New NMR tools for the analysis of two-phase liquid samples: molecular migration and parahydrogen hyperpolarisation

In biphasic samples, solutes distribute between the two immiscible phases according to their relative affinity. Characterising such samples is essential for many applications, and knowing the composition of each phase is fundamental. NMR spectroscopy is a powerful, non-invasive tool for analysing a wide range of samples. However, its intrinsic low sensitivity limits detection to concentrated or long-lived species. Several complementary methods address this limitation, including hyperpolarisation techniques, which temporarily enhance the induced signal. In this work, we focused on a hyperpolarisation method based on the reversible exchange of species to be polarised with parahydrogen on a catalyst. However, this step is more efficient in organic solvents, which are rarely compatible with biological applications. Therefore, transferring hyperpolarised substrates from an organic to an aqueous phase is a proposed solution. In this context, we studied biphasic systems, aiming to rapidly determine the composition of each phase, considering the decay of hyperpolarisation, while simultaneously analysing the entire sample. Localised NMR spectroscopy emerged as the ideal tool for this purpose. We developed a chemical shift imaging pulse sequence to map nuclear displacements based on the spatial position of solutes. To reduce acquisition time for the second dimension and avoid depolarising all spins during the first acquisition, we used radial slice selection (i.e., longitudinal slices rotating around the main axis). This allowed us to obtain 2D spectra of hyperpolarised pyridine in both phases of a water/dichloromethane system within seconds. Additionally, we introduced improvements for better phase separation after shaking (required for parahydrogen gas solubilisation) and enhanced transfer of hyperpolarised substrates to the aqueous phase of interest. Since biphasic sample analysis is not limited to hyperpolarised systems, we also proposed a method to track molecular migration across the entire sample, exemplified by acetonitrile migration from the top of the aqueous phase to the denser dichloromethane phase. A mathematical model was developed to analyse kinetic curves, enabling quantitative observation of intra- and interphase behaviours. Finally, we designed an NMR diffusion sequence to spatially measure the diffusion coefficients of substrates in biphasic systems. A calibration method for magnetic field gradients was proposed to improve measurement accuracy at the detection zone edges, where gradients are typically weaker. This work introduces novel methodologies for studying biphasic systems using NMR. Analysing solute migration between phases is of major interest in chemistry, for both synthesis and purification steps. Coupling NMR with hyperpolarisation enables the detection of low-concentration molecules in a phase, with signals enhanced by several orders of magnitude. This gain in sensitivity and contrast can be leveraged to better describe complex systems, with applications in chemistry and healthcare.