A Q-band pulse EPR/ENDOR spectrometer and the implementation of advanced one- and two-dimensional pulse EPR methodology

A versatile high-power pulse Q-band EPR spectrometer operating at 34.5--35.5 GHz and in a temperature range of 4--300 K is described. The spectrometer allows one to perform one- and two-dimensional multifrequency pulse EPR and pulse ENDOR experiments, as well as continuous wave experiments. It is equipped with two microwave sources and four microwave channels to generate pulse sequences with different amplitudes, phases, and carrier frequencies. A microwave pulse power of up to 100 W is available. Two channels form radiofrequency pulses with adjustable phases for ENDOR experiments. The spectrometer performance is demonstrated by single crystal pulse ENDOR experiments on a copper complex. A HYSCORE experiment demonstrates that the advantages of high-field EPR and correlation spectroscopy can be combined and exploited at Q-band. Furthermore, we illustrate how this combination can be used in cases where the HYSCORE experiment is no longer effective at 35 GHz because of the shallow modulation depth. Even in cases where the echo modulation is virtually absent in the HYSCORE experiment at Q-band, matched microwave pulses allow one to get HYSCORE spectra with a signal-to-noise ratio as good as at X-band. Finally, it is shown that the high microwave power, the short pulses, and the broad resonator bandwidth make the spectrometer well suited to Fourier transform EPR experiments.     

Determination of the 14N quadrupole coupling constant of nitroxide spin probes by W-band ELDOR-detected NMR

Nitroxide spin probe electron paramagnetic resonance (EPR) has proven to be a very successful method to probe local polarity and solvent hydrogen bonding properties at the molecular level. The g(xx) and the (14)N hyperfine A(zz) principal values are the EPR parameters of the nitroxide spin probe that are sensitive to these properties and are therefore monitored experimentally. Recently, the (14)N quadrupole interaction of nitroxides has been shown to be also highly sensitive to polarity and H-bonding (A. Savitsky et al., J. Phys. Chem. B 112 (2008) 9079). High-field electron spin echo envelope modulation (ESEEM) was used successfully to determine the P(xx) and P(yy) principal components of the (14)N quadrupole tensor. The P(zz) value was calculated from the traceless character of the quadrupole tensor. We introduce here high-field (W-band, 95 GHz, 3.5 T) electron-electron double resonance (ELDOR)-detected NMR as a method to obtain the (14)N P(zz) value directly, together with A(zz). This is complemented by W-band hyperfine sublevel correlation (HYSCORE) measurements carried out along the g(xx) direction to determine the principal P(xx) and P(yy) components. Through measurements of TEMPOL dissolved in solvents of different polarities, we show that A(zz) increases, while |P(zz)| decreases with polarity, as predicted by Savitsky et al.     

Sensitivity Enhancement by Matched Microwave Pulses in One- and Two-Dimensional Electron Spin Echo Envelope Modulation Spectroscopy

The concept of microwave pulse matching is applied to three-pulse electron spin echo envelope modulation and sublevel correlation (HYSCORE) spectroscopy. Matched pulses enhance the efficiency of forbidden transfers and may drastically increase the signal intensity of basic frequency and combination frequency transitions in these conventional pulse EPR experiments. The theory of matched pulses is extended to the case of strong and largely isotropic hyperfine interactions, and numerical simulations are presented to gain a deeper insight into the inner working of the matched-pulse approach. It is shown that the enhancement of combination frequencies in matched HYSCORE can be used to determine the relative sign of hyperfine coupling constants as well as the number of equivalent nuclei. The enormous capacity of the approach is demonstrated on ordered and disordered systems. In particular, it is shown that in HYSCORE experiments the signal-to-noise ratio improvement for strongly coupled nitrogens and for proton combination peaks may be considerably larger than one order of magnitude, corresponding to a reduction in measuring time of more than a factor of 100.     

A continuous-wave and pulsed electron spin resonance spectrometer operating at 275 GHz

An electron paramagnetic resonance (EPR) spectrometer is described which allows for continuous-wave and pulsed EPR experiments at 275 GHz (wavelength 1.1 mm). The related magnetic field of 9.9 T for g approximately 2 is supplied by a superconducting solenoid. The microwave bridge employs quasi-optical as well as conventional waveguide components. A cylindrical, single-mode cavity provides a high filling factor and a high sensitivity for EPR detection. Even with the available microwave power of 1 mW incident at the cavity a high microwave magnetic field B1 is obtained of about 0.1 mT which permits pi/2-pulses as short as 100 ns. The performance of the spectrometer is illustrated with the help of spectra taken with several samples.     

Single loop multi-gap resonator for whole body EPR imaging of mice at 1.2 GHz

For whole body EPR imaging of small animals, typically low frequencies of 250-750 MHz have been used due to the microwave losses at higher frequencies and the challenges in designing suitable resonators to accommodate these large lossy samples. However, low microwave frequency limits the obtainable sensitivity. L-band frequencies can provide higher sensitivity, and have been commonly used for localized in vivo EPR spectroscopy. Therefore, it would be highly desirable to develop an L-band microwave resonator suitable for in vivo whole body EPR imaging of small animals such as living mice. A 1.2 GHz 16-gap resonator with inner diameter of 42 mm and 48 mm length was designed and constructed for whole body EPR imaging of small animals. The resonator has good field homogeneity and stability to animal-induced motional noise. Resonator stability was achieved with electrical and mechanical design utilizing a fixed position double coupling loop of novel geometry, thus minimizing the number of moving parts. Using this resonator, high quality EPR images of lossy phantoms and living mice were obtained. This design provides good sensitivity, ease of sample access, excellent stability and uniform B(1) field homogeneity for in vivo whole body EPR imaging of mice at 1.2 GHz.     

Cryogenic Q-band (35GHz) probehead featuring large excitation microwave fields for pulse and continuous wave electron paramagnetic resonance spectroscopy: performance and applications

The construction and performance of a Q-band (35GHz) cryogenic probehead for pulse electron paramagnetic resonance and continuous wave electron paramagnetic resonance measurements with down-scaled loop gap resonators (LGRs) is presented. The advantage of the LGR in comparison to TE(012) resonators lies in the large B(1) microwave (mw) fields that can be generated with moderate input mw power. We demonstrated with several examples that this allows optimal performance for double-quantum electron coherence, HYSCORE, and hyperfine decoupling experiments employing matched and high turning angle mw pulses with high B(1)-fields. It is also demonstrated that with very low excitation power (i.e. 10-40 mW), B(1)-fields in LGRs are still sufficient to allow short mw pulses and thus experiments such as HYSCORE with high-spin systems to be performed with good sensitivity. A sensitivity factor Lambda(rs) of LGRs with different diameters and lengths is introduced in order to compare the sensitivity of different resonant structures. The electromagnetic field distribution, the B(1)-field homogeneity, the E(1)-field strength, and the microwave coupling between wave guide and LGRs are investigated by electromagnetic field calculations. The advantage and application range using LGRs for small sample diameters is discussed.     

A pulsed EPR study of the catalytic oxidation of CO over Cu/SnO2

The role of the Cu(II) in the catalytic oxidation of CO over Cu/SnO₂ with low Cu(II) content was studied by continuous wave EPR, electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectroscopes. Three methods were employed for introducing the copper: (i) by coprecipitation, (ii) impregnation onto SnO₂ gel and (iii) impregnation onto calcined SnO₂. Two types of Cu(II) species were identified in these calcined Cu/SnO₂ materials. Those belonging to the first type, termed B and C, exhibit highly resolved EPR spectra with well defined EPR parameters and are located within the bulk of the oxide. The other group comprises a distribution of surface Cu(II) species with unresolved EPR features and are referred to as S. While the latter were readily reduced by CO the former required long exposures at high temperatures (> 673 K). The specific interactions of the different Cu(II) species with CO were investigated through the determination of the¹³C hyperfine coupling of enriched¹³CO. The ESEEM spectra of calcined samples, generated either by coprecipitation or impregnation, show after the adsorption of CO signals at the Larmor frequencies of^{117, 119}Sn and¹³C and at twice these Larmor frequencies. Although these signals indicate that^{117, 119}Sn and¹³C are in the close vicinity of Cu(II), they cannot provide the hyperfine couplings of these nuclei. This problem was overcome by the application of the HYSCORE experiment. The 2D HYSCORE spectra show well resolved cross peaks which provide the hyperfine interaction of these nuclei. Simulations of the HYSCORE spectra yield for^{117, 119}Sn an isotropic hyperfine constant,a _iso, of ±4.0 MHz and an anisotropic component,T _?, of ±2.0 MHz. Pulsed ENDOR spectra also showed^{117, 119}Sn signals which agree with the above values. The¹³C cross peaks yielda _iso=±1.0 MHz andT _?=±2.0 MHz. Similar C cross peaks were observed in spectra of calcined Cu/SnO₂ after the adsorption of CO₂. Based on the same hyperfine couplings in the samples exposed to¹³CO and¹³CO₂ the signals were assigned to surface carbonate species generated by part of the Cu(II) S type species rather then by species B and the role of the Cu(II) in the oxidation process is discussed.     

Studying high-spin ferric heme proteins by pulsed EPR spectroscopy: Analysis of the ferric form of the E7Q mutant of human neuroglobin

In this work, the high-spin ferric form of the E7Q mutant of human neuroglobin (E7Q-NGB) is studied by X-band continuous-wave electron paramagnetic resonance (CW EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopy. It is shown that the use of matched pulses in the HYSCORE experiment is essential to observe the nitrogen spectral contributions. The validity of approximating the high-spin Fe(III) system (S=5/2) as an effectiveS=1/2 system is tested and the consequences for the HYSCORE simulations are highlighted. Comparative HYSCORE experiments combined with deuterium exchange experiments for aquometmyoglobin and ferric E7Q-NGB clearly show that the heme iron of the latter protein is pentacoordinated, lacking the distal water. Furthermore, CW EPR experiments show that, at high pH, the E10K residue is coordinating to the heme iron in this globin. These observations are corroborated by resonance Raman experiments and could also be reproduced for other E7 mutants of human and mouse neuroglobin. Finally, the proton and nitrogen hyperfine and nuclear quadrupole parameters obtained for ferric E7Q-NGB are discussed in detail.     

Microwave frequency modulation in CW EPR at W-band using a loop-gap resonator

Loop-gap resonator (LGR) technology has been extended to W-band (94GHz). One output of a multiarm Q-band (35GHz) EPR bridge was translated to W-band for sample irradiation by mixing with 59 GHz; similarly, the EPR signal was translated back to Q-band for detection. A cavity resonant in the cylindrical TE011 mode suitable for use with 100 kHz field modulation has also been developed. Results using microwave frequency modulation (FM) at 50 kHz as an alternative to magnetic field modulation are described. FM was accomplished by modulating a varactor coupled to the 59 GHz oscillator. A spin-label study of sensitivity was performed under conditions of overmodulation and gamma2H1(2)T1T2<1. EPR spectra were obtained, both absorption and dispersion, by lock-in detection at the fundamental modulation frequency (50 kHz), and also at the second and third harmonics (100 and 150 kHz). Source noise was deleterious in first harmonic spectra, but was very low in second and third harmonic spectra. First harmonic microwave FM was transferred to microwave modulation at second and third harmonics by the spins, thus satisfying the "transfer of modulation" principle. The loaded Q-value of the LGR with sample was 90 (i.e., a bandwidth between 3 dB points of about 1 GHz), the resonator efficiency parameter was calculated to be 9.3 G at one W incident power, and the frequency deviation was 11.3 MHz p-p, which is equivalent to a field modulation amplitude of 4 G. W-band EPR using an LGR is a favorable configuration for microwave FM experiments.     

Quantitative analysis of dinuclear manganese(II) EPR spectra

A quantitative method for the analysis of EPR spectra from dinuclear Mn(II) complexes is presented. The complex [(Me(3)TACN)(2)Mn(II)(2)(mu-OAc)(3)]BPh(4) (1) (Me(3)TACN=N, N('),N(")-trimethyl-1,4,7-triazacyclononane; OAc=acetate(1-); BPh(4)=tetraphenylborate(1-)) was studied with EPR spectroscopy at X- and Q-band frequencies, for both perpendicular and parallel polarizations of the microwave field, and with variable temperature (2-50K). Complex 1 is an antiferromagnetically coupled dimer which shows signals from all excited spin manifolds, S=1 to 5. The spectra were simulated with diagonalization of the full spin Hamiltonian which includes the Zeeman and zero-field splittings of the individual manganese sites within the dimer, the exchange and dipolar coupling between the two manganese sites of the dimer, and the nuclear hyperfine coupling for each manganese ion. All possible transitions for all spin manifolds were simulated, with the intensities determined from the calculated probability of each transition. In addition, the non-uniform broadening of all resonances was quantitatively predicted using a lineshape model based on D- and r-strain. As the temperature is increased from 2K, an 11-line hyperfine pattern characteristic of dinuclear Mn(II) is first observed from the S=3 manifold. D- and r-strain are the dominate broadening effects that determine where the hyperfine pattern will be resolved. A single unique parameter set was found to simulate all spectra arising for all temperatures, microwave frequencies, and microwave modes. The simulations are quantitative, allowing for the first time the determination of species concentrations directly from EPR spectra. Thus, this work describes the first method for the quantitative characterization of EPR spectra of dinuclear manganese centers in model complexes and proteins. The exchange coupling parameter J for complex 1 was determined (J=-1.5+/-0.3 cm(-1); H(ex)=-2JS(1).S(2)) and found to be in agreement with a previous determination from magnetization. The phenomenon of exchange striction was found to be insignificant for 1.     

A Dynamic Nuclear Polarization spectrometer at 95 GHz/144 MHz with EPR and NMR excitation and detection capabilities

A spectrometer specifically designed for systematic studies of the spin dynamics underlying Dynamic Nuclear Polarization (DNP) in solids at low temperatures is described. The spectrometer functions as a fully operational NMR spectrometer (144 MHz) and pulse EPR spectrometer (95 GHz) with a microwave (MW) power of up to 300 mW at the sample position, generating a MW B(1) field as high as 800 KHz. The combined NMR/EPR probe comprises of an open-structure horn-reflector configuration that functions as a low Q EPR cavity and an RF coil that can accommodate a 30-50 μl sample tube. The performance of the spectrometer is demonstrated through some basic pulsed EPR experiments, such as echo-detected EPR, saturation recovery and nutation measurements, that enable quantification of the actual intensity of MW irradiation at the position of the sample. In addition, DNP enhanced NMR signals of samples containing TEMPO and trityl are followed as a function of the MW frequency. Buildup curves of the nuclear polarization are recorded as a function of the microwave irradiation time period at different temperatures and for different MW powers.     

Impact of High-Dielectric-Loss Materials on the Microwave Field in EPR Experiments

A dielectric material distorts the microwave field inside an EPR resonator, which results in distortion of the EPR signal from spins inside the material. In this paper, the effects of a spherical bulb filled with a dielectric liquid such as water or a water–ethanol mixture were examined. EPR spectra were recorded for small samples inside and outside of the sphere. The studies include CW and ESE experiments at two microwave frequencies, X band (9.2 GHz) and L band (1.03 GHz). The double integral (area) of an EPR signal depends on[formula]at the position of the sample, causing a large difference in EPR signal intensities between samples in regions of different dielectrics. The phase of the EPR signal also is affected by the presence of the dielectric. These results were compared with three methods of calculating electromagnetic fields (quasi-static method, plane-wave-superposition method, and numerical analysis). Good agreement was found between experimental and calculated results.     

Numerical simulation of one- and two-dimensional ESEEM experiments

Numerical simulation has become an indispensable tool for the interpretation of pulse EPR experiments. In this work it is shown how automatic orientation selection, grouping of operator factors, and direct selection and elimination of coherences can be used to improve the efficiency of time-domain simulations of one- and two-dimensional electron spin echo envelope modulation (ESEEM) spectra. The program allows for the computation of magnetic interactions of any symmetry and can be used to simulate spin systems with an arbitrary number of nuclei with any spin quantum number. Experimental restrictions due to finite microwave pulse lengths are addressed and the enhancement of forbidden coherences by microwave pulse matching is illustrated. A comparison of simulated and experimental HYSCORE (hyperfine sublevel correlation) spectra of ordered and disordered systems with varying complexity shows good qualitative agreement.     

A New Q-Band EPR Probe for Quantitative Studies of Even Electron Metalloproteins

Existing Q-band (35 GHz) EPR spectrometers employ cylindrical cavities for more intense microwave magnetic fields B₁, but are so constructed that only one orientation between the external field B and B₁is allowed, namely the B B₁orientation, thus limiting the use of the spectrometer to measurements on Kramers spin systems (odd electron systems). We have designed and built a Q-band microwave probe to detect EPR signals in even electron systems, which operates in the range 2 K ≤ T ≤ 300 K for studies of metalloprotein samples. The cylindrical microwave cavity operates in the TE₀₁₁mode with cylindrical wall coupling to the waveguide, thus allowing all orientations of the external magnetic field B relative to the microwave field B₁. Such orientations allow observation of EPR transitions in non-Kramers ions (even electron) which are either forbidden or significantly weaker for B B₁. Rotation of the external magnetic field also permits easy differentiation between spin systems from even and odd electron oxidation states. The cavity consists of a metallic helix and thin metallic end walls mounted on epoxy supports, which allows efficient penetration of the modulation field. The first quantitative EPR measurements from a metalloprotein (Hemerythrin) at 35 GHz with B₁ B are presented.     

Fourier-transform EPR at high-field/high-frequency (3.4 T/95 GHz) using broadband stochastic microwave excitation

Stochastic excitation with a full-width-half-maximum bandwidth of 250 MHz was used to perform Fourier-transform (FT) high-field/high-frequency electron paramagnetic resonance (EPR) at 3.4T/95 GHz (W-band). Thereby, the required microwave peak power is reduced by a factor of tau(p)/T1 as compared to equivalent pulsed FT EPR in which the spin system with spin-lattice relaxation time T1 is excited by a single microwave pulse of length tau(p). Stochastic EPR is particularly interesting under high-field/high-frequency conditions, because the limited output power of mm microwave sources, amplifiers, and mixers makes pulse FT EPR in that frequency domain impossible, at least for the near future. On the other hand, FT spectroscopy offers several advantages compared to field-swept magnetic resonance methods, as is demonstrated by its success in NMR and X-band EPR. In this paper we describe a novel stochastic W-band microwave bridge including a bimodal induction mode transmission resonator that serves for decoupling the microwave excitation and signal detection. We report first EPR measurements and discuss experimental difficulties as well as achieved sensitivity. Moreover, we discuss future improvements and the possibility for an application of stochastic W-band FT EPR to transient signals such as those of photoexcited radical pairs in photosynthetic reaction centers.     

Low-Field, Time-Resolved Dynamic Nuclear Polarization with Field Cycling and High-Resolution NMR Detection

Dynamic nuclear polarization (DNP) effects in aqueous solutions of stable 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) radicals were studied in a pulsed mode of pumping the electron paramagnetic resonance (EPR) transitions. Our fast field cycling setup allowed us to perform the EPR pumping at low magnetic fields and to detect the enhanced nuclear magnetic resonance signals at 7 T with high spectral resolution. Pumping was performed at two different frequencies, 300 MHz and 1.4 GHz, corresponding to magnetic fields around 10 and 48.6 mT, respectively. For both frequencies, the DNP enhancements were close to the limiting theoretical values of ?110 (¹⁴N TEMPOL) and ?165 (¹⁵N TEMPOL). Our pulsed experiments exploit coherent motion of the electronic spins in the radio-frequency field as seen by an oscillatory component in the dependence of the DNP effect on the radio-frequency pulse duration. The DNP enhancement was studied in detail as a function of the pulse length, duty cycle, delay between the pulses, and applied power. The analysis of the results shows that pulsed DNP experiments provide an opportunity to achieve enhancements of about ?110 with relatively low applied power as compared to the standard continuous-wave DNP experiments. An adequate theoretical approach to the problem under study is suggested.     

Properties of the HYSCORE spin echo signal

In hyperfine sublevel correlation spectroscopy (HYSCORE), the finite duration of the microwave pulses leads to an incomplete inversion of the electron spin magnetization by the third pulse, which results in a significant admixture of stimulated ESEEM to HYSCORE ESEEM. This virtually unavoidable contribution of stimulated ESEEM seriously hampers the analysis of the modulation amplitudes in HYSCORE. In this work, we analyze the properties of the spin echo signals contributing to the composite HYSCORE signal. Based on this analysis, we propose the strategies of HYSCORE data acquisition and processing that allow one to practically eliminate the contribution of the stimulated echo and make the HYSCORE ESEEM analyzable in quantitative terms.     

ENDOR spectroscopy at 275 GHz

A pulsed ENDOR spectrometer operating at a microwave frequency of 275 GHz is described. The results demonstrate that this type of spectroscopy can now be performed routinely at this high microwave frequency. The advantages compared to conventional EPR frequencies are the high spectral resolution, time resolution, and sensitivity.     

Multifrequency (X-band to W-band) CW EPR of the organic free radical in petroleum asphaltene

Petroleum of Arabian and Colombian origin was studied by electron paramagnetic resonance (EPR) spectroscopy at X- (9 GHz), Q- (34 GHz) and W-bands (94 GHz). The experiments were performed at room temperature (about 300 K) and at 77 K (W-band only). The asymmetry in the lines corresponding to free radicals was observed more intensely in the W-band spectra. The values of the line width ΔH in the spectra increased linearly with the microwave frequency utilized in the EPR experiments. A mathematical simulation of the free radical signal for the EPR spectra in three bands with a set of parameters corresponding to a single species was attempted, but this was not exactly coincident with the experimental signals, suggesting that the hyperfine interaction of the unpaired electron with its neighborhood corresponds to more than one species of radical in the molecular structure of the petroleum asphaltene.     

Investigating magnetically aligned phospholipid bilayers with EPR spectroscopy at Q-band (35 GHz): optimization and comparison with X-band (9 GHz)

This paper presents the improvement and advantages of investigating magnetically aligned phospholipid bilayers (bicelles) utilizing electron paramagnetic resonance (EPR) spectroscopy at a microwave frequency of 35 GHz (Q-band) and at a high magnetic field strength of 1.25 T when compared to weaker magnetic fields for X-band EPR studies. The nitroxide spin label 3beta-doxyl-5alpha-cholestane (cholestane or CLS) was inserted into the bicelles and utilized to demonstrate the effects of macroscopic bilayer alignment through the measurement of orientational dependent hyperfine splittings. The effects of different lanthanide ions with varying degree of magnetic susceptibility anisotropy were examined. The requirement of minimal amounts of the Tm3+ and Dy3+ lanthanide ions for well-aligned bicelles were examined for Q-band and compared with amounts required for X-band bicelle alignment studies. At a magnetic field of 1.25 T (when compared to 0.63 T at X-band), the perpendicular and parallel orientation were aligned with lower concentrations of Dy3+ and Tm3+, respectively, and thereby eliminating/minimizing the unwanted effects associated with lanthanide-protein interactions. Thus, it is much easier to magnetically align phospholipid bilayers at Q-band when compared to X-band.