Such a process

is called spin diffusion.

Spin diffusion is slow relative to the spin-lattice relaxation in transition

metal complexes and biomolecules containing metal ions. In contrast, in the

case of radicals in organic molecular crystals (at low temperature) the spin-

lattice relaxation is slower than the spin-diffusion processes. Consequently,

the saturation is transferred to all other EPR transitions in a time short with

respect to T

, the electronic spin-lattice relaxation time. As a result, powder-

type ENDOR spectra are observed [14]. At higher temperatures, however, the

spin-diffusion may be slower, making selective saturation possible [12].

Regardless of the type of ENDOR spectra observed in disordered solids,

they are much easier to interpret than EPR powder patterns.

4.4 PULSED-ENDOR

The conventional CW-ENDOR method has a serious disadvantage: The

intensity of the ENDOR signals depends critically on the balance between the

relaxation and induced transition rates (saturation conditions for both EPR

and NMR transitions have to be satisfied). The experimental parameters [4],

in general, cannot be independently optimized. Thus, the ENDOR transitions

are often not observed because of unfavorable relaxation rates. Moreover,

even when all experimental variables are optimized, the ENDOR signal

177

4.4 Pulsed-ENDOR

intensity is only a few percent of the EPR signal. The pulsed ENDOR

techniques offer greater sensitivity, higher spectral resolution, as well as

reduced time of the measurement. The most important advantage of pulsed-

ENDOR (or sometimes called ESE-ENDOR or time-resolved ENDOR) over

CW is that there is no restriction to balance relaxation and induced transition

rates. Consequently, the temperature is not such a critical parameter in pulsed

ENDOR spectroscopy. Additionally, the pulsed technique permits a study of

transient radicals.

In a CW experiment both microwave (mw) and radio frequency fields are

applied continuously to the sample during the experiment. In a pulsed

ENDOR experiment the pulse sequence, composed of short, intense mw (of

the order of hundreds of nanoseconds) and rf (microsecond) pulses, is applied.

A pulse of an oscillating field is characterized by its frequency Z as well as its

time of duration t. Both of these parameters can be adjusted to invert the

population of the respective levels. The general scheme (Figure 4.10) for a

population transfer pulsed-ENDOR experiment (Davies sequence) is as

follows:

Figure 4.10 Schematic representation of the population transfer pulsed-ENDOR (Davies)

method.

(1) First, a microwave S pulse is applied to invert the population of the

selected EPR transition.

(2) After a time, short compared to the electron spin-lattice relaxation time,

the RF S pulse is applied. Depending on the excitation frequency of

this pulse (NMR frequency), the pulse will or will not change the

population of the nuclear transitions.

(3) The effect of the RF pulse on the EPR transition is then detected by an

electron spin-echo sequence.

While varying the NMR frequency, the whole sequence is repeated (at a

rate slower than the spin-lattice relaxation rate). A plot of the amplitude of

the spin-echo vs. the NMR frequency constitutes the ENDOR spectrum.

There is another commonly used pulsed sequence referred to as Mims

ENDOR. In this technique the S pulse at the RF frequency is applied between

the second S/2 and third S/2 mw pulse (two S/2 pulses to invert EPR

4. ENDOR Spectroscopy

178

population) in contrast to the Davies ENDOR, where the RF pulse is applied

between the first S and first S/2 microwave pulses: The Mims ENDOR

experiment is particularly effective for weakly coupled nuclei but some blind

spots do occur where frequencies cannot be observed [15]. It is often

advantageous to combine data from both the Mims and the Davies ENDOR

response [16]. For nuclei with large magnetic moments such as

H and

F, Q

at the X-band is often larger (14 MHz) than the hyperfine field and so the

ENDOR response is centered at the Larmor frequency Q

of the nucleus and is

split by the hyperfine coupling A

and by quadrupole interactions. For nuclei

with smaller magnetic moments like

H or

N, the ENDOR response is

centered at half the hyperfine coupling frequency and split into two lines that

are separated by twice the Larmour frequency. Often times, Q

= 14 MHz at

the X-band is comparable to the magnitude of many nuclear hyperfine

couplings, which results in substantial overlap of the proton ENDOR signals

with signals from other nuclei. This problem is solved by recording ENDOR

spectra at higher (fields such as 35 (Q-band) [17] or 95 (W-band) [18] GHz

microwave frequencies where Q

increases proportionally to the external field

but A

is independent of field, and results in a separation of overlapping

ENDOR lines.

Several pulsed displays have been developed. As an example, Figure 4.11

shows (a) the overlapping

H and

N resonances in the Davies-type ENDOR

spectrum and (b) the selected

N resonances obtained by the hyperfine-

ENDOR technique are given in [19–21]. Before attempting these books, an

easier-to-understand review of the basic principles in pulsed-ENDOR and the

related electron spin-echo envelope modulation (ESEEM) spectroscopy has

been given [16]. An earlier review of pulsed-ENDOR techniques is given in

[15].

A review of ESE-ENDOR techniques, tailored to the study of magnetic

nuclei coupled to paramagnetic metals and radicals, has been published with

illustrative examples from metal and radical centers in photosystem II [22].

The pulsed-Davies ENDOR experiment for S = ½ and I = ½ was developed

for W band (95 GHz) ENDOR effects observed at low temperature [23].

Two-dimensional TRIPLE experiments have been carried out on

orientationally disordered disordered samples and shown it can resolve

overlapping powder patterns. This makes it possible to resolve and determine

the relative orientation of the hyperfine tensors and the relative signs of the

hyperfine couplings [24].

4.4 Pulsed-ENDOR

selective ENDOR techniques [19]. More complete details of the pulsed-

179

Figure 4.11 Cu(pic)

/Zn(pic)

single crystal. (a) Conventional (Davies-types) pulsed-ENDOR

spectrum with

H and

N lines using the pulse sequence given above. (b) Hyperfine-selective

Commercial ENDOR spectrometers at X-, Q- and W-bands are available

from Bruker BioSpin Corp. in North America (epr@bruker.com

) and Bruker

Biospin GmbH in Europe (erp@bruker-biospin.de)

. The CW-ENDOR X-band

spectrometer is known as an ELEXSYS E-560-D, which uses a TM cavity

with vertical access. The pulsed-EPR at X-band is known as E-560-D-P using

a TE cavity (cylindrical), vertical access. The pulsed-ENDOR system at Q-

band (34 GHz) is the ELEXSYS E-580-Q and the pulsed-ENDOR system at

W-band is known as the ELEXSYS E-680, while the CW-ENDOR at W-band

is the E-600.

4.5 APPLICATIONS

ENDOR measurements can be used extensively to gain resolution in a

complex spectrum, to separate overlapping EPR spectra, and to identify the

4. ENDOR Spectroscopy

N ENDOR spectrum. Figure 4.9 shows powder spectra. From [19].

180

nuclei responsible for the coupling. This technique has been applied to

various systems. In the following section, examples are given for studying (a)

organic radicals in organic host crystals, (b) radicals trapped in xenon, argon,

or Freon matrices, (c) triplet state radicals in crystals or polycrystalline

samples, (d) free radicals in biological systems, (e) polymeric systems, (f)

inorganic radicals in irradiated inorganic single crystals, (g) paramagnetic

complexes in organic single crystals, (h) F and H centers found in inorganic

host lattices, (i) paramagnetic inorganic ions in organic crystals, (j) transition

metal ion complexes observed in frozen solution and powders, (k) defects and

complexes on surfaces such as silica alumina, zeolite, and nafion, (l) impurity

centers in semiconductor host crystals, (m) spin centers in silicon and borate

systems, (n) paramagnetic centers in cubic host crystal and (o) perovskite-type

materials. Selected examples are given in each area with references noted.

A detailed tabulation of the published ENDOR data for single crystals,

polycrystalline solids and biological materials for 58 isotropic species through

1993 has been given in the Handbook of Electron Spin Resonance [25].

Earlier ENDOR studies in disordered matrices, crystalline systems, hemes and

hemoproteins, iron-sulfur proteins, radiation biophysics, polymer studies, and

triplet state systems in solids were reviewed in Dorio and Freed (eds.) [26].

4.5.1 Organic Radicals in Organic Host Crystals

Often the crystals are irradiated at a low temperature and then the radicals

identified as the temperature is raised. Electron attachment and proton

abstraction occur at different temperatures so intermediate radicals can be

formed. An example of the principal values of the hyperfine couplings for the

D- and E-protons and the

N couplings for the radical formed [27] upon X-

irradiation of deoxycytidine-5'-phosphate-hydrate at 11 K and measured by

X-band ENDOR techniques at 6 K is given in Table 4.3. Two Mrad/hour of

irradiation was applied for 2–4 hours. Because of the increased resolution, the

couplings can be determined more precisely and it is possible to detect the

presence of several radicals. Radicals I and II

and II

were identified at 6 K.

Typically the primary radical is formed at the lowest temperature as a result of

reaction with the electrons formed from the X-irradiation. However, as the

temperature is raised, the unstable primary radical decays—forming other

products which may or may not be radicals. An example [28] where different

radicals were identified as a function of temperature occurred upon 4.2 K

X-irradiation of a single crystal of citric acid (2-hydroxy-1,2,3-propane

tricarboxylic acid), partially deuterated and warmed to 100 K. ENDOR

analysis achieved upon cooling the radicals formed at 100 K to 4.2 K, thus

quenching the reaction, are given in Table 4.4.

Radical I is formed by the loss of a carboxylic acid group from the 2-

position and radical II is due to electron attachment to the carboxylic acid

group located at the 3-position. Upon warming to 300 K, radical II decays and

181

4.5 Applications

radical III is detected where loss of the Į-proton from the 3-position is

detected. Two nonequivalent crystal sites are detected for radical III and the

Į-couplings for all sites have been determined. It was also found [28] that

radical I was stable at 300 K; demonstrating that upon decay of radical I,

radical III is an end-product of the decay of radical II. Secondary radical

products stable at room temperature have been studied to examine whether the

free radical position in the crystal is the same as that of the undamaged

molecules. The ENDOR resolution permits small couplings (on the order of

1 MHz due to distant protons) to be precisely measured. From the resulting

proton tensor, dipolar distances can be estimated and thus proton-couplings

from neighboring molecules and hydrogen-bonded protons can be assigned

based on crystal structure data.

Table 4.3 ENDOR hyperfine coupling parameters at 6 K of 11 K X-irradiated deoxycytidine

5'-phosphate-H

O (5' dCMP) single crystal) [27].

Principal Values (mT)

Radical I Radicals IIa and IIb

–2.243 ± 0.008 –2.260 ± 0.005

–1.592 ± 0.005 H

–0.732 ± 0.010

–0.651 ± 0.005 –1.184 ± 0.008

iso

–1.495 a

iso

– 1.392

1.381 ± 0.004 –2.275 ± 0.003

1.439 ± 0.004 H

–0.587 ± 0.003

1.623 ± 0.004 –1.190 ± 0.010

iso

= 1.481 a

iso

–1.350

1.520

+ N

0.615

0.011

iso

= 0.715

Radical I

Radical IIa Radical IIb

4. ENDOR Spectroscopy

182

Table 4.4 Citric acid single crystal, partially deuterated. X-irradiated at 4.2 K [28]. 4.2 K

ENDOR measured E-proton couplings for radicals I and II after crystal warmed to (100 K).

Radical I Radical II Radical I Radical III

4.2 K 4.2 K Stable Conformation Stable

Room

Room Temperature Temperature

Hyperfine Hyperfine Hyperfine

Tensors (MHz) Tensors (MHz) Tensors (MHz)

113.9 90.7 105.2

106.7 E

80.5 E

96.1 D' –94.2

102.3 79.4 92.0 (site 1) –58.6

107.5 23.1 82.0 –31.9

98.9 E

16.7 E

70.2

97.5 1.2 68.0

40.0 77.6 –92.3

26.0 70.3 D" –58.3

24.2 E

64.7 (site 2)

31.0

In Table 4.5 is given an example [29] where potassium hydrogen malonate

single crystals were Ȗ-irradiated at room temperature and

H and

––

Radical III

C–C–C–C–C

ODO

Radical II

27.1

12.7

11.3

C–C–C–C–C

–

ODO

Radical I

C–C–C–C–C

couplings were measured for the OOC HCCOO radical in the host lattice.

–

183

4.5 Applications

Table 4.5 J-irradiated potassium hydrogen malonate (KH) single crystal [29].

H and

hyperfine tensors (MHz) from ENDOR measurements at room temperature.

D-

_____________________________________________________________________

–27.4 23.2

–

OOCCHCOO

–

–86.8 33.0 radical

_____________________________________________________________________

Hyperfine tensor of the distant protons.

Principal Values (MHz)

_____________________________________________________________________

Distance

Proton

Isotropic Dipolar R

C...H

(Å)

_____________________________________________________________________

A 4.32 3.32

B 0.04 4.28 –1.83 3.33

C 0.07 1.95 4.33

D 0.10 1.79 0.66 4.45

E 1.83 0.61 4.42

_____________________________________________________________________

The signs of the couplings are not obtained from the experiment.

the crystal structure data.

In this study, it was found that upon loss of an Į-hydrogen, the resulting

free radical retained the same position as the parent molecule. Often this is

not the case, and considerable movement upon radical formation occurs, all

dependent on the intermolecular packing arrangements of the original crystal.

ENDOR measurements of nuclei other than

H and

C can also be carried

out. Studies of deuterium [30], nitrogen [27]

Na [31], and chlorine-35 [32]

hyperfine coupling constants, as well as their?coupling constants have been

reported. From such data, structural relationships can be deduced. For

example the relationship between deuterium quadrupole constants and the N-

D and D…O bond lengths in a perdeuterates sulphamic acid (ND

) single

crystal were reported [30]. Example of the couplings for D and

Cl deduced

are given in Table 4.6.

ENDOR measurements at 1.6 K have been used to deduce the exchange-

able dissociation proton coupling, the D-proton, the exchangeable O-H proton

in the undamaged structure and in the alkoxy radical, as well as the Jand E

proton, and G-methyl protons in a crystal of rhamnose X-irradiated at

4. ENDOR Spectroscopy

–1.14 –2.36 –1.96

Protons of neighboring molecules and hydrogen-bonded protons; protons assignment refers to

–2.32

–0.89 –1.07

–1.21

–56.3 207.8

–1.14

–0.04

184

guanosine 5'-monophosphate single crystals have been reported [34]. 4.2 K

[33]. Examples of radicals formed at 10 K and 65 K in X-irradiated

Table 4.6 Perdeuterated sulphamic acid (ND

) single crystal.

H ENDOR at 4.2 K [30].

X-irradiated phenancyl chloride:

H and

Cl hyperfine coupling constants and

Cl quadrupole

coupling constant (MHz), ENDOR at 123 K [32].

Deuteron Quadrupole Tensors Phenancyl chloride Single Crystal

Deuteron Eigenvalues of Q

z 257.4 (–)26.5

y –169.6 A(

H) (–)52.5 a

iso

= –5.30

x –87.8 (–)80.0

qQ/h = 171.6

K = 0.370

z 273.0 (–)08.3

y –163.9 A(

Cl) (–)15.8 a = 7.3

x –109.1 45.6

qQ/h = –182.0

K = 0.201

z 266.0 5.3 O

y –182.2 Q(

Cl) 5.9 »»

x –83.8 –11.2 CCHCl

qQ/h = 177.3 radical

K = 0.318

_________________________________________________________

Errors in tensor components are ± 2 kHz.

Radicals formed in J-irradiated organic single crystals often exhibit

internal motions, methyl groups rotate rapidly and t-butyl group rotate slowly

on an ENDOR time scale. This motion is reflected in the temperature

dependence of the electron spin relaxation times as detected by pulsed-EPR

measurements [35]. Typical examples are given by the study of irradiated

4-methyl-2,6-di-t-butylphenol [35] and irradiated 10-nonadecanone in urea

[36] where wobbling of the methylene groups and rotational motion of the

radical in the channel were detected. ENDOR studies can be useful for

determining the radiation chemistry of co-crystallized DNA base pairs [37,

38]. Varying temperature can be useful to follow the interconnecting of the

planar and puckered conformers of the adduct pyrroline ring [39] by ENDOR

measurements.

(kHz)

iso

185

4.5 Applications