Pulse EPR of Triarylmethyl Probes: A New Approach for the

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Pulse EPR of Triarylmethyl Probes: New Approach for Investigation of Molecular Motions in Soft Matter Andrey A. Kuzhelev, Olesya A. Krumkacheva, Mikhail Yu. Ivanov, Sergey A. Prikhod'ko, Nicolay Yu Adonin, Victor M. Tormyshev, Michael K Bowman, Matvey V. Fedin, and Elena G. Bagryanskaya J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b07714 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 23, 2018

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The Journal of Physical Chemistry

Pulse EPR of Triarylmethyl Probes: New Approach for Investigation of Molecular Motions in Soft Matter Andrey A. Kuzhelev,1,2,3 Olesya A. Krumkacheva,2,3,* Mikhail Yu. Ivanov,2,3 Sergey A. Prikhod’ko,4 Nicolay Yu. Adonin,4 Victor M. Tormyshev1,3, Michael K. Bowman,1,5 Matvey V. Fedin,2,3,* Elena G. Bagryanskaya1,3,*

1

N.N. Vorozhtsov Institute of Organic Chemistry SB RAS, Novosibirsk 630090, Russia

2

International Tomography Center SB RAS, Novosibirsk 630090, Russia

3

Novosibirsk State University, Novosibirsk 630090, Russia

4

Boreskov Institute of Catalysis SB RAS, Novosibirsk, 630090, Russia

5

University of Alabama, Tuscaloosa, Alabama 35487-0336, USA

ABSTRACT Triarylmethyl (TAM) radicals have become widely-used free radicals in the past few years. Their electron spins have long relaxation times and narrow electron paramagnetic resonance (EPR) lines, which make them an important class of probes and tags in biological applications and materials science. In this work we propose a new approach to characterize librations by means of TAM radicals. The temperature dependence of motional parameter τc, where is the mean-squared amplitude of librations and τc is their characteristic time, is obtained by comparison of the 1/Tm phase-relaxation rates at X- and Q-band EPR frequencies. We study three soft matrices, viz. glassy trehalose and two ionic liquids, using TAMs with optimized relaxation properties OX063D and a dodeca-n-butyl homologue of Finland trityl (DBT). The motional parameters τc obtained using TAMs are in excellent agreement with those obtained by means of nitroxide radicals. At the same time, the new TAM-based approach has: (1) greater sensitivity due to the narrower EPR spectrum; (2) greater measuring accuracy and broader temperature range due to longer relaxation times. The developed approach may be fruitfully implemented to probe low-temperature molecular motions of TAM-labeled biopolymers, membrane systems, polymers, molecules in glassy media and ionic liquids.

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INTRODUCTION Molecular motion in glassy organic solvents and biological media, such as saccharides and membranes, are very important for understanding their properties and functions.1,2 Modern physical methods allow study of the dynamics of molecules and diffusion in porous media over a range of frequency and temperature.3,4 Low-temperature dynamics of molecules in soft matter can be studied by neutron scattering,5,6 nuclear magnetic resonance (NMR)7,8 and dielectric relaxation measurements.9,10 Paramagnetic probes and pulsed EPR can investigate matrix properties that control the dynamics of solutes.11–13 EPR spectroscopy is a simple and quick approach that typically uses nitroxide radicals, whose g-factors and hyperfine interactions (HFIs) are anisotropic and make their EPR spectra sensitive to the motion. The anisotropy also broadens the EPR spectrum, so that selective pulses applied at different spectral position make it possible to investigate the anisotropy of the electron spin dynamics and extract information about the dynamics of the surrounding matrix. The motion of nitroxides was extensively studied by EPR and revealed important information about the molecular properties of glasses, about the structural organization of biological membranes,14–16 ionic liquids,17–20 alginate gels and liquid crystals.21,22 However, the phase memory time (Tm) of a nitroxide can be shortened by modulation of the hyperfine anisotropy by molecular librations to such an extent that pulse EPR signals are not observable, limiting the ability to investigate molecular motion at high temperatures using nitroxide probes. Triarylmethyl radicals (TAMs) represent a class of spin probes/tags possessing narrow EPR lines and long electron spin relaxation times.23,24 Consequently, TAM radicals are widely used in EPR tomography,25 oxymetry,26 and pH measurements.27 Recently, TAM radicals began to be used as spin labels in structural EPR studies of biomolecules.28–32 Their unique properties allow single-frequency pulsed dipolar EPR techniques31,33–35 even at room temperatures.28,29,36,37 TAM radicals, such as Finland trityl and OX063, have insignificant HFI anisotropy, and their gtensor anisotropy is also negligible at EPR frequencies of 10 GHz or less.23 Therefore, the standard EPR approaches to investigate molecular dynamics, which were developed for nitroxides, are not suitable for TAMs. However, at EPR frequencies greater than 10 GHz, the g-tensor anisotropy of TAM radicals24,33,38 produces field-dependent phase memory times induced by small-angle radical motions, also called stochastic molecular librations. In this work, we demonstrate a new method to study molecular motion in soft matrices using TAM radicals and analysis of the temperature dependence of 1/Tm at X- and Q-bands. In particular, we use TAMs to investigate molecular motion in glassy trehalose, an attractive immobilizer for room temperature dipolar EPR measurements,36,37,39,40 and in ionic liquids, low-temperature glasses retaining great molecular mobility.19 The new TAM-based approach is compared to the previously used method 2 ACS Paragon Plus Environment

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employing spirocyclohexane-substituted nitroxide radicals. For this sake, reference experiments using nitroxides are performed in the same matrices, and advantages of using TAMs are seen.

Figure 1. Chemical structures of the studied nitroxide and TAM radicals (OX063D, DBT and Finland), glassy trehalose and ionic liquids ([C10mim]BF4 and [Bmmim]BF4). Molecular frame (x, y, z) of the TAM and Nitroxide radicals, where gzz is parallel to the z axis.

EXPERIMENTAL The synthesis of the TAM and nitroxide radicals was described in detail previously.24,41 TAM and nitroxide radicals were prepared in glassy trehalose (purchased from Sigma-Aldrich and used as received) by lyophilization. An 80 µL drop of aqueous solutions of 0.65 M trehalose and 1.2 × 10−5 M for TAM radicals or 1 × 10−4 M for the nitroxide radical were placed in 2 mL Eppendorf tubes. Each solution was shock-frozen in liquid nitrogen, quickly transferred to a desiccator and dried under pressure of ~10−4 bar for 3 h. Then, the samples were placed into quartz tubes (outer diameter of 2.8 mm, internal diameter 1.8 mm) and dried at room temperature using a turbomolecular pump (~5 × 10−9 bar) for 78–90 h. The ionic liquids 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmmim]BF4) and 1-decyl-3methylimidazoliumtetrafluoroborate ([C10mim]BF4) were used in our previous work19,42 and synthesized using similar procedures. The TAM radicals were dissolved in the ionic liquids at concentrations of 0.1 mM. Next, each solution was placed in a quartz tube with outer diameter 2.8 mm and was evacuated (10-2 mbar pressure) for 3 hours to reduce the amount of water remaining in the ionic liquids and to eliminate oxygen. Then each sample was treated with 3–5 freeze–pump–thaw cycles, and finally the tube was sealed off under vacuum. Pulse EPR experiments were made on a commercial X/Q-band Bruker Elexsys E580 spectrometer equipped with an Oxford flow helium cryostat and temperature control system. Tm and echo-detected 3 ACS Paragon Plus Environment


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EPR spectra were measured using a two-pulse electron spin echo (ESE) sequence. The π-pulse of 200 ns was used at both X/Q-band for TAM radicals, and the π-pulse of 100 ns was used for nitroxide. Phase memory times were estimated as a delay at which the echo signal decayed by a factor of e. Free induction decay (FID)-detected spectra were recorded at 80 K with /2 pulse of 1000 ns. Simulation of FID-detected EPR spectra were performed using the Easyspin toolkit for MATLAB.43

THEORETICAL BACKGROUND Experimental pulse EPR data for nitroxides in soft matrices usually can be described well by the “isotropic” libration model, in which librational motion occurs around three mutually-perpendicular axes: x, y, and z.17,44 In the fast motional limit 〈∆ 〉 ≪ 1, Tm is described by

≈ 〈∆ 〉

(1)

where 〈∆ 〉 is the mean-squared fluctuation of the angular resonance frequency, which is

proportional to the mean-squared amplitude of libration ; c is the correlation time of the fluctuations. Nitroxide radicals have an

14

N nucleus with relatively strong HFI anisotropy, which

results in anisotropically-broadened EPR spectra. The shift of the resonance frequency caused by librations, and thus the 1/Tm value, depends on the position within the nitroxide spectrum. For instance, for libration around the x-axis of nitroxide, ∆ is:12

∆ ≈ [ − + ℏ

where 56 is the projection of the

14

"!! # "$$

% "&& '(" )* "!! (+," -* "$$ '(" -

]/01234/2

(2)

N-nuclear spin (I = 1), and θ and φ are polar coordinates of the

magnetic field direction in the molecular frame. As a result, librational motions of nitroxide radicals manifest themselves in a dependence of Tm on the field position within the nitroxide spectrum. The comparison of Tm values across the nitroxide spectrum allows one to deduce the motional parameter τc. For nitroxides, the HFI anisotropy exceeds the g-anisotropy at both X- and Q-bands; therefore,

〈∆ 〉 is virtually independent of the microwave frequency in this range. For instance, the 1/Tm values

for nitroxide radicals are nearly the same at X- and Q-band in lipid bilayers45 and in glassy trehalose.46 Model calculations estimate that the difference (∆7 ) in phase memory relaxation rates in different parts of the spectrum at X-band is:20

∆7 =

II − I ≈ 9 · 10 = 〈 〉

(3)

where (II) is the position in the ED spectrum of the nitroxide close to 56 = − 1; and (I) is the field

position close to 56 = 0 (Figure 2).


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Compared to a nitroxide, the trityl radical has virtually no HFI anisotropy,24,33,38 therefore the fielddependent term in ∆ becomes dominant. The 1/Tm values for the trityl radical in glassy matrix can be described as:

≈ ?

∆@ @

∙ 〈 〉 + B

(4)

where γ is a gyromagnetic ratio for the electron, ∆g ≈ gzz − gxx (as the maximal difference between components of g-tensor for TAM) and C accounts for contributions of all field-independent terms. Therefore, for the TAM radicals, comparison of Tm measured at two significantly different magnetic

fields of X- and Q-bands, provides the possibility to evaluate the motional parameter 〈 〉 :

∆@

∆7 = C − D = E @ F ?C − ?D 〈 〉 = G〈 〉

(5)

where D is the g-anisotropy containing factor in units of s-2.

RESULTS AND DISCUSSION In this study we used TAM radicals having different chemical properties: highly polar probe OX063D (also known as OX071) and nonpolar probe DBT (Figure 1). Both TAMs are free of methyl groups with pronounced proton HFI. Therefore, Tm does not experience drastic shortening in the temperature range of 120-200 K, which occurs in some other TAMs owing to rotation of methyl group and consequent modulation of electron-proton hyperfine interactions47 (Figure S1 in SI). Thus, the selected TAMs allow measurements over a broad temperature range, for example, from 80 K to 298 K achieved in this work. For investigation of molecular dynamics we have chosen glassy trehalose and two well-known ionic liquids having different properties: [Bmmim]BF4 which is a good solvent for polar TAM OX063D, and [C10mim]BF4 having long alkyl chain (decyl) and able to dissolve only nonpolar TAM DBT (Figure 1).



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Figure 2. (A) X-band ED EPR spectrum of nitroxide in trehalose. (B) X/Q-band FID-detected EPR spectra of OX063D in trehalose. (C) X/Q-band FID-detected EPR spectra of OX063D in [Bmmim]BF4 (D). X/Q-band FID-detected EPR spectra of DBT in [C10mim]BF4. Blue lines in (B-D) refer to X-band, black lines to Q-band, and red lines to simulations which neglect the 13C satellites. All experiments were done at 80 K.

High-field EPR spectra of TAM radicals may have different shapes depending on their chemical structure, ranging from symmetric to noticeably asymmetric lines.24,33,47 The width and shape of the EPR spectrum of TAM radical are dictated by its g-tensor anisotropy. Therefore, small modifications in the chemical structure of TAM can produce significant changes in the observed EPR spectrum and the principal values of the g-tensor.24 Figure 2 shows FID-detected EPR spectra of OX063D and DBT at 80 K at X- and Q-bands. The EPR linewidth is different for OX063D in glassy trehalose and in [Bmmim]BF4. This indicates that the g-tensor is affected by both chemical structure of TAM and by surrounding matrix. Table 1. Simulation parameters for the studied TAMs in soft matrices. ∆g is gzz – gxx. The g-anisotropy containing factor D was estimated using eq.(5). Scaling factor is introduced for comparison the data with nitroxide. Anisotropic g-strains do not exceed 0.0007. TAM in matrix OX063D in trehalose OX063D in [Bmmim]BF4 DBT in [С10mim]BF4

giso

gxx

gyy

gzz

(∆g/g)2 [·10-7]

D [·1016] / s-2

scaling factor

2.00267

2.00207

2.00274

2.0032

3.18

1.30

3.55

2.0028

2.00245

2.00285

2.0031

1.05

0.43

3.76

2.0027

2.00225

2.0028

2.00305

1.67

0.68

3.15

We simulated the FID-detected spectra to obtain ∆g ≈ gzz – gxx from eq.(5). The simulations were done simultaneously using the same parameters of modeling at the X- and Q-bands; they took into account g-strain, as well as the broadening caused by HFI, as Gaussian or Lorentzian functions for ionic liquids and glassy trehalose, correspondingly. The isotropic g-value giso was measured at room temperature at X-band frequency related to FD in water (more detailed in ref. 48). Therefore the values of gzz and gxx were the principal variables in simulations, gyy was recalculated from gzz, gxx and experimental giso, and the broadenings were additionally adjusted. During the fitting we tried to 6 ACS Paragon Plus Environment

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maximize the difference between gzz and gxx and minimize broadenings. Next, we calculated (∆g/g)2 and estimated the factor D (eq.(5)). Table 1 lists the parameters yielding the best fits. Figure 3 shows the temperature dependence of the phase memory time (Tm) of the TAM radicals in each matrix. At temperatures below 120 K for glassy trehalose and below 80 K for [Bmmim]BF4 the Tm values are practically the same at X- and Q-bands. This indicates that the matrices are essentially rigid. At the onset of matrix movement, the Tm of the TAM probes is then influenced by g-anisotropy modulated by molecular librations (eq.(4)). The sharp increase in the relaxation curve at temperatures greater than 230 K in the ionic liquid is caused by softening of the matrix, hence the libration model is no longer valid.

Figure 3. Upper panel: Temperature dependence of 1/Tm for TAMs in trehalose and ionic liquids. X-band (black squares) and Q-band (red circles). Lower panel: Temperature dependence of ∆W = 1/Tm(Q)-1/Tm(X) for TAM (red circles) and ∆W = 1/Tm(II)-1/Tm(I) for nitroxide (black triangles) in each matrix (indicated in figure).

The temperature dependence of ∆7 for the TAM probes was obtained by subtraction of 1/Tm at Q-

and X-band frequencies. These ∆7H dependences were compared with those for the nitroxide radical in the same matrices. We used the nitroxide with spirocyclohehane substituents adjacent to the NO-moiety (Figure 1) instead of TEMPO or another nitroxide with tetramethyl substituents in order to prevent the enhancement of electron spin relaxation by rotation of methyl groups.41 Librational motion of nitroxides was studied earlier in glassy trehalose,49 and the mobility was found to be strongly dependent on content of residual water. Therefore, we prepared samples with nitroxide in trehalose using the same preparation methods as those implemented for TAM probes. Relaxation curves for nitroxide in trehalose were measured at field positions marked by I and II (Figure 2). We note that the X-band EPR spectra of nitroxide are practically independent of the matrix used (Figure S2 in SI), because the spectrum is mainly determined by HFI anisotropy. The temperature 7 ACS Paragon Plus Environment


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dependences ∆7H = 1/Tm(II) – 1/Tm(I) for nitroxide probes were obtained in this work for trehalose and ionic liquid [C10mim]BF4, whereas the data for [Bmmim]BF4 were taken from 50. The temperature dependences of ∆7 for nitroxide have the same shapes but larger amplitudes compared to those for TAMs, which is assigned to the larger HFI and g-anisotropies in nitroxide. The observed shape of the ∆7H dependence and the temperature at which the rising trend begins are in good agreement for TAM and nitroxide probes in each matrix. This confirms that the molecular mobility of glassy matrices is their intrinsic property, which does not depend on the size and nature of the embedded paramagnetic probe. We stress that herewith we report the first measurement of molecular dynamics using carboncentered radicals of such a large size as ~1 nm.

Figure 4. Upper panel: Temperature dependence of motional parameter τc = ∆W/D for TAM (see Table 1) (red circles) and motional parameter τc=∆W/(9·1016s-2) for nitroxide (black triangles) in trehalose and in ionic liquids. Lower panel: Temperature dependence of the same motional parameters as in upper panel, but using additional scaling coefficient (see text) for TAM.

The temperature dependence of the motional parameter τc was calculated for TAMs (Table 1) as ∆7/(1.3·1016s-2); ∆7/(4.3·1015s-2); ∆7/(6.8·1015s-2) for glassy trehalose, [Bmmim]BF4 and

[С10mim]BF4, respectively, and for the nitroxide probe as ∆7/(9·1016s-2) in each matrix (Figure 4,

top). However, the temperature dependences are not the same for TAM and nitroxide probes. This is not very surprising because the simple librational model does not take into account the differences in molecular size, molecular shape, or intermolecular interactions of the spin probes and the matrix molecules. However, for each matrix, the different probe radicals seem to have the minima and maxima at similar temperatures. Therefore we compared the results for nitroxide and TAM probes by multiplying the TAM-based data by a constant to make them match the data of the nitroxide probe. 8 ACS Paragon Plus Environment

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Surprisingly, this simple scaling turned out to be extremely effective and led to a very good coincidence for the temperature dependences of τc obtained using TAM and nitroxide probes (Figure 4, bottom, and Table 1). Application of the scaling factor shows that TAM and nitroxide probes show the same motional behavior when they are in the same matrix. At the same time, τc in each matrix has its own individual behavior (Figure 4, bottom). The scaling factor for each matrix is approximately the same and lies in the range of 3.0 - 4.0. This suggests that the factor D correctly accounts for differences in gtensor anisotropy for the TAM probes, but, in addition, a correction is needed to account for the difference in molecular size and shape between the nitroxide and the TAMs. The motional parameter in glassy trehalose increases linearly above ~125 K, while in the ionic liquids the roughly linear increase reverses about 150 K where the matrix molecules begin to reorganize.50 TAM probes have several significant advantages for the investigation of molecular dynamics in soft media. The first is the high sensitivity due to their narrow EPR spectrum. This allows one to use lower concentrations or lower mole fractions of the probe compared to nitroxides, and in this way to diminish the perturbation of the matrix properties by probes. In this work, the concentration of TAMs used was about one order of magnitude lower than that of nitroxides. The second advantage of TAMs is the high accuracy of measurement resulting from their long phase memory time. The Tm of nitroxides is very sensitive to surrounding magnetic nuclei, particularly to methyl groups of the matrix.51 The relatively small size of a nitroxide allows matrix nuclei to approach radical spin by ~0.25 nm, thus producing significant dipolar HFIs. Nuclear spin diffusion among the matrix nuclei also modulates the dipolar HFIs, shortening the Tm furthermore. TAMs are much larger than nitroxides, and the radical center is more ‘shielded’ from the surrounding matrix, therefore matrix nuclei can approach it no closer than by ~0.6 nm.52 This is why the dipolar HFIs with matrix nuclei are about an order of magnitude weaker in TAMs than in nitroxides, leading to roughly two orders weaker influence on Tm. As a result, the Tm of nitroxide in ionic liquids was found to be less than a microsecond at T>120 K (Table S1-3 in SI), whereas the Tm of TAMs exceeded a microsecond throughout the whole temperature range even at Q-band. The third advantage of TAM radicals becomes evident when the libration amplitude is high. The smaller anisotropy of TAMs provides that their Tm decreases more slowly compared to nitroxides when librations have large amplitudes, therefore ESE signals of librating TAMs can be measured at much higher temperatures than for nitroxides. Consequently, in general, nitroxide probes are more sensitive to low-amplitude librations due to their greater anisotropy, whereas TAMs are advantageous for investigation of large amplitude molecular motions at high temperatures. Thus, TAMs are a valuable complement to nitroxide probes, as they extend the range of motion that can be studied by pulse EPR techniques. TAMs, as well as nitroxides, can be used for a 9 ACS Paragon Plus Environment


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wide range of matrices by selecting an appropriate probe. In particular, in this work we have demonstrated that OX063D is suitable for polar glasses and DBT is suitable for non-polar glasses. This work was mainly focused on obtaining information on librational motion. However, it also shows that librations influence relaxation differently at different microwave frequencies bands. This occurs because large changes in magnetic field affect the contribution of g-anisotropy to the librationinduced relaxation. The effect is quite relevant for high-field pulsed dipolar EPR spectroscopy (PDS) experiments, e.g., in membranes, where the librations become active at temperatures as low as ~100 K.18,20 Because of their pronounced influence on electron spin relaxation, stochastic molecular librations affect the amount of time required for PDS measurements, and do it to a different extent at different microwave bands. Last but not least, librations are able to provide additional information about the surroundings and localization of the TAM probes in complex systems. CONCLUSION TAM radicals are very unusual molecules due to their slow spin relaxation and narrow EPR spectrum, which make them attractive for applications in structural biology and materials science.53 In this work, we have demonstrated that the Tm of TAMs in soft matrices provides a convenient way to characterize stochastic molecular librations. Relaxation measurements at different EPR frequency bands reveal different contributions of librations to the observed phase memory time. The latter owes to the field-dependent contribution from g-anisotropy modulated by librations. The absence of methyl groups and the strong field dependence of 1/Tm make OX063D and DBT good probes for this purpose. We proposed and demonstrated a new approach for determining motional parameter of librations 〈 〉 and its temperature dependence, based on 1/Tm measurements at X- and Q-bands. We investigated three soft matrices: glassy trehalose and two ionic liquids. The motional parameters 〈 〉 obtained using TAM probes are in excellent agreement with those obtained using nitroxides. The TAM probes have significant advantages compared to nitroxides, which include high sensitivity, high accuracy of the obtained motional parameters, and wider range of available temperatures. The proposed approach may be implemented to probe low-temperature molecular motions of TAM-labeled biopolymers, TAM-doped glasses, membrane systems and ionic liquids.

ASSOCIATED CONTENT Supporting Information available: Electron spin relaxation of Finland trityl at X- and Q-band frequency; Echo detected EPR spectra for nitroxide in trehalose and ionic liquid; The values of phase memory times; Algorithm for study of librations with TAM probe. AUTHOR INFORMATION 10 ACS Paragon Plus Environment

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Corresponding Authors: [email protected] (E.G.B.), [email protected] (O.A.K.), [email protected] (M.V.F.)

NOTES The authors declare that they have no competing financial interests.

ACKNOWLEDGEMENT This work was supported by the Ministry of Education and Science of the Russian Federation (state contract no. 2017-220-06-7355). OAK is grateful for RF President’s Grants (MK-3214.2017.3). This work was supported by program of fundamental research of the Siberian Branch Russian Academy of Sciences "Interdisciplinary Integration Studies" for 2018-2020. The study of libration in trehalose was supported by RSF (No. 14-14-00922). We are indebted to Dr. Igor Kirilyuk (NIOCh SB RAS) for providing us with the nitroxide spin probes.

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Figure 1. Chemical structures of the studied nitroxide and TAM radicals (OX063D, DBT and Finland), glassy trehalose and ionic liquids ([C10mim]BF4 and [Bmmim]BF4). Molecular frame (x, y, z) of the TAM and Nitroxide radicals, where gzz is parallel to the z axis. 74x67mm (300 x 300 DPI)

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Figure 2. (A) X-band ED EPR spectrum of nitroxide in trehalose. (B) X/Q-band FID-detected EPR spectra of OX063D in trehalose. (C) X/Q-band FID-detected EPR spectra of OX063D in [Bmmim]BF4 (D). X/Q-band FID-detected EPR spectra of DBT in [C10mim]BF4. Blue lines in (B-D) refer to X-band, black lines to Q-band, and red lines to simulations which neglect the 13C satellites. All experiments were done at 80 K. 66x53mm (300 x 300 DPI)



Figure 3. Upper panel: Temperature dependence of 1/Tm for TAMs in trehalose and ionic liquids. X-band (black squares) and Q-band (red circles). Lower panel: Temperature dependence of ∆W = 1/Tm(Q)-1/Tm(X) for TAM (red circles) and ∆W = 1/Tm(II)-1/Tm(I) for nitroxide (black triangles) in each matrix (indicated in figure). 86x44mm (300 x 300 DPI)


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Figure 4. Upper panel: Temperature dependence of motional parameter τc = ∆W/D for TAM (see Table 1) (red circles) and motional parameter τc=∆W/(9•1016s-2) for nitroxide (black triangles) in trehalose and in ionic liquids. Lower panel: Temperature dependence of the same motional parameters as in upper panel, but using additional scaling coefficient (see text) for TAM. 91x49mm (300 x 300 DPI)



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Pulse EPR of Triarylmethyl Probes: A New Approach for the

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