Matthew W.
Dale
a,
Daniel J.
Cheney
b,
Claudio
Vallotto
a and
Christopher J.
Wedge
*ab
aDepartment of Physics, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK
bDepartment of Chemical Sciences, University of Huddersfield, Huddersfield, HD1 3DH, UK. E-mail: c.wedge@hud.ac.uk
First published on 8th December 2020
Spin hyperpolarization can dramatically increase signal intensities in magnetic resonance experiments, providing either improved bulk sensitivity or additional spectroscopic detail through selective enhancements. While typical hyperpolarization approaches have utilized microwave irradiation, one emerging route is the use of optically generated triplet states. We report an investigation into the effects of solution viscosity on radical–triplet pair interactions, propose a new standard for quantification of the hyperpolarization in EPR experiments, and demonstrate a significant increase in the optically generated 1H NMR signal enhancement upon addition of glycerol to aqueous solutions.
Optically generated triplet states often have a large initial electron spin polarization which has been utilized directly in both EPR and NMR studies.9 In the solid-state application of triplet polarization to enhance sensitivity in EPR distance measurements using pulsed dipolar spectroscopy have recently attracted significant interest,10–13 whereas in NMR impressive enhancements have been observed for matrix nuclei but are much reduced upon transfer to a solution-state substrate.14,15 In solution although some triplet states can still be relatively long-lived they undergo rapid spin relaxation, hence their EPR spectra are not normally observed and direct exploitation of their initial polarization is difficult. It is however possible for state mixing in the encounter pair formed by a radical and unpolarized triplet to lead to electronic hyperpolarization of a radical.16 The distinguishing feature of this radical triplet pair mechanism (RTPM)17 as compared to other spin chemical mechanisms generating hyperpolarized electron spin states, such as the radical pair, relaxation and triplet mechanisms,9,18 is that hyperpolarization can be generated on a persistent rather than transient radical species. The RTPM therefore has great potential to provide hyperpolarization that can be utilized more generally for sensitivity enhancement, without the complication of radical reaction or decay.
In the RTPM photoexcitation of a suitable dye molecule followed by rapid intersystem crossing generates an excited triplet state which undergoes collisional encounters with a stable doublet radical. During these encounters the energy levels of the combined radical–triplet pair are best described as quartet and doublet states, which are separated by the electron exchange interaction (J), Fig. 1. The doublet levels are rapidly depopulated by spin-allowed transitions to the ground state of the dye molecule making the radical an efficient triplet quencher by the so-called enhanced intersystem crossing (EISC) mechanism.19 Due to spin-state mixing driven by the zero-field splitting of the triplet molecule this process results in generation of net polarization of the radical, which is emissive in the case of a positive exchange interaction.18
Fig. 1 Schematic energy level diagram of a radical and dye molecule. (i) At large separations the spin states of the two molecules are independent. Shortly after photoexcitation triplet sub-levels (T+,T0,T−) of the dye are equally populated with only a small Boltzmann polarization difference (not visible) of the radical levels (α and β). (ii) Upon diffusive encounter the resultant radical–triplet complex occupies exchange split quartet and doublet states. Spin-allowed quenching by the process known as enhanced intersystem crossing (EISC) selectively depopulates the doublet states to produce radical–singlet pair character (iii). Finally the pair separates, with passage through the level anti-crossing (LAC) region resulting in generation of emissive electron spin polarization of the radical (iv), with overpopulation of the |T+α〉 and |T0α〉 states.18 The diagram assumes a constant applied magnetic field resulting in equal Zeeman splitting of the radical and triplet energy levels (gR ≈ gT ≈ 2). |
The majority of early studies of the RTPM focussed on polyaromatic triplet sensitizers in organic solvents, but recently the electron spin polarization efficiency of the RTPM was investigated for a range of xanthene dyes and nitroxide radicals in aqueous solution.20 Not only is generation of hyperpolarization in aqueous solution essential to applications in biological systems, but unusually large electronic polarizations were observed in these systems; in the case of Rose Bengal and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) the net electron spin polarization (Pnet) generated by each radical triplet encounter was reported to be −150 times the thermal electron spin polarization (Peq).20 This large polarization was attributed to slow rotational and translational diffusion of the dianionic Rose Bengal molecule at the neutral pH used. Having already demonstrated transfer of this large electronic hyperpolarization to nuclei,3 in the present study the scope to further enhance the polarization level through increasing the solvent viscosity is considered, and the effects on both hyperpolarized EPR and NMR spectra are reported.
Samples were illuminated in the EPR resonator using a frequency-doubled Nd:YAG (Continuum Surelite SLI-20 Hz) whose beam diameter of 6 mm filled the optical window of the resonator. For synchronisation with the microwave pulse sequence the laser was operated in DAT mode with a fixed Q-switch delay set using a digital delay generator (Quantum Composer QC9512) triggered by the EPR spectrometer. Adjustment of laser power was achieved using a Glan-laser polarizer and half-wave plate (Thorlabs GL10A and WPMH05M-532) in rotation mounts. Laser power levels were checked using a volume absorbing head (Gentec UP19K-15S-VR).
Samples were illuminated using a diode-pumped solid-state laser at 532 nm (MGL-F-532-2W), gated by an optical shutter (Lasermet LS-10) controlled by a TTL pulse from the NMR console. A pair of N-BK7 lenses were used to expand the beam diameter before a multi-order half-wave plate in rotation mount and polarizing beamsplitter (Thorlabs WPMH05M-532 and CCM1-PBS251/M) used to adjust laser power. This was measured using a surface absorbing thermal sensor (Thorlabs S310C). A cylindrical lens was used to fill the optical window of the resonator.
Fig. 2 Delay after flash EPR experiments for (a) solutions with varying amounts of glycerol in the solvent with percentage by volume as indicated (unbuffered) and (b) water solutions with and without pH 7.2 phosphate buffer (two replicates of each condition). Sample composition 0.2 mM Rose Bengal, 0.2 mM TEMPO, deoxygenated by N2 bubbling; illumination 6 mJ per pulse at 532 nm. Dashed lines are fits to the data. As described in the text, triplet concentration was determined based on the buffered solution for which Pnet/Peq = −150 has been reported,20 and was held constant in the other fits to permit the relative value of Pnet/Peq to be determined. The uncertainty in the determined fit parameters is provided in the main text and Table 1. |
All triplets may be considered to form instantaneously on the EPR timescale as intersystem crossing from the singlet to triplet excited state of the dye takes places on a picosecond timescale, and the laser pulse width of approximately 5 ns is comparable to normal microwave EPR pulse widths. The peak emissive signal intensity is reached rapidly as the triplets are quenched by radicals which become spin-polarized, followed by a slower decay to thermal equilibrium governed by electron spin lattice relaxation (timescale T1e). Addition of glycerol notably alters the time profile, lowering the peak magnetization obtained but also significantly extending the lifetime of the polarization.
Molecular oxygen as a ground state triplet is an effective quencher of the triplet state of the dye molecule, competing with quenching by the nitroxide radicals. As shown in Fig. S1a (ESI†) the removal of oxygen from the sample therefore significantly increases the peak magnitude of the magnetization detected. The decay kinetics are also slower in the deoxygenated case as a result of (i) an increase in the effective triplet lifetime 1/kd and (ii) an increase in the electronic relaxation time T1e of approximately 50% in the absence of the Heisenberg exchange contribution from dissolved oxygen (values in ESI†). Sample deoxygenation by nitrogen bubbling was therefore carried out in all cases as described in the Experimental section. This is in contrast to previous work on the Rose Bengal/TEMPO system, and along with the fact that we chose not to use a pH buffer (see below), results in a larger and longer lived magnetization in Fig. 2a than reported in ref. 20.
In order to extract information on the efficiency of generation of electron spin-polarization the time evolution of the magnetization may be modelled using a kinetic approach based on the Bloch equations.20,22,23 While previous work used a simple kinetic scheme we have found that excellent agreement between simulations and experiment can be obtained by using a more detailed photochemical reaction scheme arising from a recent comprehensive study of the photochemistry of Rose Bengal in water.24 The full scheme is as follows:
(1) |
(2) |
(3) |
(4) |
(5) |
(6) |
(7) |
(8) |
(9) |
(10) |
(11) |
(12) |
(13) |
The time-profile of the magnetization 〈SZ(t)〉 was simulated by numerically solving eqn (10)–(13) to produce a fit to the experimental data. For standard aqueous solutions the kinetic parameters relating to Rose Bengal were taken from ref. 24 and the electron spin–lattice relaxation rate T1e was measured using an inversion recovery pulse sequence (Table 1). With this information the MATLAB25 solver ode45 was used to compute the EPR time profiles, with variable parameters as described below determined by using the lsqcurvefit routine to minimise the sum of squares deviation between the simulated and experimental transients. The kinetic parameters used are given in Table S1 (ESI†).
Volume fraction glycerol/% | P net (relative) | Max. seff | T 1e/ns |
---|---|---|---|
0 | 1.92 | 13.6 | 620 ± 20 |
10 | 2.4 ± 0.2 | 12.9 | 670 ± 10 |
20 | 2.29 ± 0.09 | 10.8 | 745 ± 3 |
30 | 1.92 ± 0.04 | 7.4 | 807 ± 2 |
If the initial triplet concentration generated by the laser pulse is known the only remaining variable parameter in fitting the experimental data is the polarization ratio Pnet/Peq. It is theoretically possible to calculate triplet concentrations using the laser illumination intensity, extinction coefficient of the dye and quantum yield of triplet formation. A global fit across multiple illumination intensities ought then to produce a unique value for the polarization ratio,20 however incomplete and non-uniform illumination of the total sample volume contributing to the EPR signal complicates this approach, and calculated triplet concentrations are prone to errors arising from slight variations in laser focus and unquantified optical losses such as refraction from the curved surface of the capillary sample tube. To avoid these difficulties and as the polarization ratio for the Rose Bengal/TEMPO system has already been determined for an aqueous solution to be Pnet/Peq = −150 (ref. 20), this value was taken as a fixed reference and all polarization values are given relative to this system. Delay after flash EPR data for the Rose Bengal/TEMPO system in aqueous solution with the same pH 7.2 phosphate buffer as in this previous work were recorded in duplicate and fitted with the triplet concentration as the sole variable parameter (Fig. 2b), providing a convenient way to determine the initial Rose Bengal triplet concentration as 0.143 ± 0.005 mM in our experimental arrangement with 532 nm illumination at 6 mJ per pulse. When the experiment was repeated under identical conditions but without pH buffer the polarization was longer lived. A fit to this data can only be obtained by reducing the radical quenching rate kq from 1.7 × 109 M−1 s−1 to (8.9 ± 0.3) × 108 M−1 s−1, which holding the previously determined triplet concentration constant requires that Pnet/Peq = −287.6 ± 0.1, an increase of 1.92 times relative to the same sample in a buffered solution. The work described below was therefore carried out using deoxygenated aqueous solutions without pH buffer. The origin of the buffer effect remains unknown but is not thought to be a simple pH effect, the nitrogen bubbled solutions being at close to neutral pH (see ESI†); investigations into pH effects in these samples are ongoing and will be reported elsewhere.
Data were collected for laser pulse energies from 1 to 6 mJ. In order to fit these time profiles while holding all other parameters constant a non-linear variation in triplet concentration with laser pulse energy was necessary. This implies a non-linear scaling of triplet concentration with photon flux, which may be rationalised by considering the known tendency of Rose Bengal to form intermolecular aggregates at concentrations above 1 μM.26,27
Measurements across the same range of laser powers were repeated for each of the solvent mixtures containing added glycerol, without adjusting the laser alignment. The 6 mJ per pulse data are shown in Fig. 2a, with full laser power dependent data sets in Fig. S2 (ESI†). As noted above addition of glycerol to the aqueous solvent leads to a reduction in the peak magnetization and a lengthening of the magnetization decay profile. The prolonged magnetization is a result of a substantial increase of the electron spin–lattice relaxation time with glycerol addition (Table 1). Spin relaxation mechanisms in nitroxides have been studied extensively by Eaton and Eaton, and found to have contributions from spin rotation, modulation of the large g-factor and hyperfine anisotropies of the nitroxide, and a thermally activated process.28,29 Slower spin relaxation in glycerol containing solutions (up to 69% added glycerol) was previously shown to arise from an increase in the tumbling correlation time of the radical, which affects both the spin rotation and modulation of g- and hyperfine anisotropies.29 While the reproducibility of solid-state DNP studies has been reported to be affected by polymorphism of water–glycerol solutions upon freezing to below 150 K,30 to our knowledge there are no reports of polymorphism influencing relaxation properties at ambient temperatures. As our measurements are carried out exclusively at room temperature (∼295 K), over 100 K above the glass transition temperature of the water–glycerol mixtures used and in a composition region not subject to reported liquid–liquid transitions,31 polymorphism is not expected to affect our study. We also note that any clustering of radicals in the water–glycerol mixtures would increase the relaxation rate (T1e)−1 of the radicals in the glycerol containing solutions, which is the opposite behaviour to that we observe.
Increasing the volume fraction of glycerol also affects the triplet lifetime by a reduction in the rate of radical quenching kq and other bimolecular decay processes arising from slower diffusion in the more viscous media. The initial polarization generation from the Rose Bengal triplet therefore occurs over a longer timescale, permitting electron spin relaxation to begin to contribute such that the peak magnetization obtained is reduced. Fits to the data in Fig. S2b–d (ESI†) are obtained assuming the initial triplet concentration for each laser power to be invariant upon addition of glycerol, and using the increased values of T1e measured by inversion recovery. The reduction in the rates of the reaction steps are accounted for by scaling the relevant rate constants by the ratio of viscosities (Table S1, ESI†). In this way excellent fits to the data are obtained with the only free parameter being the polarization ratio Pnet/Peq. The magnitude of the polarization shows a moderate increase upon addition of small amounts of glycerol but at 30% glycerol by volume is identical within error to the sample without glycerol (Table 1), although the peak polarization is lower and polarization lifetime significantly extended. This illustrates the inadequacy of the polarization ratio in isolation for comparison of the polarization characteristics of different systems.
The increase in Pnet with addition of 10% glycerol is in line with earlier experimental results for the 1-chloronaphthalene (1CN)/TEMPO system for which the increase in net polarization with solvent viscosity upon cooling has been extensively studied.32–34 This effect has been related to reduced translational diffusion, enabling the radical–triplet pair to spend more time in the crossing region of the quartet and doublet states. This has led to attempts to increase polarization by chemically linking the dye and radical to further restrict motion.35 The unusually large dynamic electron polarization of the xanthene dyes has also been attributed to the slow diffusional motion of the strongly hydrated dianionic dye molecules, and slow rotational diffusion highlighted as critical to polarization generation to prevent rotational averaging of the anisotropic zero-field splitting interaction that drives state mixing.20
Analytical expressions relating Pnet to the relative diffusion coefficient Dr have been derived for the weak and strong exchange limits,34 with the strong-exchange result Pnet ∝ 1/Dr when Dr → 0 applicable to xanthene/nitroxide systems at X-band.20 Deviations from this simple linear relationship between Pnet and viscosity have previously been observed for 1CN/TEMPO in highly viscous media (η > 20 mPa s). Kobori et al. were able to model the observed viscosity dependence numerically and attributed the deviation to neglected back transitions during slow passage through the level-crossing region.33 Shushin revised the earlier analytical expressions to account for Kobori's data resulting in a simple expression of the form Pnet ∝ 1/(constant + Dr).36 This cannot reproduce the present result of decreasing polarization in the more viscous mixtures, which in fact exhibit Dr values of 2.1 to 6.2 × 10−10 m2 s−1, spanning the linear dependence range of the earlier work. Shushin's analytical expression has been used to calculate the polarization efficiency of a range of dye and radical systems in benzene and water solutions, but we note experimentally observed variations in Pnet between Rose Bengal and Erythrosine-B or between different radicals with Eosin-Y (which we also see in the Rose Bengal case, Fig. S1b, ESI†) were not reproduced.20 At present the range of chemical systems studied experimentally is rather limited with further investigations needed to test the validity of the existing theory and determine the reasons for numerous discrepancies when treating xanthene dyes. Such work is beyond the scope of the present study, so having identified that glycerol addition can alter the magnitude and timescale of electron spin-polarization in the Rose Bengal/TEMPO system we now move on to consider the resultant effects on the optically generated nuclear hyperpolarization arising from cross-relaxation of the electron–nuclear spin system.
Fig. 3 Optically generated enhancement in NMR signal intensity with varying glycerol concentration. (a) Optimization of illumination power for 10 s continuous illumination. (b) NMR spectra recorded in the presence and absence of illumination (4.0 s at 2.0 W) for aqueous solvent with or without added glycerol. (c) Optimization of illumination time for 2.0 W illumination. (d) Variation in enhancement with glycerol content for 4.0 s illumination at powers as indicated. All samples 0.2 mM Rose Bengal and 1.0 mM TEMPO, deoxygenated by nitrogen bubbling and illuminated at 532 nm. As discussed in the text with 2.0 W illumination data for samples containing 30% glycerol were unreliable, hence these points are omitted in (a) and (d) and no data for this mixture appears in (c). The illumination time dependence for all mixtures with 1.0 W illumination can be found in Fig. S3 (ESI†). |
An upgraded optical system permitted preliminary investigation into optimal illumination conditions. Whereas our earlier study with a relatively divergent 520 nm laser diode source was power limited,3Fig. 3a shows that with a collimated 532 nm laser the NMR enhancement of glycerol containing solutions drops for powers over 1.0 W. Given sample flow is used to reduce photodegradation this is likely due to heating effects and shorter illumination periods were therefore trialled to reduce the heating duty cycle. As shown in Fig. 3c the enhancement achieved is increased further by using a higher laser power of 2.0 W gated to 4 s of illumination immediately prior to the NMR acquisition which was repeated at a fixed rate of 0.1 Hz. It can be seen that even for 2 s illumination (20% duty cycle) a significant enhancement is achieved, and such conditions will significantly reduce the likelihood for sample heating and potentially permit recovery of a reversibly photobleached sample. Data for lower illumination power (1.0 W) also showed significant enhancement with reduced duty cycle, and signs of a plateau for longer illumination times (Fig. S3, ESI†). The scope for maintaining enhancements using a pulsed laser to produce much lower duty cycle trains of pulses has been discussed elsewhere and remains a target for future experimental investigation.23
As noted elsewhere the build-up time for the optical enhancement is expected to be governed by the nuclear relaxation time T1n,23,37 values for which are given in Table 2. While one might therefore expect maximal polarization to be achieved with illumination for ∼5T1n, corresponding to 3.5–7.0 s in the present case, illumination longer than 4 s reduced the enhancement obtained. This is likely the result of unwanted heating effects when using a high power illumination source, similar to observations of Liu et al. who noted polarization build up at twice the expected rate.37 Laser induced heating was particularly problematic in the most viscous solvent mixture when using the highest illumination power of 2.0 W, with data showing poor reproducibility under these conditions. For this reason only data recorded with illumination powers of up to 1.5 W are presented for samples containing 30% glycerol by volume.
Volume fraction glycerol/% | T 01n/s | T 1n/s | f | ξ |
---|---|---|---|---|
0 | 2.59 ± 0.05 | 1.39 ± 0.05 | 0.46 ± 0.02 | 0.37 |
10 | 2.11 ± 0.06 | 1.19 ± 0.03 | 0.44 ± 0.02 | 0.34 |
20 | 1.72 ± 0.05 | 0.96 ± 0.02 | 0.44 ± 0.02 | 0.30 |
30 | 1.13 ± 0.04 | 0.70 ± 0.01 | 0.38 ± 0.01 | 0.24 |
To rationalize the variation in NMR enhancement with addition of glycerol, Fig. 3d, it is necessary to introduce the Overhauser formula:38
(14) |
Before considering the change in electronic hyperpolarization with glycerol addition the effect on the other parameters in eqn (14) must be accounted for. The leakage factor f = 1 − T1n/T01n, where the superscript indicates the relaxation time in the absence of radical, was obtained from 1H NMR inversion recovery data. Both the nuclear relaxation time and leakage factor decrease upon addition of glycerol (Table 2). The coupling factor is also expected to decrease with increasing viscosity, being strongly dependent on the tumbling rate of the molecules. Using the force free hard sphere (FFHS) approximation introduced by Hwang and Freed39 it is possible to calculate the spectral density and hence coupling factor, from the correlation time τc of the radical.40,41 While the Han group use this approach to determine local water dynamics from enhancement values in microwave-driven Overhauser DNP,41 here we estimate the variation in coupling factor upon glycerol addition (Table 2) by using literature values of the closest approach distance and diffusion constants for the radical and water molecules,40,41 and scaling these according to the changing solution viscosity. Details of the spectral density function used and discussion of this choice are provided elsewhere.23
Considering the leakage and coupling factors both reduce on addition of glycerol it is clear that the increased NMR enhancement must arise from a significantly increased saturation factor. By rearranging eqn (14) we determined the effective saturation factor in optical DNP experiments (Fig. 4a). With 1.0 W illumination the saturation factor increases across the series as the glycerol content is increased, implying that the maximal enhancements recorded in the 20% glycerol case arise from the interplay of increasing saturation factor with the decreasing leakage and coupling factors. A similar compromise between saturation and leakage factors was previously recognised as determining optimal radical concentrations for the optical DNP method.3
Looking at the absolute value of the saturation factor in all cases seff < 0.1, with only a small increase upon doubling the illumination power from 1.0 W to 2.0 W, hence the optical DNP method does not yet compete with microwave driven methods for which seff ∼ 1 has been achieved.42–45 Further optimization of the polarizing system is therefore required, for which it will be useful to quantify the extent of electronic hyperpolarization achieved in different systems. It is notable that the saturation factor for 1.0 W illumination is higher in the 30% glycerol case than in the absence of added glycerol, despite the relative electronic polarization per quenching event Pnet being within experimental uncertainty identical for these two volume fractions (Table 1). An alternative measure of the efficiency of optical polarization is therefore needed.
The time integral of seff obtained from delay after flash EPR measurements is shown in Fig. 4b as a function of glycerol content. The value of this integral increases with the volume fraction of glycerol because although the peak magnetization progressively decreases, the polarization is also becoming longer lived. In this case the qualitative trend appears to mirror that in seff obtained from optical DNP measurements (Fig. 4a), though there is a discrepancy for the highest glycerol content. The approximation that the viscous solvent mixtures can be treated according to a single bulk diffusion constant in calculating the coupling factor and hence optical DNP saturation factor is a potential source of this discrepancy, and it should be recalled that for this volume fraction the polarization Pnet deviated from predicted trends. A quantitative correspondence between the saturation factors obtained from optical DNP and pulsed EPR studies has not yet been established and these measurements necessarily correspond to different illumination conditions and radical concentrations. It is, however, proposed that as a directly measurable quantity that reflects a combination of both the magnitude and persistence timescale of RTPM generated electronic hyperpolarization, the time-integrated electronic magnetization is a parameter that should be considered in the search for optimal polarization systems for the future implementation of the optical DNP method.
To increase the effective saturation factor in optical DNP the electronic polarization step must be optimized. Reliance on diffusive encounters between the dye and radical was already identified as a limiting factor, leading the saturation factor to be maximised at low radical concentrations that give a sub-optimal leakage factor.3 This may be alleviated by linked dye–radical systems, with development of such polarizers targeted at improved liquid-state optical DNP performance already underway.35,48,49 As we have shown such efforts should consider not only the magnitude of the electronic polarization generated but also the persistence timescale of this polarization, as this significantly impacts upon the electronic saturation factor achieved under DNP conditions. At present a ten-fold increase in the saturation factor is needed to allow optical pumping to match the theoretical enhancement limit of microwave driven Overhauser DNP, provided the leakage and coupling factors can be simultaneously maximised to ensure efficient transfer of the optically generated electronic polarization to nuclei. The coupling factor for any new system requires direct measurement as has been demonstrated using the pulsed electron–electron double resonance (ELDOR) method for the fullerene-nitroxide polarizing agents developed by Bennati and co-workers, although ultimately the optical polarization in these systems was low.37,44 To simultaneously optimize all of these requirements in a single photo-stable system is undeniably challenging, but reflecting on the significant advances made in rational design of bi-radical polarizing agents for solid-state DNP illustrates what might be achieved through concerted synthetic, spectroscopic and theoretical efforts.50
Footnote |
† Electronic supplementary information (ESI) available: Further experimental details, simulation parameters and additional data. See DOI: 10.1039/d0cp04012f |
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