Saturation recovery EPR and ELDOR at W-band for spin labels

Abstract

A reference arm W-band (94 GHz) microwave bridge with two sample-irradiation arms for saturation recovery (SR) EPR and ELDOR experiments is described. Frequencies in each arm are derived from 2 GHz synthesizers that have a common time-base and are translated to 94 GHz in steps of 33 and 59 GHz. Intended applications are to nitroxide radical spin labels and spin probes in the liquid phase. An enabling technology is the use of a W-band loop-gap resonator (LGR) [J.W. Sidabras, R.R. Mett, W. Froncisz, T.G. Camenisch, J.R. Anderson, J.S. Hyde, Multipurpose EPR loop-gap resonator and cylindrical TE(011) cavity for aqueous samples at 94 GHz, Rev. Sci. Instrum. 78 (2007) 034701]. The high efficiency parameter (8.2 GW(-1/2) with sample) permits the saturating pump pulse level to be just 5 mW or less. Applications of SR EPR and ELDOR to the hydrophilic spin labels 3-carbamoyl-2,2,5,5-tetra-methyl-3-pyrroline-1-yloxyl (CTPO) and 2,2,6,6,-tetramethyl-4-piperidone-1-oxyl (TEMPONE) are described in detail. In the SR ELDOR experiment, nitrogen nuclear relaxation as well as Heisenberg exchange transfer saturation from pumped to observed hyperfine transitions. SR ELDOR was found to be an essential method for measurements of saturation transfer rates for small molecules such as TEMPONE. Free induction decay (FID) signals for small nitroxides at W-band are also reported. Results are compared with multifrequency measurements of T(1e) previously reported for these molecules in the range of 2-35 GHz [J.S. Hyde, J.-J. Yin, W.K. Subczynski, T.G. Camenisch, J.J. Ratke, W. Froncisz, Spin label EPR T(1) values using saturation recovery from 2 to 35 GHz. J. Phys. Chem. B 108 (2004) 9524-9529]. The values of T(1e) decrease at 94 GHz relative to values at 35 GHz.

Figure 1

Spin labels used in this study: (I) is known as CTPO and (III) as TEMPONE. Spin label (II) is discussed in the text.

Figure 2

Energy level diagrams (a) ¹⁵N and (b) ¹⁴N spin labels.

Figure 3

Block diagram of the W-band SR EPR bridge configuration. Numerous isolators and interconnection components are not shown for clarity.

Figure 4

Timing diagram.

Figure 5

W-band EPR spectrum of ¹⁵N CTPO.

Figure 6

Free induction decays from 0.1 mM ¹⁵N CTPO under nitrogen at 93.80 GHz, data sets are 2048 points, pulse width 0.3 µs, power level ~3 mW: (a) approximately on resonance 2.1 × 10⁶ averages on and off resonance; (b) 10 G offset 3.5 × 10⁶ averages on and off resonance.

Figure 7

Representative (a) SR EPR and (b) SR ELDOR signals for 0.5 mM ¹⁵N CTPO. For SR 1024 points per record, 6.5 × 10⁵ averages on resonance minus 6.5 × 10⁵ averages off resonance, total acquisition time 26 s, pulse width 1 µs, pump power 3 mW, observe power is 0.015 mW, high field line observed, 94.02 GHz. For SR ELDOR parameters were the same except 8192 per record, 2.2 × 10⁶ averages both on and off resonance, total acquisition time 91 s, pump width 0.3 µs on low field line. Separation of pump and observe 62.17 MHz. Best fit is overlaid and residuals are shown. See Table 1 and Table 2 for relaxation times and rates.

Figure 8

Microwave frequency dependence of the electron spin-lattice relaxation rate ${\mathrm{T}}_{1\mathrm{e}}^{-1}$ of ¹⁴N CTPO (circles) and TEMPONE (triangles) in water. The W-band relaxation rates and standard deviations are T_1e⁻¹ (CTPO) = 0.80 ± 0.03 (µs)⁻¹ and T_1e⁻¹ (TEMPONE) = 1.18 ± 0.03 (µs) ⁻¹.