Hyperpolarization
Parahydrogen Induced Polarisation (PHIP) refers to a set of techniques which utilise para-enriched hydrogen as a source of nuclear spin hyperpolarisation to enhance the sensitivity of NMR. The origin of the NMR signal enhancement in PHIP lays in the singlet order of molecular hydrogen, i.e. the imbalance between the populations of the symmetric (ortho) and antisymmetric (para) spin configurations relative to thermal equilibrium. When transferred to substrate molecules by a chemical reaction (e.g. hydrogenative PHIP) or transient association (i.e. SABRE), this singlet order can determine a large increase of nuclear spin polarization and, consequently, of NMR signal.
nhPHIP chemosensing
Over the last years, we have developed a non-hydrogenative PHIP (nhPHIP) method to detect and quantify analytes in complex mixtures such as biofluids and food extracts down to sub-micromolar concentrations, well below typical NMR limits of detection. This technique is based on the reversible association at high magnetic field of analyte molecules to a complex of iridium with an N- heterocyclic carbene ligand, such as the Ir-IMes catalyst, in the presence of para-enriched hydrogen (p-H2) and of an excess of co-substrate.
Figure nhPHIP-NMR chemosensor: at high magnetic field the Ir-Imes catalyst reversibly interacts with dilute analytes (sub), an excess of co-ligand (co-sub) and para-enriched hydrogen in solution. Red font for H2 in solution indicates singlet order, while black font indicates thermal hydrogen. Red font for the dihydride indicates longitudinal spin order.
Upon association to the catalyst, the singlet order from p-H2 is transferred to the iridium dihydride in the transient complex, converted to enhanced hydride magnetization by a proper RF-excitation scheme and NMR detected. Fast repolarization of the hydrides can be achieved by bubbling p-H2 through the solution, which allows the acquisition of multiscan, multidimensional hyperpolarized NMR spectra.
For each analyte that associates with the iridium catalyst, two hyperpolarized hydride signals at well-defined chemical shifts are observed. These hyperpolarized hydride signals act, therefore, as probes to indirectly reveal the presence of specific analytes in solution. Importantly, since hydrides resonate well below −20 ppm, a region of the 1H spectrum that is generally empty, they do not overlap with the signals originating from the sample matrix. Submicromolar sensitivity and the absence of matrix spectral background render the nhPHIP-NMR technique highly suitable for the detection of dilute analytes in complex mixtures, with minimal sample treatment.
Enhanced hydride resonances can be resolved by 2D zero-quantum (ZQ) spectroscopy: hydride signals corresponding to different analytes can be spread in the indirect dimension, according to their zero-quantum frequency (i.e. the frequency difference within the dihydride).
We have applied this 2D nhPHIP-NMR technique to investigate the dilute, heteroaromatic metabolites in human urine.
Figure (left) 2D nhPHIP-Zero-Quantum hydride spectrum for a solid phase extract (SPE) of human urine in methanol-d4, in the presence of 0.8 mM of iridium catalyst, 15 mM of mtz (co-substrate) and 5 bar of 51% p-H2. The 1D trace displays the hydrides’ signals of nicotinamide. (right) Quantification of nicotinamide in the SPE by standard addition.
2D nhPHIP-NMR has also been used for the detection and enantiomeric discrimination of -amino acids in biofluids and food extracts without any fractionation, purification or functionalization step.
Figure Region of a 2D nhPHIP-Zero-Quantum hydride spectrum recorded on a sample of goat yoghurt, in the presence of 0.8 mM of iridium catalyst, 15 mM of (S)-nicotine (co-substrate) and 5 bar of 51% p-H2. The assignment of alpha-aminoacid is indicated.