Loss of Volatile Metabolites during Concentration of Metabolomic Extracts.

Volatile metabolites can be lost during the preanalytical stage of metabolomic analysis. This work is aimed at the experimental and theoretical study of mechanisms of volatile substance evaporation and retention in the residues during the drying of extract solutions. We demonstrate that solvent evaporation leads to the unavoidable loss of nondissociating volatile metabolites with low boiling points and high vapor pressures (such as acetone and ethanol). The retention of dissociating volatile compounds (primarily organic acids RH) during the evaporation depends on the presence of buffer salts in solution, which are responsible for maintaining the neutral pH. An acid remains in the solution as long as it is present predominantly in the dissociated R- state. At the very last stage of solvent evaporation, buffer salts precipitate, forming a solid matrix for metabolite trapping in the residue. At the same time, buffer precipitation leads to a decrease of the solution pH, increase of the portion of RH in associated state, and acceleration of RH volatilization. The RH recovery is thus determined by the competition between the solute volatilization in the associated RH form and metabolite trapping in the solid matrix. The retention of volatile acids in the residue after extract drying can be improved either by adding buffer salts to maintain high pH or by incomplete sample drying.

Magnetic field dependence of the para-ortho conversion rate of molecular hydrogen in SABRE experiments.

The use of parahydrogen - the isomer of molecular hydrogen with zero nuclear spin - is important for promising and actively developing methods for spin hyperpolarization of nuclei called parahydrogen induced polarization (PHIP). However, the dissolved parahydrogen in PHIP experiments quickly loses its spin order, resulting in the formation of orthohydrogen and reduction of the overall nuclear polarization of the substrate. This process is due to the difference of chemical shifts of hydride protons, as well as spin-spin couplings between nuclei, in the intermediate catalytic complexes, and it has not been rigorously explained so far. We proposed a new experimental technique based on magnetic field cycling for measuring the rate of molecular hydrogen para-ortho conversion in solution and applied it for non-hydrogenative PHIP Signal Amplification By Reversible Exchange (SABRE) experiments. The para-ortho conversion rate was measured over a wide range of magnetic field from 0.5 mT to 9.4 T. It was found that the conversion rate strongly depends on the magnetic field in which the reaction occurs, as well as on the concentrations of reactants. The rate decreases with increasing the concentration of pyridine ligand and increases with increasing the concentration of iridium catalyst. The model, which takes into account the reversible exchange of molecular hydrogen with the catalyst, nuclear spin-spin interaction of hydride protons with nuclei of ligands within catalytic complex and nuclear Zeeman interactions, qualitatively describes the experimental data. Two types of complexes with different spin system symmetry contribute to the molecular hydrogen conversion. In asymmetric complexes possessing hydride protons with different chemical shifts due to the presence of chlorine anion ligand the para-ortho conversion rate increases with magnetic field, while for symmetric complexes this mechanism is not operable. In the magnetic field where level anti-crossing occurs the resonant feature for the rate of para-ortho conversion is found. The results of this work can be utilized for finding the optimal conditions for obtaining the maximum hyperpolarization in the experiments employing parahydrogen.

The influence of hindered rotation on electron transfer and exchange interaction in triarylamine-triptycene-perylene diimide triads.

Stretched electron-donor-bridge-acceptor triads that exhibit intramolecular twisting degrees of freedom are capable of modulating exchange interaction (J) as well as electronic couplings through variable π-overlap at the linear bond links, affecting the rate constants of photoinduced charge separation and recombination. Here we present an in-depth investigation of such effects induced by methyl substituents leading to controlled steric hindrance of intramolecular twisting around biaryl axes. Starting from the parent structure, consisting of a triphenyl amine donor, a triptycene (TTC) bridge and a phenylene-perylene diimide acceptor (Me0), one of the two phenylene linkers attached to the TTC was ortho-substituted by two methyl groups (Me2, Me3), or both such phenylene linkers by two pairs of methyl groups (Me23). Photoinduced charge separation (kCS) leading to a charge-separated (CS) state was studied by fs-laser spectroscopy, charge recombination to either singlet ground state (kS) or to the first excited local triplet state of the acceptor (kT) by ns-laser spectroscopy, whereby kinetic magnetic field effects in an external magnetic field were recorded and analysed using quantum dynamic simulations of the spin dependent kinetics of the CS state. Kinetic spectra of the initial first order rate constants of charge recombination (k(B)) exhibited characteristic J-resonances progressing to lower fields in the series Me0, Me2, Me3, Me23. From the quantum simulations, the values of the parameters J, kS, kT and kSTD, the singlet/triplet dephasing constant, were obtained. They were analysed in terms of molecular dynamics simulations of the intramolecular twisting dynamics based on potentials calculated by density functional theory. Apart from kT, all of the parameters exhibit a clear correlation with the averaged cosine square products of the biaryl angles.

Marcus Cross-Relationship Probed by Time-Resolved CIDNP.

The time-resolved CIDNP method can provide information about degenerate exchange reactions (DEEs) involving short-lived radicals. In the temperature range from 8 to 65 °C, the DEE reactions of the guanosine-5'-monophosphate anion GMP(-H)- with the neutral radical GMP(-H)•, of the N-acetyl tyrosine anion N-AcTyrO- with a neutral radical N-AcTyrO•, and of the tyrosine anion TyrO- with a neutral radical TyrO• were studied. In all the studied cases, the radicals were formed in the reaction of quenching triplet 2,2'-dipyridyl. The reorganization energies were obtained from Arrhenius plots. The rate constant of the reductive electron transfer reaction in the pair GMP(-H)•/TyrO- was determined at T = 25 °C. Rate constants of the GMP(-H)• radical reduction reactions with TyrO- and N-AcTyrO- anions calculated by the Marcus cross-relation differ from the experimental ones by two orders of magnitude. The rate constants of several other electron transfer reactions involving GMP(-H)-/GMP(-H)•, N-AcTyrO-/N-AcTyrO•, and TyrO-/TyrO• pairs calculated by cross-relation agree well with the experimental values. The rate of nuclear paramagnetic relaxation was found for the 3,5 and ß-protons of TyrO• and N-AcTyrO•, the 8-proton of GMP(-H)•, and the 3,4-protons of DPH• at each temperature. In all cases, the dependences of the rate of nuclear paramagnetic relaxation on temperature are described by the Arrhenius dependence.

Magnetic Field Effect in Bimolecular Rate Constant of Radical Recombination.

The influence of magnetic fields on chemical reactions, including biological ones, has been and still is a topical subject in the field of scientific research. Experimentally discovered and theoretically substantiated magnetic and spin effects in chemical radical reactions form the basis of research in the field of spin chemistry. In the present work, the effect of a magnetic field on the rate constant of the bimolecular spin-selective recombination of radicals in the bulk of a solution is considered theoretically for the first time, taking into account the hyperfine interaction of radical spins with their magnetic nuclei. In addition, the paramagnetic relaxation of unpaired spins of the radicals and the non-equality of their g-factors that also influence the recombination process are taken into account. It is found that the reaction rate constant can vary in magnetic field from a few to half a dozen percent, depending on the relative diffusion coefficient of radicals, which is determined by the solution viscosity. It is shown that the consideration of hyperfine interactions gives rise to the presence of resonances in the dependence of the rate constant on the magnetic field. The magnitudes of the magnetic fields of these resonances are determined by the hyperfine coupling constants and difference in the g-factors of the recombining radicals. Analytical expressions for the reaction rate constant of the bulk recombination for magnetic fields larger than hfi (hyperfine interaction) constants are obtained. In general, it is shown for the first time that accounting for hyperfine interactions of radical spins with magnetic nuclei significantly affects the dependence of the reaction rate constant of the bulk radical recombination on the magnetic field.

Analytical solution for the inverting pulses with constant adiabaticity.

The exact solution was found for inverting pulses with constant adiabaticity for spin ½. The analytical relationship between the time-varying frequency of the microwave resonant field (or RF field in the case of NMR) and its amplitude time dependence such that the adiabaticity parameter remains constant for the single isochromat throughout the pulse is found. Comparison with EPR (hyperbolic tangent)-(hyperbolic secant) pulse method was carried out. On the basis of the analytical solution the pulses with different dependences of the microwave field amplitude conserving the constant adiabaticity have been constructed. The pulses exhibit rather sharp inversion selectivity that can be used in the field of EPR, NMR and MRI.

Readout of spin quantum beats in a charge-separated radical pair by pump-push spectroscopy.

Spin quantum beats prove the quantum nature of reactions involving radical pairs, the key species of spin chemistry. However, such quantum beats remain hidden to transient absorption­based optical observation because the spin hardly affects the absorption properties of the radical pairs. We succeed in demonstrating such quantum beats in the photoinduced charge-separated state (CSS) of an electron donor­acceptor dyad by using two laser pulses­one for pumping the sample and another one, with variable delay, for further exciting the CSS to a higher electronic state, wherein ultrafast recombination to distinct, optically detectable products of singlet or triplet multiplicity occurs. This represents a spin quantum measurement of the spin state of the CSS at the time instant of the second (push) pulse.

Exchange interaction in short-lived flavine adenine dinucleotide biradical in aqueous solution revisited by CIDNP (chemically induced dynamic nuclear polarization) and nuclear magnetic relaxation dispersion.

Flavin adenine dinucleotide (FAD) is an important cofactor in many light-sensitive enzymes. The role of the adenine moiety of FAD in light-induced electron transfer was obscured, because it involves an adenine radical, which is short-lived with a weak chromophore. However, an intramolecular electron transfer from adenine to flavin was revealed several years ago by Robert Kaptein by using chemically induced dynamic nuclear polarization (CIDNP). The question of whether one or two types of biradicals of FAD in aqueous solution are formed stays unresolved so far. In the present work, we revisited the CIDNP study of FAD using a robust mechanical sample shuttling setup covering a wide magnetic field range with sample illumination by a light-emitting diode. Also, a cost efficient fast field cycling apparatus with high spectral resolution detection up to 16.4 T for nuclear magnetic relaxation dispersion studies was built based on a 700 MHz NMR spectrometer. Site-specific proton relaxation dispersion data for FAD show a strong restriction of the relative motion of its isoalloxazine and adenine rings with coincident correlation times for adenine, flavin, and their ribityl phosphate linker. This finding is consistent with the assumption that the molecular structure of FAD is rigid and compact. The structure with close proximity of the isoalloxazine and purine moieties is favorable for reversible light-induced intramolecular electron transfer from adenine to triplet excited flavin with formation of a transient spin-correlated triplet biradical F⚫--A⚫+. Spin-selective recombination of the biradical leads to the formation of CIDNP with a common emissive maximum at 4.0 mT detected for adenine and flavin protons. Careful correction of the CIDNP data for relaxation losses during sample shuttling shows that only a single maximum of CIDNP is formed in the magnetic field range from 0.1 mT to 9 T; thus, only one type of FAD biradical is detectable. Modeling of the CIDNP field dependence provides good agreement with the experimental data for a normal distance distribution between the two radical centers around 0.89 nm and an effective electron exchange interaction of -2.0 mT.

Giant magnetic field effects in donor-acceptor triads: On the charge separation and recombination dynamics in triarylamine-naphthalenediimide triads with bis-diyprrinato-palladium(II), porphodimethenato-palladium(II), and palladium(II)-porphyrin photosensitizers.

A series of triads consisting of a triarylamine donor, a naphthalenediimide acceptor, and a palladium photosensitizer bridge was investigated for the photoinduced electron transfer processes and the spin chemistry involved. In this series, the ligand in the palladium photosensitizer was varied from bis-dipyrrinato to porphodimethenato and to a porphyrin. With the porphyrin photosensitizer, no charge separated state could be reached. This is caused by the direct relaxation of the excited photosensitizer to the ground state by intersystem crossing. The bis-dipyrrinato-palladium photosensitizer gave only a little yield (7%) of the charge separated state, which is due to the population of a metal centered triplet state and a concomitant geometrical rearrangement to a disphenoidal coordination sphere. This state relaxes rapidly to the ground state. In contrast, in the porphodimethenato-palladium triads, a long lived (µs to ms) charge separated state could be generated in high quantum yields (66%-74%) because, here, the population of a triplet metal centered state is inhibited by geometrical constraints. The magnetic field dependent transient absorption measurement of one of the porphodimethenato triads revealed a giant magnetic field effect by a factor of 26 on the signal amplitude of the charge separated state. This is the consequence of a magnetic field dependent triplet-singlet interconversion that inhibits the fast decay of the charge separated triplet state through the singlet recombination channel. A systematic comparative analysis of the spin-dependent kinetics in terms of three classical and one fully quantum theoretical methods is provided, shedding light on the pros and cons of each of them.

Magnetic field effect on recombination of radicals diffusing on a two-dimensional plane.

Magnetic Field Effects (MFEs) on the recombination of radicals, which diffuse on an infinite plane, are studied theoretically. The case of spin-selective diffusion-controlled recombination of Radical Pairs (RPs) starting from a random spin state is considered assuming uniform initial distribution of the radicals. In this situation, reaction kinetics is described by a time-dependent rate coefficient K(t), which tends to zero at long times. Strong MFEs on K(t) are predicted that originate from the Δg and hyperfine driven singlet-triplet mixing in the RP. The effects of spin relaxation on the magnetic field are studied, as well as the influence of the dipole-dipole interaction between the electron spins of the RP. In the two-dimensional case, this interaction is not averaged out by diffusion and it strongly affects the MFE. The results of this work are of importance for interpreting MFEs on lipid peroxidation, a magnetosensitive process occurring on two-dimensional surfaces of cell membranes.

Multifrequency Nuclear Magnetic Resonance as an Efficient Tool To Investigate Heterospin Complexes in Solutions.

We report a multifrequency nuclear magnetic resonance (NMR) study of heterospin complexes [Eu(SQ)3Ln], where SQ is 3,6-di(tert-butyl)-1,2-semiquinone, L is tetrahydrofuran (THF), pyridine (Py), or 2,2'-dipyridyl (Dipy), and n is the number of diamagnetic ligands. Multifrequency NMR experiments allowed us to determine the effective paramagnetic shifts of the ligands (L = THF or Py) and the chemical equilibrium constant for [Eu(SQ)3(THF)2]. In addition, we have found a strong magnetic field effect on the NMR line broadening, giving rise to very broad NMR lines at high magnetic fields. We attribute this effect to broadening under fast exchange conditions when the NMR spectrum represents a homogeneously broadened line with a width proportional to the square of the NMR frequency difference of the free and bound forms of L. Consequently, the line width strongly increases with the magnetic field. This broadening effect allows one to determine relevant kinetic parameters, i.e., the effective exchange time. The strong broadening effect allows one to exploit the [Eu(SQ)3(THF)2] complex as an efficient shift reagent, which not only shifts unwanted NMR signals but also broadens them, notably, in high-field NMR experiments. We have also found that [Eu(SQ)3Dipy] is a thermodynamically stable complex; hence, one can study [Eu(SQ)3Dipy] solutions without special precautions. We report an X-ray structure of the [Eu(SQ)3Dipy]·C6D6 crystals that have been grown directly in an NMR tube. This shows that multifrequency NMR investigations of heterospin compound solutions not only provide thermodynamic and kinetic data for heterospin species but also can be useful for the rational design of stable heterospin complexes and optimization of synthetic approaches.

Magnetic field and orientation dependence of solid-state CIDNP.

The magnetic field dependence of Chemically Induced Dynamic Nuclear Polarization (CIDNP) in solid-state systems is analyzed theoretically with the aim to explain the puzzling sign change of polarization found at low fields [D. Gräsing et al., Sci. Rep. 7, 12111 (2017)]. We exploit the analysis of polarization in terms of level crossings and level anti-crossings trying to identify the positions of features in the CIDNP field dependence with specific crossings between spin energy levels of the radical pair. Theoretical treatment of solid-state CIDNP reveals a strong orientation dependence of polarization due to the spin dynamics conditioned by anisotropic spin interactions. Specifically, different anisotropic CIDNP mechanisms become active at different magnetic fields and different molecular orientations. Consequently, the field dependence and orientation dependence of polarization need to be analyzed together in order to rationalize experimental observations. By considering both magnetic field and orientation dependence of CIDNP, we are able to explain the previously measured CIDNP field dependence in photosynthetic reaction centers and to obtain a good qualitative agreement between the experimental observations and theoretical results.

Magnetic field effects in rigidly linked D-A dyads: Extreme on-resonance quantum coherence effect on charge recombination.

Charge recombination in the photoinduced charge separated (CS) state of a rigidly linked donor/bridge/acceptor triad with a triarylamine (TAA) donor, a 1,3-diethynyl-2,5-dimethoxy benzene bridge (OMe), and a perylenediimide (PDI) unit as an acceptor, represents a spin chemical paradigm case of a rigid radical ion pair formed with singlet spin and recombining almost exclusively to the locally excited PDI triplet state (3PDI). The magnetic field dependence of the CS state decay and 3PDI formation kinetics are investigated from 0 to 1800 mT by nanosecond laser flash spectroscopy. The time-resolved magnetic field affected reaction yields spectra of the CS state population and 3PDI population exhibit a sharp and deep resonance at 18.9 mT, indicating level crossing of the S and T+ levels separated by an exchange interaction of J = 18.9/2 mT at zero field. The kinetics are biexponential around the resonance field and monoexponential outside that range. The monoexponential behavior can be simulated by a classical kinetic model assuming a single field dependent double Lorentzian function for the energy gap dependence of all spin conversion processes. The full field dependence of the kinetics has been simulated quantum theoretically. It has been shown that incoherent and coherent hyperfine coupling contribute to S/T+ spin conversion at all fields and that the biexponentiality of the kinetics at resonance is due to a partitioning of the overall kinetics into 2/3 of the singlet hyperfine states exhibiting strong isotropic coupling to T+ and 1/3 of the singlet hyperfine states that do not or only weakly couple isotropically to T+.

Exploiting adiabatically switched RF-field for manipulating spin hyperpolarization induced by parahydrogen.

A method for precise manipulation of non-thermal nuclear spin polarization by switching a RF-field is presented. The method harnesses adiabatic correlation of spin states in the rotating frame. A detailed theory behind the technique is outlined; examples of two-spin and three-spin systems prepared in a non-equilibrium state by Para-Hydrogen Induced Polarization (PHIP) are considered. We demonstrate that the method is suitable for converting the initial multiplet polarization of spins into net polarization: compensation of positive and negative lines in nuclear magnetic resonance spectra, which is detrimental when the spectral resolution is low, is avoided. Such a conversion is performed for real two-spin and three-spin systems polarized by means of PHIP. Potential applications of the presented technique are discussed for manipulating PHIP and its recent modification termed signal amplification by reversible exchange as well as for preparing and observing long-lived spin states.

Theoretical description of spin-selective reactions of radical pairs diffusing in spherical 2D and 3D microreactors.

In this work, we treat spin-selective recombination of a geminate radical pair (RP) in a spherical "microreactor," i.e., of a RP confined in a micelle, vesicle, or liposome. We consider the microreactor model proposed earlier, in which one of the radicals is located at the center of the micelle and the other one undergoes three-dimensional diffusion inside the micelle. In addition, we suggest a two-dimensional model, in which one of the radicals is located at the "pole" of the sphere, while the other one diffuses on the spherical surface. For this model, we have obtained a general analytical expression for the RP recombination yield in terms of the free Green function of two-dimensional diffusion motion. In turn, this Green function is expressed via the Legendre functions and thus takes account of diffusion over a restricted spherical surface and its curvature. The obtained expression allows one to calculate the RP recombination efficiency at an arbitrary magnetic field strength. We performed a comparison of the two models taking the same geometric parameters (i.e., the microreactor radius and the closest approach distance of the radicals), chemical reactivity, magnetic interactions in the RP and diffusion coefficient. Significant difference between the predictions of the two models is found, which is thus originating solely from the dimensionality effect: for different dimensionality of space, the statistics of diffusional contacts of radicals becomes different altering the reaction yield. We have calculated the magnetic field dependence of the RP reaction yield and chemically induced dynamic nuclear polarization of the reaction products at different sizes of the microreactor, exchange interaction, and spin relaxation rates. Interestingly, due to the intricate interplay of diffusional contacts of reactants and spin dynamics, the dependence of the reaction yield on the microreactor radius is non-monotonous. Our results are of importance for (i) interpreting experimental data for magnetic field effects on RP recombination in confined space and (ii) for describing kinetics of chemical reactions, which occur predominantly on the surfaces of biomembranes, i.e., lipid peroxidation reactions.

Exploiting level anti-crossings (LACs) in the rotating frame for transferring spin hyperpolarization.

A method of transferring hyperpolarization among scalar-coupled nuclear spins is proposed, which is based on spin mixing at energy Level Anti-Crossing (LAC) regions. To fulfill LAC conditions a resonant RF-field was applied with properly set frequency and amplitude. In this situation LACs occur between the nuclear spin levels in the rotating doubly tilted reference frame. The validity of the approach is demonstrated by taking as an example the transfer of para-hydrogen induced polarization in a symmetric molecule, whose coupled spin network can be modeled as a four-spin AA'MM'-system with two pairs of 'isochronous' spins. For this spin system LAC positions have been identified; rules for the sign of spin polarization have been established. The dependence of the polarization transfer efficiency on the RF-field parameters and on the time profile of switching off the RF-field has been studied in detail; experimental results are in excellent agreement with the theory developed. In general, exploiting LACs in the rotating doubly tilted frame is a powerful tool for manipulating hyperpolarization in multispin systems.

Highly Efficient Polarization of Spin-1/2 Insensitive NMR Nuclei by Adiabatic Passage through Level Anticrossings.

A method is proposed to transfer spin order from para-hydrogen, that is, the H2 molecule in its singlet state, to spin-1/2 heteronuclei of a substrate molecule. The method is based on adiabatic passage through nuclear spin level anticrossings (LACs) in the doubly rotating frame of reference; the LAC conditions are fulfilled by applying resonant RF excitation at the NMR frequencies of protons and the heteronuclei. Efficient conversion of the para-hydrogen-induced polarization into net polarization of the heteronuclei is demonstrated; the achieved signal enhancements are about 6400 for (13)C nuclei at natural abundance. The theory behind the technique is described; advantages of the method are discussed in detail.

Manipulating spin hyper-polarization by means of adiabatic switching of a spin-locking RF-field.

We propose a technique for transferring the multiplet spin polarization (CIDNP or PHIP, or one created by any other method), which is the mutual entanglement of spins, into net hyper-polarization with respect to the direction of a high magnetic field by slowly (adiabatically) switching-off a strong external RF-field with a specially selected frequency. The net hyper-polarized molecules can then be used in NMR spectroscopy or imaging for strong signal enhancement.

Consistent treatment of spin-selective recombination of a radical pair confirms the Haberkorn approach.

In the present work, we have shown that consistent derivation of the kinetic equations describing the electron spin-selective recombination of radical pairs confirms the conventional Haberkorn approach. The derivation has been based on considering the interaction of the reactive system (radical pair and product state) with the thermal bath. The consistency of this approach has also been substantiated by numerical simulations performed for the purely quantum mechanical model of the recombining radical pair. Finally, we have shown that the quantum Zeno effect on radical pair recombination is not an exclusive feature of the approach recently proposed by Kominis, as it should be present at any rate of the singlet-triplet dephasing in the radical pair, which always accompanies the recombination process.