Tarragona 2007

Triple frequency technique for resolving congested ENDOR and ELDOR-detected NMR spectra. Alexey Potapov and Daniella Goldfarb. Department of Chemical Physics, Weizmann Institute of Science, Rehovot, Israel.

Introduction

1. ELDOR-ENDOR scheme

NMR1

ELDOR-detected (electron-electron double resonance) and ENDOR (electron-nuclear double resonance) are pulse EPR techniques that allow to measure nuclear frequencies from which electron-nuclear interactions can be determined. However, in many cases the spectra obtained from these techniques are congested and not easy to interpret. Moreover, they do not provide the signs of the hyperfine interactions. These are usually important because they are closely related to the electronic structure. The TRIPLE experiment is a variant of ENDOR which provides the relative signs of the hyperfine couplings and can be used to assign signals to their respective paramagnetic centers.

ENDOR

FID

Inverted echo

|α1β2 |α1α2>>

ν0

allowed

|ββN>

|ββ1β2>

|ββN>

νrf

forbidden |β1β2> |β1α2> |α1β2> |α1α2>

Long microwave pulse transfers polarization via a forbidden transition.

|ββ1α2> |βα1β2>

νrf1

|α1β2 |α1α2>>

|β1β2> |β1α2> |α1β2> |α1α2>

RF

RF

|βα1α2>

ν1

ν0

ν0

|αβN> |ααN>

ν0

|αβN>

|αβ1β2>

Combines ELDORdetected NMR and ENDOR.

|αβ1α2> |αα1β2>

|ααN>

νrf2

|αα1α2>

Schosseler P, Wacker T, Scheweiger A, Chem. Phys. Lett. 244 (319-324)

|β1β2> |β1α2> |α1β2> |α1α2> allowed nuclear transitions RF

MW

ν1

ν1

|β1β2> |β1α2>

FID

ν0

ν0

ν0

Proposed scheme: ELDOR-ENDOR

TRIPLE ENDOR

Inverted echo

|βαN>

Here we present a new correlation experiment which combines the ELDOR-detected NMR and ENDOR methods. It provides the same information as the TRIPLE-experiment but unlike TRIPLE is not a difference technique. In addition it allows to correlate NMR frequencies that are not easily observed in ENDOR. 1

ELDOR detected NMR

|β1β2> |β1α2>

The resulting spectrum shows lines belonging to the electron manifold opposite the excited NMR transition. This is in contrast to TRIPLE where lines belonging to the same manifold as the excited one appear

|β1β2> |β1α2> |α1β2> |α1α2> allowed nuclear RF pulse transfers the polarization within transitions

the nuclear subsystem. The resulting polarization is detected on one of the allowed transitions by application a long selective pulse (marked in red on the diagram).

A1

A2

Schematic ENDOR spectrum

νΙ2

νΙ1

Schematic ENDOR-ELDOR spectrum

This line is excited with ν1

νΙ2

νI1

2. ELDOR-ENDOR on Cu(II) in a single crystal A single crystal of Cu(II)-doped L-histidine was used. Experiments were performed at W-band (95 GHz) where the nuclear resolution is high.

2.1 Echo-detected EPR

2.2 Energy diagram for Cu(II)

A=154 MHz

3/2

The field was set to the second line of Cu(II) (I=3/2) quartet. Hence in ENDOR and ELDOR we expect to observe the nuclear lines arising from mI -3/2<->-½ and ½<->-½ transition. The latter are less affected by the nuclear quadrupole interaction. The latter are expected at 50 and 120 MHz

29000 29200 29400 29600 29800 30000 30200 30400

Magneticfield, G

Regular ENDOR

3/2

+

1/2 -1/2 -3/2

+

1/2 -1/2 -3/2

-

ENDOR TRIPLE at 49.6 MHz Ms=α

+

The echo-detected EPR spectrum shows the presence of several sites ( and crystals) in the sample. The observed copper hyperfine splitting is about 154 MHz.

-3/2 -1/2 1/2 3/2

-3/2

-

+

-1/2 1/2 3/2

Energy level diagram for Cu(II), showing that ELDOR-detected NMR of single crystal is not symmetric about ∆ν=ν1-ν0. Exciting the MI=3/2 component leads to two lines on the positive side of ELDOR-detected NMR spectrum (∆ν >0). Excitation of MI=1/2 component yields four lines in the spectrum, two of which are on the positive side the other two on the negative side (∆ν <0)

ENDOR TRIPLE at 120.5 MHz Ms=β

2.4 Assigning the protons.

2.3 ENDOR , ELDOR. + side - side ENDOR

Variable Mixing Time (VMT) ENDOR and TRIPLE ENDOR allow to determine the absolute signs of the couplings and assign them to their respective paramagnetic species.

VMT mixing time 1 ms

excitation

ELDOR-ENDOR at 49.6 MHz

Ms=α

ELDOR-ENDOR at 120.5 MHz triple @ 122.25 MHz

Ms=α triple @ 121.79 MHz

118 120 122 124 126 128 130 132 134 136 138

RF, MHz

triple @ 122.21 MHz 20

30

40

50

60

70

80

90

100 110 120 130 140 150

regular ENDOR

Frequency, MHz

118

120

122

124

126

128

130

132

134

136

138

RF, MHz

ELDOR-detected NMR (positive and negative sides) compared with the ENDOR spectrum. Lines chosen for excitation in the ELDOR-ENDOR experiments are tentatively assigned to α and β lines of the 63Cu at the transition MI=1/2<->-1/2. The conditions for ELDOR-detected NMR: excitation pulse =3 us detection=2 us.

118 120 122 124 126 128 130 132 134 136 138

RF, MHz

The 1H-ENDOR . Line attributed to different paramagnetic sites are marked with triangles and squares. α-manifold of paramagnetic site #1 β-manifold of paramagnetic site #1 α-manifold of paramagnetic site #2

ELDOR-ENDOR spectra obtained by selecting 63Cu ELDORdetected NMR lines and sweeping the RF frequency in the 1Hregion. The experiment allows to assign the proton and the Cu lines belonging to the complementary manifold as predicted. The lines in TRIPLE spectra with excitation at the same frequency belong to same manifold. This yields the positive sign for the Cu coupling. The ELDOR-ENDOR shows a better selection.

β-manifold of paramagnetic site #2

3. ELDOR-ENDOR on Cu(II) in a frozen solution. Echo-detected field sweep EPR spectrum.

The experiment has been tested on a frozen solution of Cu(II)-histidine in D2O/glycerol-d8

Observer field g⊥

1,6 1,4 1,2

Temperature : 8.5 K

Temperature : 12K

BLUE – regular ENDOR spectrum. BLACK - ELDOR-ENDOR experiment with ELDOR-pulse fixed at the line in ELDOR-detected NMR spectrum. RED – ELDOR-ENDOR experiment with no ELDOR-pulse.

a.u.

1,0 0,8 0,6 0,4 0,2 0,0 30000

30500

31000

31500

32000

32500

33000

33500

Magnetic field, G

ELDOR-detected NMR spectrum 14N

The excited nitrogen line

lines 2H

The experiment as been performed with ELDOR-frequency set on the high frequency nitrogen lines in the ELDORdetected NMR spectrum. Measurements carried out at 8.5 K show only partial selection and signals were observed also without the first excitation MW pulse. We attribute this to non-complete relaxation of the nuclei which occurs if the nuclear relaxation time is longer than repetition rate of the experiment. Increasing the temperature to 12 K suppressed the effect. 128 130 132 134 136 138 140 142 144 146 148 150 152

63Cu, 65Cu

-250

-200

-150

-100

-50

0

50

Frequency, MHz

100

150

200

250

-120 -100

-80

lines

-60

-40

-20

0

20

frequency, MHz

40

60

80

100

120

The experiment clearly allows to resolve the relative hyperfine of the line in ELDOR-detected NMR spectrum and also allows to determine the relative signs of the protons in Cu-L-histidine complex. Since the hyperfine coupling of α-proton is positive (largest proton isotropic coupling) then apparent line on the left is αline, hence the excited nitrogen line is β.

RF, MHz

128 130 132 134 136 138 140 142 144 146 148 150 152

RF, MHz

Acknowledgements. We thank Yaakov Lipkin and Yehoshua Gorodetzky for their efforts in the design and building of the W-band bridge and Boris Epel for his constant support of Specman. This work was supported by the Binational USA-Israel Science Foundation (BSF) ( Grant number 2002175).