# Hyperpolarised \({}^{1}\)H-\({}^{13}\)C Benchtop NMR Spectroscopy
## 2 Materials and Methods  
SABRE-hyperpolarised NMR spectra are acquired by dissolving H\({}_{2}\) gas, enriched in the _para_ state, in a solution containing the active SABRE catalyst and the target analyte within a weak polarisation transfer field (PTF) typically in the range of 0-20 mT [59; 60]. The introduction of H\({}_{2}\) into the solution is achieved either manually, via sample shaking, or via bubbling. In the so-called shake-and-drop approach, the headspace of an NMR tube fitted with a Young's tap is filled with several bars of _p_-H\({}_{2}\). The sample is shaken vigorously within the desired polarisation transfer field (PTF) for a period of a few seconds, allowing for the buildup of hyperpolarised analyte in solution. This polarisation transfer step is followed by rapid manual transfer of the sample into the NMR spectrometer for signal detection. In the automated flow-based approach, _p_-H\({}_{2}\) is bubbled through the solution for a period of seconds in a reaction chamber that sits within a small electromagnet that provides the desired PTF [61; 62]. Following polarisation transfer, the sample is flowed pneumatically under a pressure of N\({}_{2}\) gas into the NMR spectrometer for detection. In general, the automated approach provides a lower SABRE enhancement due to a combination of engineering limitations including less efficient mixing, longer sample transfer times, and lower levels of _p_-H\({}_{2}\) enrichment in solution during bubbling. However, the flow system provides control over parameters such as the duration of polarisation, PTF, and sample transfer time and therefore can generate the level of reproducibility required to achieve SABRE-enhanced 2D NMR spectroscopy. The level of reproducibility of this system has been assessed previously which gave experimental variation of \(\sim\)5% [49]. In this work, the 1D NMR spectra acquired to investigate the efficacy of different \({}^{13}\)C NMR detection strategies were carried out using the manual shaking approach and the SABRE-enhanced 2D spectroscopy was achieved using an automated flow system. For manual SABRE experiments, the sample was made up in a NMR tube fitted with a Young's valve. The sample contained 260 mM of 4-methylpyridine (4-MP) and 5.2 mM of [IrCl(COD)(IMes)] pre-catalyst (where COD is 1,5-cyclodextrinadiene and IMes is 1,3-bis(2,4,6-trimethyl-phenyl)-imidazolium) made up to 0.6 mL with methanol-\(d_{4}\). The sample was de-gassed under vacuum using a freeze-pump-thaw method (detailed by [PERSON] et al. [63] but with liquid N\({}_{2}\) replaced with a dry-ice acetone bath) to allow the sample to be placed under an atmosphere of _p_-H\({}_{2}\) during SABRE experiments. Repeat shake-and-drop experiments were performed on a single sample by evacuating the head space and refilling with _p_-H\({}_{2}\) between experiments. _Parahy_drogen was generated by cooling H\({}_{2}\) gas over a paramagnetic catalyst based on activated charcoal at 28 K (with a conversion efficiency of 99%). The design of this generator has been described previously in Reference [64]. A handheld magnetic array with a 6.1 mT field strength was used to supply the necessary PTF during SABRE transfer [65]. The sample shaking time was 10 s and the sample transfer time was 2.0 \(\pm\) 0.5 s in all experiments. On the addition of _p_-H\({}_{2}\) to the solution, the pre-catalyst will convert to the active form,[Ir(IMes)(H)\({}_{2}\)(4-MP)\({}_{3}\)]Cl. Full conversion to the active form is required prior to achieving quantitative SABRE results. The activation was monitored by acquiring 6 repeat \({}^{1}\)H shake-and-drop experiments over a typical period of 10 min, with the addition of fresh \(p\)-H\({}_{2}\) to the headspace of the NMR tube between each experiment. Automated SABRE experiments were performed on a 3 mL solution containing 4-methylpyridine (260 mM) and 5.2 mM of the pre-catalyst [IrCl(COD)(IMes)] in methanol-\(d_{4}\).